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Mechanisms of Central Pressor and Tachycardic Effects

of Prostaglandin E2 in Conscious Sheep

presented by

Babetta A. Breuhaus

has been accepted towards fulfillment
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ilECHANISMS OF CENTRAL ERESSOR.AND TACHYCARDIC EETECTS
CE'PRDSIAGLAEDIE’Eé IN CONSCIOUS SHEEP

By
Babetta Ann Breuhaus

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Physiology

1985

 

ABSTRACT

OF CENTRAL PRESSOR AND TACHICARDIC EEEECTS
OF PROSTAGLANDIN E2 IN CONSCIOUS SHEEP

By
Babetta Ann Breuhaus

Conscious, chronically—instrumented, mature female sheep were used
to investigate the central cardiovascular effects of prostaglandin Eb
(PGEZ). Although PGE2 is a potent vasodilator in the periphery,

intracarotid (IC) administration of PGE 1O ng/kg/min, causes blood

2,
pressure and heart rate to increase. These effects of IC PGE2 are not
due to direct action of PGE2 at carotid sinus baroreceptors. The
increase in blood pressure is caused by an increase in alpha—adrenergic
tone in the periphery (Hull and Chimoskey 1984). The increase in heart
rate is caused by a combination of cardiac betaradrenergic activation
and withdrawal of parasympathetic tone to the heart. In conscious
sheep, IC PGE2 (10 ng/kg/min or 100 ng/kg/min) has no effect on arterial
P002 or plasma renin activity (Hull and Chimoskey 1984), plasma
osmolality, or plasma concentrations of sodium, epinephrine,
norepinephrine, or vasopressin. Packed cell volume (PCV) is elevated by

100 ng/kg/min IC PGE but not by 10 ng/kg/min IC PGE

2’ 2'
When PGE2 is given into the lateral cerebral ventricles of conscious
sheep, the maximum increase in blood pressure which can be obtained at

any dose is less than that produced by 10 ng/kg/min IC PGE2, and heart

in.

 

rate is unchanged. The increase in blood pressure can be prevented by
pretreatment with phentolamine in the presence of propranolol. Despite
its less potent cardiovascular action, intracerebroventricular (IVT)
PGE2 has certain effects that are not observed during IC administration
of PGE2. These include pupillary constriction, vocalization, and
production of a dry, hacking cough. At 300 ng/kg/min, IVT PGE2
increases REV and plasma vasopressin concentration, and decreases plasma
sodium concentration. Plasma osmolality and catecholamines are
unchanged.

Intracerebroventricular angiotensin II (All), 50 ng/kg/min,
increases blood pressure, total peripheral resistance, right atrial
pressure, stroke volume, and plasma vasopressin concentration in
conscious sheep. Blockade of the brain renin—angiotensin system with
[Sar1Thr8]AII (sarthran), 1000 ng/kg/min IVT, does not prevent the
pressor responses to IC PGE2 or IVT PGEZ. Conversely, prostaglandhi
synthesis blockade with indomethacin or flunixin. meglumine does not
alter the pressor response to IVT AII. Thus, central PGE2 and All act
independently to increase blood pressure in conscious sheep.

Prostaglandin E2 may participate in cardiovascular regulation
through action within the central baroreflex pathway. Endogenous PGE2
probably' comes from cerebral vascular endothelial cells or is reds
localky in neural tissue; it is unlikeky that it comes from cerebral

spinal fluid.

To GUS

For whom I was too busy

ii

ACKNCHIEDGHENTS

My heartfelt thanks go to John, who created an environment in which the
only limit was my imagination.

Imagination became reality, thanks to "The Girls" (they know who they
are), especially Dana and Helga. They were my best instructors. They
patiently (most of the time) endured my mistakes. They taught me more
than anyone about research in general and about neural control of the
circulation specifically. They worked many long hours with me. Without
them, none of these experiments would have been possible.

Thankyou to the rest of my committee: Dr. George Eyster, Dr. N.
Edward Robinson, Dr. Harvey V. Sparks, Jr. , and Dr. Robert B.
Stephenson.

From Michigan State University, East Lansing, I also want to thank Dr.
Greg Fink for his continued advice and support; Dr. Keith Demarest for
developing and running the catecholamine assay; and Dr. Richard Heisey
for initial help in placing cerebroventricular catheters. From the
University of Michigan, Ann Arbor, I want to thank Dr. Richard Malvin,
who sparked the idea for the method of ventricular catheterization
developed for the studies in this document; and Dr. Roger Grekin, who
assayed my plasma samples for vasopressin concentration.

iii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
I. GENERAL INTRODUCTION

II. GENERAL LITERATURE REVIEW
A. Discovery of PGE
B. Pertinent Peripheral Actions of PGE
1. Effects on vascular Smooth Muscle
2. Renal EEfects
C. Central Actions of PGE
1. Behavior
2. Fever
3. Blood Pressure
4. Circulating Hormones / Body Fluid Regulation
D. Localization and Synthesis of PGE in Brain Tissue
1. PGE Stimulation of Central Neurons
2. Endogenous Formation of Prostagdandins'by Central
Nervous Tissue
3. Transport of Exogenous PGE into Central Nervous
Tissue

III. CHAPTER ONE: PGE DOES NOT ACT AT THE CAROTID SINUS TO
RAISE ARTERIAL PRESSURE IN CONSCIOUS SHEEP
INTRODUCTION
METHODS
Instrumentation
Experimental Procedures
PGE Infusions
Caraiac Output Determinations
verification of Denervation
Statistical Analysis
RESULTS
DISCUSSION

IV. CHAPTER TWO: PARASYMPATHETIC NERVOUS SYSTEM RESPONSE TO

INTRACAROTID PGE IN CONSCIOUS SHEEP
INTRODUCTION2

iv

vii

xii

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21

 

V.

VI.

VII.

METHODS

Instrumentation

Experimental Procedures
PGE Infusions
Muscarinic Receptor Blockade
Beta—Adrenoceptor Blockade
Combined Blockade

Statistical Analysis

TS

Muscarinic Receptor Blockade

BetarAdrenoceptor Blockade

Combined Blockade
DISCUSSION

CHAPTER THREE: INTRACEREBROVENTRICULAR METHOD AND RESPONSES
TO CENTRAL AND PERIPHERAL INFUSIONS OF ANGIOTENSIN II
INTRODUCTION
NEEHODS
Instrumentation
Experimental Procedures
Vasopressin Assay
Statistical Analysis
Verification of Catheter Placement
RESULTS
Angiotensin II Infusions
DISCUSSION

CHAPTER FOUR: COWPARISONS OF INTRACAROTID VS
INTRACEREBROVENTRICULAR INFUSIONS OF PGE2
INTRODUCTION
NETHODS
Instrumentation
Experimental Procedures
Intracarotid PGE Infusions
Intracerebroventricular PGE2 Infusions
Intravenous PGE Infusions
Adrenergic Blocéade of IVT PGE
Catecholamine Assay
vasopressin Assay
Statistical Analysis
Verification of Catheter Placement
RESULTS
Intracarotid PGE Infusions
Intracerebroventricular PGE2 Infusions
Intravenous PGE Infusions
Adrenergic Biocfiade of IVT PGE2
DISCUSSION

2

CHAPTER FIVE: CENTRAL ANGIOTENSIN II AND PGE ACT
INDEPENDENTLY TO INCREASE BLOOD PRESSURE IN CONSCIOUS SHEEP
INTRODUCTION

 

SSEQSESRSSSSESSQQBS

88

METHODS
Instrumentation

'Experimental Procedures
Intracarotid PGE Infusions

Intracerebroventricular Infusions

AIIéReceptor Blockade of Central PGE

Prostaglandin Synthesis Blockade of Central AII
Statistical Analysis
verification of ventricular Catheter PLacement

RESULTS

AIIéReceptor BloCkade of Central PGE
Prostaglandin Synthesis Blockade of

DISCUSSION
VIII. SUMMARY AND CONCLUSIONS
APPENDICES
APPENDIX.A
APPENDIX B
APPENDIX C
APPENDIX D
APPENDIX E
APPENDIX F
APPENDIX G
LIST OF REFERENCES

vi

Central AII

115
117
118
124
131
136
149
159

Table

IIST OF TABLES

Blood Pressure and Heart Rate Responses to Infusion of
PGE , 1O ng/kg/min, either into the External Carotid
Artery Beyond the Carotid Sinus or into the Common
Carotid Artery Past a Denervated Carotid Sinus

Muscarinic blockade with atropine met I bromide (1 mg/kg
IV) during IC PGE2 infusion (10 ng/k min) in eight
conscious sheep

Betaradrenoceptor blockade with propranolol (1 mg/kg +
0.025 ms/kg/min IV) during IC PGE infusion (10
ng/kg/min) in seven conscious she p

Heart rate increases to test boluses of isoproterenol, 5
ug before and 50 ug after experimental agents, during
four experimental protocols in seven conscious sheep

Composition of Artificial Cerebral Spinal Fluid (CSF)

Effct of Intracerebroventricular (IVT) Infusion of
Angiotensin II (AII), 50 ng/kg/min, on Packed Cell
Volume, Plasma Sodium Concentration, Plasma Osmolality,
and Plasma Vasopressin Concentration in Conscious Sheep

Blood Pressure, Heart Rate, and Drinkin Responses to
Peripheral Infusions of Angiotensin II AII)

Hemodynamic, Hematologic, and Humoral Responses to
Centrally-Administered PGE2

Blood Pressure and Heart Rate Before and After Infusion
of PGE2, 10 ng/kg/min, into the Common Carotid Artery
Past an Intact Carotid Sinus or Past a Denervated Sinus,
or into the External Carotid Artery

vii

 

Page

33

47

53

66

76

90

117

Table

C—1.

C—2.

C—5.

C-4.

C-6.

IFJ.

E—2.

 

Page

Blood Pressure and Heart Rate Data from Individual Sheep

from Three Separate Experiments in which Intravenous

Atropine Methyl Bromide was Given Alone (1 mg/ ),

Intracarotid PGE was Given Alone (10 ng/kg/min , and

Atropine Was Given During PGE2 Infusion 118

Blood Pressure and Heart Rate Data from Individual Sheep
from Three Separate Experiments in which Atropine Methyl
Bromide Was Given Alone (1 mg/kg), Intracarotid PGE Was
Given Alone (10 ng/kg/min), and Atropine was Given fore

the Start of PGE2 Infusion 119

Blood Pressure and Heart Rate Data from Individual Sheep
from Three Separate Experiments in which Intravenous
Propranolol was Given Alone (1 mg/kg + .025 mg/kg/min),
Intracarotid PGE Was Given Alone (10 ng/kg/min), and

Propranolol Was Given During PGE2 Infusion 120

Blood Pressure and Heart Rate Data from Individual Sheep
from Three Separate Experiments in which Intravenous
Propranolol Was Given Alone (1 mg/kg + .025 mg/kg/min),
Intracarotid PGE was Given Alone (10 ng/kg/min), and

Propranolol Was Given Before the Start of PGE2 Infusion 121

Blood Pressure and Heart Rate Data from Individual Sheep in
which Intravenous Pro ranolol (1 mg/kg/min + .025 mg/kg/min)
and Atropine (1 mg/kg Were Given During Intracarotid

Infusion of PGE2, 1O ng/kg/min 122

Blood Pressure and Heart Rate Data from Individual Shee in
which Intravenous Propranolol (1 mg/kg + .025 mg/kg/min§
and Atropine (1mg/kg) Were Given Before the Start of

Intracarotid PGE2 Infusion, 1O ng/kg/min 125

Verification of Ventricular Catheter Placement 124

Blood Pressure and Heart Rate Responses of Individual Sheep
to Intracerebroventricular Angiotensin II, 50 ng/kg/min 131

Hemodynamic Responses of Individual Sheep to
Intracerebroventricular Angiotensin II, 50 ng/kg/min,

in Sheep with Electromagnetic Flowmeters on Their Main
Pulmonary Arteries 132

viii

Table
E—5.

E—5.

F—1.

F—2.

F—3.

F—4.

F—5.

F-6.

F—7.

PLB.

IL9.

F—10.

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracarotid Angiotensin II, 10 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intravenous Angiotensin II, 10 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intravenous Angiotensin II, 50 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracarotid PGE2, 0 ng/kg/min (Vehicle)

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracarotid PGE2’ 5 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracarotid PGEZ, 10 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracarotid PGEZ, 30 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracarotid PGEZ, 60 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual

Sheep to Intracarotid PGE2, 100 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual

Sheep to Intracerebroventricular PGEZ, 0 ng/kg/min
(Vehicle)

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracerebroventricular PGEZ, 1O ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracerebroventricular PGEZ, 30 ng/kg/min

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracerebroventricular PGE2, 100 ng/kg/min

ix

 

 

Page

133

134

135

136

137

139

140

141

145

Table
F—11.

F—12.

F—13.

G—2.

G—3.

G—5.

G—7.

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracerebroventricular PGE2, 300 ng/kg/min

Blood Pressure and Heart Rate Responses to
Intracerebroventricular PGE , 750 ng/kg/min, in
Sheep that did not Respond go Lower Doses

Blood Pressure and Heart Rate Responses to
Intracerebroventricular PGE , 1000 ng/kg/min, in
Sheep that did not Respond go Lower Doses

Blood Pressure Responses of Individual Sheep to
Intracerebroventricular Sarthran, 1000 ng/kg/min

Heart Rate Responses of Individual Sheep to
Intracerebroventricular Sarthran, 1000 ng/kg/min

Blood Pressure Responses of Individual Sheep to
Intracerebroventricular (IVT) Sarthran, 1000
ng/kg/min, + IVT Angiotensin II, 50 ng/kg/min

Heart Rate Responses of Individual Sheep to
Intracerebroventricular (IVT) Sarthran, 1000
ng/kg/min, + IVT Angiotensin II, 50 ng/kg/min

Blood Pressure Responses of Individual Sheep to
Intracerebroventricular Sarthran, 1000 ng/kg/min,
+ Intracarotid PGEZ, 10 ng/kg/min

Heart Rate Responses of Individual Sheep to
Intracerebroventricular Sarthran, 1000 ng/kg/min,
+ Intracarotid PGE2, 1o ng/kg/min

Blood Pressure Responses of Individual Sheep to
Intracerebroventricular Sarthran, 1000 ng/kg/min,
+ IVT PGE2, 300 ng/kg/min

Heart Rate Responses of Individual Sheep to
Intracerebroventricular (IVT) Sarthran, 1000 ng/kg/min,
+ IVT PGE2, 300 ng/kg/min

Page

146

147

149

150

151

152

153

154

155

156

Table
G—9.

G—10.

 

Page

Blood Pressure and Heart Rate Responses of Individual

Sheep to Intracerebroventricular Angiotensin II, 50

ng/k min, Two Hours After Subcutaneous Indomethacin,

4 kg 157

Blood Pressure and Heart Rate Responses of Individual
Sheep to Intracerebroventricular Angiotensin II,
50 ng/kg/min, One Half Hour After Intravenous Flunixin

Meglumine, 3 mg/kg 158

xi

 

Figure

 

 

LIST OF FIGURES

Anatomy of the carotid sinus region of the sheep and
placement of the external carotid artery catheter. 27

Verification of carotid sinus denervation by unilateral

carotid artery occlusion. Figure 2 shows arterial

pressure (mmHg) in ri t carotid artery (above) and left
carotid artery (belOW'. The right carotid sinus has been
denervated. Occlusion of the common carotid artery

proximal to the intact left carotid sinus causes a rise

in systemic blood pressure (measured in the right carotid
artery). Occlusion of the common carotid artery proximal

to the denervated right carotid sinus fails to elicit a

carotid sinus baroreflex. 30

A. Hemodynamic changes during control and during external
carotid infusion of PGE , 10 ngykgdmin, in three conscious
Sheep (9 replicates). . Hemodynamic changes during
control and during infusion of PGE2, 1O ng/kg/min, past a
denervated carotid sinus in three conscious sheep (6
replicates). The increase in blood pressure is due to an
increase in total peripheral resistance in both cases.
Asterisks indicate which variables are significantly
different from control 25 minutes after the start of PGE

infusion. 2

34

BloCked comparison of blood ressure during control and

during PGE infusion, 10 n g/min, by three routes:

into the cghmon carotid artery past an intact carotid sinus,
into the common carotid artery past a denervated carotid

sinus, and into the external carotid artery beyond the

carotid sinus (4 conscious sheep/16 replicates per route. 35

xii

Figure

Page

Atropine + , 8 conscious sheep. Blood pressure and
heart rate in ree se arate experiments (all experiments
performed in all sheep comparing the responses to 1 m
atropine IV given alone, to 10 ng/kg/min PGE IC given
alone, and to atropine + PGE given together. Open bars
represent control values. Sgaded bars represent test
values 30 minutes after atropine alone (Atropine), 25
minutes after the start of PGE infusion alone (PGE ),

and 30 minutes after atropine and 25 minutes after he
start of PGE infusion when the two drugs were given
together (Atgopine + PGE ). A. Blood pressure: *=p<.05 re
Control; =p<.05 re Atropine. B. Heart rate: *=p<.05 re
Control; o=p<.05 re Atropine; +=p<.05 re PGEZ.

Propranolol + PGEZ, 7 conscious sheep. Blood pressure

and heart rate in three separate experiments (all
experiments performed in all sheep) comparing the
responses to 1 mg/kg + .025 mg/kg/min propranolol IV given
alone, to 10 ng/kg/min PGE IC given alone, and to
propranolol + PGE given ogether. Open bars represent
control values. Shaded bars represent test values 35
minutes after propranolol alone (Propranolol), 25 minutes
after the start of PGE infusion alone (PGE ), and 35
minutes after propranoIol and 25 minutes er the start
of PGE infusion when the two drugs were given together
(Propranolol + PGE ). A. Blood pressure: *=p<.05 re
Control and Propranolol. B. Heart rate: *=p<.05 re
Control and Propranolol; +=p<.05 re PGEZ. 49

Propranolol (1 mg/kg + 0.025 mg/kg/min IV) + Atropine

(1 IV) + (10 ng/kg/min IC), 7 conscious sheep.
Bars represent values of blood pressure and heart

rate during consecutive administration of substances

during one experiment. Note that administration of
propranolol did not change heart rate or blood pressure.
Addition of atropine five minutes later caused a
significant increase in heart rate with no change in blood
pressure. Subsequent infusion of IC PGE2 caused no further
increase in heart rate and raised blood pressure.

* = p<.05 re Control and Propranolol. + = p<.05 re
Propranolol + Atropine. 50

xiii

 

Figure
8.

10.

10

11.

Page

(10 ng/kg/min 10) + Propranolol (1 lug/kg + 0.025

min IV) +-Atropine (1 mg/kg IV), 7 conscious Sheep.
A. Comparison of changes in blood pressure.
the that the Observed change in blood pressure at 25
minutes PGE infusion + 10 minutes propranolol + 5 minutes
atropine (PGE+PROP+ATRO) is significantly less than the
change in blood pressure calculated (PGE+PROP+ATRO
calculated) from the sum of the observed changes in blood
pressure during PGE alone (PGE), propranolol alone (PROP),
and atropine alone IATRC) . "PROP+ATRO calculated" is the
sum Of PROP + ATRO. *:p<.05 re (PGE+PROP+ATRO calculated).
B. Comparison of changes in heart rate. the that the
observed change in the heart rate at 25 minutes PGE2
infusion + 10 minutes propranolol + 5 minutes atropine
(PGE+PROP+ATRO Observed) is significantly less than the
change in heart rate calculated (PGEHPROP+ATRO calculated)
from the sum of observed changes in heart rate during P
alone (PGE), propranolol alone (PROP), and atropine alone
(ATRO). "PROP+ATRO calculated" is the sum of PROP + ATRO.
*=p<.05 re PGE+PROP+ATRO calculated. 52

Position of ventricular catheter in lateral cerebral
ventricle. 61

Placement of the ventricular catheter. A. s = Skin, co =
coronal suture, m = muscle, and cp = cornual process.

IB. Trephine hole centered between the midline and an oblique
imaginary' line passing through the cornual process. 63

(cont'd.). C. Trephine hole made and screws placed.
D. Catheter (with infusion line attached) ready to
be advanced into brain. 64

Time course of blood pressure response to IVT AII, 50
ngykgymin, in eleven conscious sheep. All points differ

from control and each other (p<.05) except 15 and 20

minutes, which do not differ from each other, and 25 and

30 minutes, which do not differ from each other. 75

xiv

Figure

12.

13.

14.

15.

Intracarotid PGE at three doses. .A. Time course of

blood pressure rSSponses. 'B. Time course of heart

rate responses. Blood pressure and heart rate are
significantly greater than control at all time points

for all doses. At 10 ng/kg/min, PGE reaches steady

state 15 minutes after the start of Infusion. At 30

ng/kg/min, PGE2 reaches steady state at 10 minutes.

At 100 ng/kg/mln, PGE reaches steady state at 5

minutes. (Control values for blood pressure and heart

rate are not significantly different from each other). 89

Time course of blood pressure responses to IVT PGE at

four doses in eleven conscious sheep. For all dos s,

all time points are significantly greater than control

but are not different from eaCh other. Steady state

was reached 5 minutes after the start of infusion.

(Control values for all doses are not significantly

different from each other). 92

Comparison of log dose response data for intracarotid

PGE (solid circles) and IVT PGE (open circles).

Intracarotid PGE produces signigicantly greater pressor

and tachycardic Fesponses than IVT PGE at all doses.

(IVT PGE does not change heart rate). A. Changes in

blood pr ssure. iB. Changes in heart rate. 93

Changes in blood pressure during three experimental
protocols in the same feur sheep. The first bar shows

the change in blood pressure 45 minutes after the
administration of propranolol and 35 minutes after the
administration of phentolamine. The asterisk indicates

that blood pressure at this time point is significantly

less than control, p<.05. The second bar shows the pressor
response of these sheep to IVT PGE , 300 ng/kg/min, 3O
ndnutes after the start of infusiog. The asterisk indicates
that blood pressure at this time point is significantly
greater than control, p<.05. The last bar shows that when
propranolol and pentolamine are given prior to PGE

infusion, there is no change in blood pressure 30 minutes
after the start of PGE2 infusion. 95

Figure
16.

17.

18.

19.

Page

Blood pressure responses to IVT AII, 50 k:g/min from

O to 30 minutes; to IVT sarthran, 1000 min from

-15 to 30 minutes; and to the combination of IVT sarthran
started at -15 minutes and IVT AII started at 0 minutes.

During IVT AII infusion alone, blood pressure is greater

than control after time 0, and reaches steady state at 20
minutes (ie. pressures at times 20—30 minutes are not

different from each other). IVT sarthran and IVT sarthran

+ IVT AII do not alter blood pressure and are not different
from each other. 105

Blood pressure responses to IC PGE , 1O nngg/min, from O
to 30 minutes; and to the combinat on of IVT sarthran, 1000
nngg/min, started at -15 minutes, and IC PGE started at O
ndnutes. There is no difference in the blood pressure
response to IC PGE with or without sarthran pretreatment.
For IC PGE infusign alone, blood presure is greater than
control (pg.05) from 5-30 minutes after the start of
infusion, and reaches steady state at 15 minutes (ie.
pressures at times 15—30 minutes are not different from
each other). For sarthran + IC PGE , blood pressure is
greater than control (p<.05) from 5-30 minutes after the
start of infusion, and reaches steady state at 10 minutes. 106

Blood pressure responses to IVT PGE , 300 ng/kg/min from

O to 30 minutes, and to the combine ion of IVT sarthran,
1000 ng/kg/min started at —15 minutes and IVT PGE started
at 0 minutes. There is no difference in the blood pressure
response to IVT PGE ‘with or without sarthran pretreatment.
For IVT PGE2 alone,2blood pressure is greater than control
at all time points (p<.05) and reaches steady state 15
ndnutes after the start of infusion (ie. pressures from
15—30 minutes are not differint from each other). For
sarthran + IVT PGE , blood pressure is greater than

control (p<.05) at 10—30 minutes. 10'!

Comparison of changes in blood pressure caused by IVT AII
alone, 50 ngfkg/min; caused by IVT AII after pretreatment

with indomethacin, 4 mg/kg subcutaneously; and caused by

IVT AII after pretreatment with flunixin.meglumine, 3

mg/kg intravenously. All increases in blood pressure

differ significantly from control (*:p<.05), but are not
different from each other. 108

I . m INTRODUCTION

When given into the cerebral blood supply or into cerebral spinal
fluid, prostaglandins of the E series (PGES) cause blood pressure and
heart rate to increase (Kaplan et al. 1969; Gyang et al. 1973; Andersson
and Ieksell 1975; Ieksell 1976; Hoffman and Schmid 1979; Hoffman and
Valigura 1979; Kondo et al. 1979; Hoffman et a1. 1981; Skarnes et al.
1981; Takahashi and Bunag 1981b; Okuno et al. 1982; Wu and Wei 1982;
Breuhaus and Chimoskey 1983; Hull and Chimoskey 1984). This observation
has attracted attention for at least two reasons. First, the ability of
PGE8 to raise blood pressure is paradoxical, since PGEs are potent
vasodilators when infused into the arterial inflow of most peripheral
vascular beds (Moncada et al. 1980) . Second, the increase in blood
pressure is accompanied by tachycardia, not bradycardia, suggesting some
form of interference with the baroreflex. Peripheral mechanisms by
which centrally—administered PGEs raise blood pressure have been studied
in a variety of species and will be discussed in the general literature
review which follows.

This thesis extends what is known about peripheral changes in
response to centrally-administered PGE2 in conscious chronically—
instrumented sheep, and then focuses on possible central mechanisms of
the PGE2 pressor response. The questions answered are divided by
chapters, outlined below. Each chapter has its own introduction which
reviews literature specific to that subject, a methods section which
builds on methods introduced in previous chapters, a results section,

1

and a discussion specific to those results. A general discussion of the
possible significance of PGE2 in arterial pressure regulation is

included in the summary and general conclusions .

Chapter 1 examines whether intracarotid PGE2 increases blood

 

pressure by direct action at carotid sinus baroreceptors.
Chapter 2 examines whether withdrawal of parasympathetic tone to
the heart contributes to the increase in heart rate caused by

intracarotid infusion of PGE2.

Chapter 3 discusses development of a method of chronic

 

catheterization of the cerebral ventricles for repeated
infusions in conscious sheep and the use of a drinking response
to intracerebroventricular (IVT) infusion of angiotensin II to
verify proper catheter placement premortem. Changes in blood
pressure , heart rate , plasma vasopress in and sodium
concentrations, plasma osmolality, and packed cell volume during

infusions of angiotensin II also are reported.

Chapter 4 compares hemodynamic and hormonal responses to

 

intracarotid (IC) and IVT PGE2.

Chapter 5 examines whether centally-administered PGE2 raises blood
pressure by causing central release of angiotensin II or whether
centrally—administered angiotensin II raises blood pressure by
stimulating central prostaglandin biosynthesis.

II. GENERAL LITERATURE REVIE

A. Discovery of PGE

Prostaglandins were discovered by Goldblatt (1933, 1935) and von
Euler (1936) in fluid from male accesory sex glands. Prostaglandin E
was first isolated by Bergstrdm and devall (1960) , and its structure
was identified by Bergstrbm, Ryhage, Samuelsson, and devall in 1962.
Prostaglandins are unsaturated fatty acid autacoids formed from
arachidonic acid by a series of enzymatic reactions. The types of
prostaglandins produced by a given tissue are determined by the terminal
enzymes present in the tissue. Since most prostaglandins have very
short half lives (on the order of seconds to minutes) they are not
stored, but are produced as needed. Prostaglandins of the E series are
metabolized rapidly by the lungs (Ferreira and Vane 1967; Moncada et al.
1980), making it possible to separate peripheral effects of PGE fran

central effects .

B. Pertinent Peripheral Actions of PGE
1 . Effects on Vascular Smooth Muscle

 

Although there are some exceptions (primarily in the rat), in
most species and in most vascular beds PGEs relax vascular smooth
muscle. Bergstrdm et al. (1964, 1965) and Carlson and Oro (1966) were
the first to report that peripherally—administered PGE1 decreases blood

pressure and increases heart rate in man and in dog. The hypotensive

3

action of PGE1 in the periphery was greater when given intra—arterially
than when given intravenously.

In anesthetized dogs, Nakano and McCurdy (1967) showed that the
tachycardia produced by intravenous PGE1 is caused by reflex stimulation
of the sympathetic nervous system in response to the decrease in blood
pressure. Administration of PGE1 into the arterial inflow of a variety
of vascular beds increased flow in those beds without changing systemic
blood pressure. These investigators concluded that PGE1 lowers blood
pressure by peripheral vasodilation caused by a direct action of PGE1 on
vascular smooth muscle. The depressor effect of PGE1 is not prevented
by atropine (von Euler 1936; Bergstrdm et al. 1959), antihistamine
(Bergstrbm et al. 1959) . Propranolol (Carlson and Oro 1966; Nakano and
Kusakari 1966), reserpine, or ganglionic blockade (Carlson and Oro
1966). In vitro studies have confirmed a direct relaxant action of PGEs
on vascular smooth muscle (Strong and Bohr 1967) .

In addition to their direct action to relax vascular smooth muscle,
PGE: also affect vascular smooth muscle tone by modulating adrenergic
activity. Although there is some variation by species and by tissue, in
most tissues studied PGEs inhibit adrenergic neurotransmission by acting
presynaptically to decrease norepinephrine release (Hedqvist 1970, 1976;
Kadowitz 1972; Bergstrbm et al. 1973; Frame and Hedqvist 1975; Malik and
McGiff 1975; Malik 1978; Hillier and Templeton 198)). Prostaglandins of
the E series also may act postsynaptically to attenuate pressor re-
sponses to infused norepinephrine (McGiff et al. 1972; Okuno et al.
1982) -

2 . Renal Effects

 

Acute intrarenal infusion of PGE2 increases renal blood flow
without changing systemic blood pressure, increases urine flow, and
increases sodium excretion (Lee 1973). Chronic (7 days) intrarenal
administration of PGE2 in conscious unilaterally-nephrectonized dogs on
a fixed sodium intake increases urine output and water intake without
changing glomerular filtration rate, increases blood pressure, and
increases sodium excretion despite increased plasma renin activity and
increased plasma aldosterone concentration. The increase in blood
pressure can be reversed by blockade of the renin-angiotensin system
(Hockel and Cowley 1979 , 1980) .

There are several mechanisms by which renal PGE2 increases sodium
excretion. Prostaglandin E2 increases renal sodium excretion directly
by acting on renal tubular cells to inhibit sodium reabsorption (Kauker
1977; Stokes and Kokko 1977) . Prostaglandin E2 increases renal sodium
excretion indirectly by changing intrarenal blood flow distribution
(Feigen et al. 1976) , by interfering with vasopressin action on the
distal nephron (Grantham and Orloff 1968; Fulgraff and Brandenbush 1974)
and by interactions with the renin-angiotensin and kallikrein—kinin

systems (Mills et al. 1978).

0. Central Actions of PGE

1 . Behavior

 

The first report of prostaglandins acting in the central nervous
system appeared in 1964. Horton found that PGEs injected into the
lateral cerebral ventricles of conscious cats cause sedation and stupor

lasting as long as 24 hours and signs of catatonia lasting up to 4

hours. Moderate pupillary dilation occurred, although pupillary re—
flexes were normal. The doses of PGE used in this study were high (7-10
ug/kg) . Intravenous injection of PGE into conscious chicks (10—400
ug/kg) also caused profound sedation. At the higher doses the chicks
lost righting reflexes. Horton believed that these results in chicks
were caused by a central action of PGEs, since the blood—brain barrier

is not yet developed in chicks of that age.

2. Fever
Prostaglandins of the E series, but not PGAs or PGFs, cause
hyperthermia accompanied by shivering, skin vasoconstriction, and pilo—
erection when injected into the cerebral ventricles of conscious cats
and rabbits (Milton and Wendlandt 1970 and 1971 ) . The doses of PGEs
used in these studies (10 ng - 10 ug) were lower than those used by
Horton (1964). Signs of catatonia were not observed, although some of
the cats were unresponsive to external stimuli and appeared stuporous
while shivering vigorously. Body temperature began to rise within
minutes after injection of PGE and this increase was not prevented by
the cyclo-oxygenase inhibitor paracetamol. Similar results were ob-
served in sheep by Hales et al. (1973). One hundred ug of PGE1 or PGE2
injected into the lateral ventricles of sheep exposed to cool, thermo—
neutral, or warm environments increased rectal temperature 0.5-1 .20 C.
Shivering occurred in cool and thermoneutral environments. Panting was
decreased in warm environments. Prostaglandins of the F series were

less potent.
The studies of Milton and Wendlandt described above also provided

early evidence for a role of endogenous prostaglandins in the production

of fever. In some experiments there was a late rise in body temperature
within hours after injection of any substance tested (including isotonic
saline). This late rise was prevented or reversed with paracetamol.
Dey et al. (1974) confirmed that fevers which occur nonspecifically
after perfusion of cat cerebral ventricles with various salt solutions
are abolished by paracetamol or indomethacin, and are accompanied by
increased PGE-like activity in cerebrospinal fluid.

The role of PGE as the mediator of fever produced by bacterial
pyrogen or endotoxin has been studied in a variety of species (Eeldberg
and Gupta 1973; Feldberg et al. 1973; Feldberg and Milton 1973; infers et
al. 1974; Phillip-Dormston and Siegert 1974; Veale and Cooper 1974;
Feldberg 1975; Skarnes and McCracken 1980; and Skarnes et al. 1981 ) .
Intracerebroventricular injection of endotoxin in conscious sheep pro—
duces a biphasic fever response (Skarnes et al. 1981). The initial, but
not the secondary, rise in body temperature is preceded by an increase
in systemic blood pressure accompanied by increased concentrations of
PGE and PCP in jugular venous and carotid arterial blood. Pretreatment
with indomethacin prevents the initial pressor effect of endotoxin as
well as both phases of the fever response. In that same study,
intracarotid infusions of PGE2,
(10-2576 beginning 1-3 minutes after the start of infusion) and caused

20—55 ng/kg/min, raised blood pressure

monOphasic fevers (0.9—1.2O C. beginning 3—5 minutes after the start of

infusion).

3 . Blood Pressure

 

The first evidence of a central pressor effect of PGE was re-

ported by Kaplan et al. in 1969. Using cross-circulation procedures,

these investigators found that intracarotid PGE1 increases blood
pressure in neurale-intact vascularly—isolated trunks of recipient dog
with denervated carotid sinuses and carotid bodies. This increase in
blood pressure was prevented by administration of the ganglionic
blocking drug hexamethonium into the recipient dog's trunk. Blood
pressure decreased in response to intracarotid PGE1 in the donor dog.
Blood pressure also decreased in recipient dog trunks with intact
carotid sinuses and carotid bodies. The authors concluded that, in
addition to its depressor action on vascular smooth muscle, PGE1 acts in
the brain to increase blood pressure and acts at the carotid sinus to
decrease blood pressure.

Central pressor effects of PGE1 since have been demonstrated by
infusion into the vertebral arteries of chloralose—anesthetized cats
(Gyang et al. 1973), by administration into cerebral spinal fluid of
conscious goats (Andersson and Ieksell 1975; Leksell 1976), and by
intracarotid infusion in urethane—anesthetized rats (Rinchuse and Deubin
1976) . More recently, PGE2 also has been shown to cause centrally-
mediated increases in blood pressure when given into cerebral spinal
fluid of conscious rats (Hoffman and Schmid 1979; Kondo et al. 1979;
Hoffman et al. 1981; Takahashi and Bunag 1981b; Okuno et al. 1982; Wu
and Wei 1982) or into carotid blood of conscious sheep (Skarnes et al.
1981; Breuhaus and Chimoskey 1983; Hull and Chimoskey 1984), dogs, and
calves (Hull and Chimoskey 1984). Note that pressor effects of
centrally-administered PGE2 have been demonstrated only in conscious
animals; anesthesia inhibits this response (Vilhardt and Hedqvist 1970;
Yamamoto et al. 1976; Takahashi and Bunag 1981b; Hull and Chimoskey
1984).

One exception to this last statement must be mentioned, since it is

the only study in which PGE has been injected into discrete

2
hypothalamic nuclei. In this study (Feurstein et al. 1982), rats were
prepared surgically 3-4 days before use with guide cannulae either into
a lateral cerebral ventricle, into the dorsomedial nucleus of the hypo-
thalamus, or into the posterior hypothalamic nucleus. 0n the day of the
experiment the rats were anesthetized with halothane and oxygen only,
and during injections of PGE2 they were maintained on 0.8% halothane,
which is below the concentration of halothane required for surgical
anesthesia (Minimal Alveolar Concentration, MAC, for halothane is 0.95-
1.1%: Eger 1974). Control blood pressures in these rats were not high
(approximately 90 mmHg) . Intracerebroventricular or intrahypothalamic
injections of PGE2 in these rats caused increases in blood pressure and
heart rate.

Since centrally-administered PGEs cause fever, it is possible that
their pressor effect is simply a by-product of peripheral vasocon-
striction produced as part of the febrile response. It also is possible
that the increase in body temperature results from or is augnented by an
independent effect of PGE on central cardiovascular centers to increase
total peripheral resistance. In studies designed to disassociate the
blood pressure and body temperature responses to intracerebroventricular
PGE

2
PGEZ—induced increase in body temperature is not caused by peripheral

in conscious rats, Hoffman and Valigura (1979) Proved that the

vasoconstriction. Pretreatment with phenoxybenzamine prevented the
pressor response but not the febrile response to PGE2. The reverse
study has not been done.

Although centrally—administered PGrE2 does cause a small increase in

 

cardiac output, the pressor response is mainly caused by an increase in
total peripheral resistance (Breuhaus and Chimoskey 1983; Hull and
Chimoskey 1984) mediated by increased sympathetic nerve activity
(Takahashi and Bunag 1981b), and can be inhibited or reversed by alphas
adrenoceptor blockade (Hoffman and Schmid 1979; Okuno et al. 1982; Hull
and Chimoskey 1984) or cervical section of the spinal cord (Takahashi
and Bunag 1981b). Decreased blood flow in renal, mesenteric, and iliac
beds contributes to the increase in total peripheral resistance (Hull
and Chimoskey 1984)-

The central pressor effect of PGE2 is accompanied by tachycardia
(Hoffman and Schmid 1979; Hoffman. et al. 1981; Takahashi and Bunag
1981b; Feurstein et al. 1982; Okuno et al. 1982; Breuhaus and Chimoskey
1983; Hull and Chimoskey 1984), not bradycardia, suggesting that the
baroreflex's ability to control blood pressure is interfered with. Hull
and Chimoskey (1984) have shown that, over a limited range of blood
pressure, the baroreflex is reset upward with no change in sensitivity.

Further studies on the effect of PGE on the baroreflex will be

2
discussed in Chapter 1.

The PGEz—induced tachycardia is inhibited, but not always abolished,
by the 'betasadrenoceptor antagonist propranolol (Hoffman and Schmid
1979; Hull and Chimoskey 1984). The increase in heart rate which
remains in calves and sheep after adrenergic blockade may be caused by
parasympathetic withdrawal; atropine can attenuate heart rate and blood

pressure responses to intracerebroventricular PGE2 in rats (Hoffman and

Schmid 1979). This possibility will be examined in Chapter 2.

4. Circulating Hormones / Body Fluid Regulation

 

The response of circulating catecholamines to central adminis-
tration of PGE2 has been measured only in rats. In one study, peak
plasma norepinephrine increased approximately 2.5K after intracerebro-

ventricular or intrahypothalamic PGE (Control values ranged from 186

2.
to 294 pg/ml.) Plasma epinephrine did not change significantly
(Feurstein et al. 1982).

In addition to measuring circulating catecholamines before and
during intracerebroventricular injection of PGE2 in conscious rats,
Okuno et al. (1982) also assessed the effect of bilateral adrenahnedul-
lectomy on the PGE2 response. Control values of plasma norepinephrine
(356 i 40 pg/ml) and epinephrine (413 i 90 pg/ml) in this study were
slightly higher than those reported by Feurstein et al. (1982). Intra—
cerebroventricular PGE2 in intact rats increased plasma norepinephrine
2X and plasma epinehrine 2.6X. After adrenalmedullectany, control and
PGE2—induced plasma norepinephrine levels were similar to those found
before adrenalmedullectomy. Plasma epinephrine concentration decreased
to 19 i 6.6 pg/ml after adrenalmedullectomy, and did not change in
response to intracerebroventricular PGEZ. Bilateral adrenalmedullectcmy
also prevented a PGE2—induced rise in plasma renin activity (to be
discussed next). Loss of epinephrine and renin responses to PGE2 in
these rats, however, had no effect on PGEz—induced pressor responses.
Whether there was any effect on the heart rate response was not
discussed.

As alluded to above, centrally—administered PGE‘2 increases plasma
renin activity (Okuno et al. 1982; Hull and Chimoskey 1984), probably by

renal beta—adrenoceptor stimulation from circulating epinephrine.

 

Bilateral renal denervation did not affect the renin response to
centrally-administered PGEZ, propranolol attenuated it, and bilateral
adrenalmedullectomy (which prevented the increase in plasma epinephrine)
prevented the renin response (Okuno et al. 1982). Although this
increase in plasma renin activity may influence longrterm blood pressure
control through fluid balance mechanisms, it does not mediate the acute
pressor effect of centrally—administered PGE2, since intravenous
pretreatment with the angiotensin—converting enzyme inhibitor captopril
does not change the PGEZ-induced increase in blood pressure in rats
(Okuno et al. 1982) or in sheep (Hull and Chimoskey 1984).

Unlike changes in plasma renin activity, changes in plasma vaso—
pressin concentration may participate in the pressor response to
centrally-administered PGEZ. Hypophysectomy halved the pressor response
to intracerebroventricular PGE2 in rats (Takahashi and Bunag 1981b) and
the vasopressin antagonist d(CH2)5Tyr(Me)AVP attenuated this response
(Okuno et al. 1982). In the latter study, alpha—adrenoceptor blockade
alone only attenuated the PGEZ-induced pressor response; the combina—
tion of both vasopressin blockade and alpha—adrenoceptor blockade were
necessary to abolish it. If vasopressin is released during intracarotid
infusion of PGE2 in conscious sheep or calves, it must not participate
in the pressor responses, since adrenergic blockade alone completeLy
abolishes the increases in blood pressure.

Early evidence for the release of vasopressin in response to
centrally—administered PGEs was reported before vasopressin could be
detected by radioimmunoassay. As early as 1970 Vilhardt and Hedqvist
showed that intracarotid PGE2 decreases urine volume and increases urine

osmolality without changing blood pressure in anesthetized, water—loaded

 

 

 

13

rats. Hoffman and Schmid (1979) also reported an antidiuretic effect of
intracerebroventricular PGE2 in conscious water—loaded rats. Median
emminence lesions abolished the PGEZ—induced antidiuretic responses
without changing the cardiovascular responses.

In in vitro studies, Gagnon et al. (1973) demonstrated that PGE2 can
stimulate vasopressin release from isolated rat neurohypopheses. Vasc—
pressin concentration was determined in this study by bioassay; blood
pressure responses to extracts of the hypophyseal bathing medium were
compared to pressor responses to synthetic vasopressin in anesthetized
assay rats.

Rat bioassay also was used to measure increases in urinary concen—
tration of vasopressin in response to intracerebroventricular infusions

of PGE in conscious, water—loaded goats (Leksell 1976). In this and in

1
an earlier study (Andersson and Ieksell 1975) PGE1 also decreased
urinary free water clearance, increased urinary sodium excretion,
increased blood pressure, and weakly stimulated drinking behavior. Body
temperature did not change.

Yamamoto et al. (1976) used radioimmunoassay to measure changes in
plasma vasopressin concentration in response to ventriculocisternal
perfusion with PGE‘2 at two different rates in morphine—chloralose—
urethane anesthetized dogs. Plasma vasopressin was unchanged at the
lower dose. At the higher dose (152.6 ng/min or approximately 8—15
ng/kg/min) , plasma vasopressin concentration doubled by ten minutes
after the start of infusion and was increased 4.5X by thirty minutes
after the start of infusion. There were no significant changes in
arterial pressure, rectal temperature, plasma sodium or potassium

concentrations, or plasma osmolality. Hematocrit decreased 9%, but a

 

similar decrease with time also occurred during vehicle perfusion.
Despite the increase in plasma vasopressin concentration, there also
were no significant changes in urinary volume, osmolar and free water
clearances, or urinary excretion of sodium or potassium. Some of the
negative results in this study may have been caused by anesthesia.
Nevertheless, it should be noted that in this and in previous studies
cited, it is possible for PGE2 to increase plasma vasopressin
concentration without reaching levels high enough to have a pressor
effect.

In contrast to the last study, the contribution of vasopressin
release to the pressor response of intracerebroventricular injections of
PGE2 in conscious rats has been discussed previously (Takahasi and Bunag
1981b; Okuno et al. 1982). Okuno et al. also measured plasma
vasopressin concentrations by radioimmunoassay. Plasma vasopressin
increased 11.6X from 2.5 i 0.5 pg/ml to 29 i 8 pg/ml. Plasma osmolality

did not change .

D. Localization and Synthesis of PGE in Brain Tissue
1. PGE Stimulation of Central Neurons

 

Avanzino et a1. (1966) examined the effects of prostaglandins on
brain stem neurons from the medulla and caudal pons (0-7 mm rostral to
the obex and within 3 mm from the midline) of decerebrate, unanesthe—
tized cats. Applied by iontophoresis, PGE1 excited 18.5% and inhibited
4% of the 341 spontaneously-firing neurons tested. Prostaglandin E2
excited 27.5% of 69 neurons tested, but had no inhibitory effect.
Although no attempt was made to correlate spontaneous nerve firing to

body function, it is possible (due to the location of the neurons

15
tested) that at least some of them could have been involved in cardio—
vascular regulation. In any event , the study shows that PGEs can stimu-

late central neurons .

2. Endogenous Formation of Prostaglandins by Central Nervous Tissue

 

In order for endogenous mm to influence central cardiovascular
regulation, they must be synthesized by brain tissue, by vascular tissue
supplying the brain, or by blood cells, since prostaglandins are not
stored and since PGEs are removed rapidly from blood and metabolized by
the lungs (Moncada et al. 1980). In early in situ studies, perfusion
fluid from cat cerebral cortex (Ramwell and Shaw 1966) and from frog
spinal cord (Ramwell et al. 1966) contained material which stimulated
smooth muscle and which was identified by thin—layer chromatography as a
mixture of PGE and PGE. Direct electrical and peripheral nerve
stimulation increased the release of this active material.

In vitro measurements of prostaglandins in various brain tissue
preparations have not yielded consistent results, both in terms of
absolute concentrations measured and in terms of distribution. This is
probably due to many things, including variations in species, tissue
collection and preparation, and assay techniques. All studies agree,
however, that PGEs are present in central nervous tissue. Several of
these studies are discussed below.

The concentration of PGE2 alpha in cat forebrain (rostral to the
intercollicular level), whole chicken brain, and chicken spinal cord was
approximately 10 ng/g wet weight tissue in one study (Horton and Main
1967). By comparison, PGE (probably PGE1) was relatively low in cat

forebrain (5 ng/g) , but PGE2 was relatively high in chicken brain and

spinal cord (100 ng/g and 400 ng/g respectively). The authors suggested
that uneven distribution of prostaglandins in the central nervous system
might indicate that prostaglandins have a role in the
specializedfunction of the region where their concentrations are
highest.

To determine the distribution of prostaglandins in the brain, Holmes
and Horton (1968b) measured prostaglandins in homogenates of dog cortex,
hippocampus, caudate nucleus, hypothalamus, cerebellum, medulla and
pons, cortical white matter, and spinal cord. In whole brain, the
following concentrations were found: PGE1 35 ng/g, PGE2 62 ng/g,
PGII‘1 alpha 63 ng/g, and PGE2 alpha 56 ng/g. (All values are uncorrected
for recoveries, which probably were somewhere between 65 and 80%.) All
four prostaglandins also were found in approximately equal concentra-
tions in all regions of the central nervous system tested separately,
although the medulla and pons and the cortical white matter tended to
contain less PGE.

Prostaglandins E2 and F also have been isolated from slices of

2alpha

rat cerebral cortex (Wolfe et al. 1976a and 1976b). These investigators
showed that the trauma involved in collecting the brain tissue to be
analyzed stimulates prostaglandin synthesis. When whole cerebral hemi—
spheres from rats were frozen immediately in liquid nitrogen, levels of
PGFZalpha were very low (approximately 0.1 rig/g tissue) and PGE2 was
undetectable. Merely slicing the cortex increased prostaglandin syn—
thesis approximately 8X. In incubated slices of cortex, synthesis of
PGE and PGE increased almost linearly with respect to time for the first
60 minutes, and then decreased slightly. The ratio of PGE2 alpha.

formation to PGE formation was 3.5. In incubated homogenates of

2

 

 

cortex, PGF

2alpha synth681s reached a nmx1mum in 60 minutes, whereas
PGE2 formation peaked in 15 minutes. Formation of PGE2 was much more
rapid, and relatively more PGE2 was formed, in homogenates than in
slices; the ratio of PGFZalpha to PGE2 in homogenates was 2.1.
Steady—state values of prostaglandin synthesis in homogenates and slices

were on the order of 2—3 ng/g initial tissue weight for PGE and 6—8

2
ng/g for PGF

2alpha'

Wolfe et al. (1976a. and 1976b) also reported that prostaglandin
synthesis in cat cortex is 3X greater than in rat cortex and that synr
thesis in human cortex is 2/3 less than in rat cortex. In all three
species, synthesis of PGF2alpha was greater than that of PGE2, except in
cat cerebellum. These investigators showed, however, that although
brain tissue does not catabolize tritiated PGF2alpha to any great extent
((10%), it can convert PGE2 to PGF2alpha’ suggesting that it has PGE
9—keto reductase activity. Although this transformation may account for
part of the preponderance of PGF2alpha compared to PGE2 in brain tissue,
the authors considered this unlikely to be significant at the low level
of prostaglandin synthesis occurring spontaneously.

Abdel—Halhm and Angggrd (1979) confirmed that brain tissue does not
catabolize prostaglandins, but they found no evidence for interconver—
sion. In contrast to the suggestion of Horton and Main that prosta—
glandin synthesis may' vary' in. different regions of the brain, they
showed that, although total amounts of prostaglandins varied, the relay
tive amounts of prostaglandins in various brain regions studied (cortex,
cerebellum, hippocampus, striatum, pons—medulla, amd nucleus accumbens)
were constant. Relative prostaglandin concentrations varied by species.

The main prostaglandin(s) produced by species were: rat— PGDZ, guinea

18

pig— PGD and PGE ha’ rabbit— PGF , and cat— PGE .

2 2alp 2alpha 2
In a more recent study, Abdel-Halim et al. (1980) measured prosta-

glandin synthesis in homogenates of mouse, rat, and rabbit whole brain,
cerebral cortex, and cerebral blood vessels (choroid plexus and internal

cerebral vessels). After thirty minutes of incubation, PGE2, PGF2 alpha’

PGD2, and 6-keto-PGF1 alpha (the stable metabolite of PGI2) were detected

in whole brains of all three species. In rat and mouse brain PGD2
predominated (approximately 5 ng/ g tissue), with levels of PGE2 alpha’
and PGE2 slightly lower. In rabbit brain PGF2 alpha (7.5 ng/g) and PGE2
(6 ng/g) predominated. In rat cerebral blood vessels 6-keto—PGF1 alpha
was by far the most abundant prostaglandin formed (15 ng/g) . Prosta-
glandin F2 alpha
of detection.

also was formed , but PGE and PGD were below the level

2 2

In contrast to the study described above, Hagen et al. (1979) showed
that exogenous administration of labelled arachidonic acid to bovine
cerebral arteries results in synthesis of PGE , PGFZalpha’ and 6—keto-
PGrF1 alpha in approximately equal amounts. Differences in results
between the two studies may be due to differences in species or in size
of blood vessels incubated, or may reflect differences in spontaneous
vs. stimulated production of prostaglandins. In this same study,
also was measured and

unstimulated production of PGE and PGE

2 2alpha
found to be equal , but unstimulated production of 6—keto—PGF1 alpha was
not measured.

Quantitative interpretation of prostaglandin synthesis results after
administration of exogenous precursors may be unreliable for several

reasons. In the kidney, the products resulting from stimulation of

prostaglandin synthesis by exogenously—administered arachidonic acid

 

differ from those produced in response to endogenous arachidonic acid
release. Exogenous arachidonic acid causes formation of both PGE2 and
PGIZ, whereas endogenous arachidonic acid released by bradykinin forms
only PGE2 (Blasingham and Nasjletti 1979). Failure to isolate labelled
prostaglandins in brain tissue after administration of labelled
arachidonic acid (Coceani and Wolfe 1965; Pace—Asciak and Nashat 1976)
has been attributed to dilution of the labelled arachidonic acid in a
large pool of unlabelled endogenous arachidonic acid (Pace—Asciak 1976;
Wolfe et al. 1976a, 1976b). As described above, Hagen et al. (1979)
were able to detect labelled prostaglandins in extracts of incubation
medium from cerebral arteries preincubated with labelled arachidonic
acid, but most of the radioactivity measured was still associated with
arachidonic acid; radioactivity associated with individual labelled
prostaglandins was only about 5% of the total radioactivity for each
prostaglandin. These investigators made no attempt to identify
radioactive products within the tissue itself. Thus, interpretation of
relative amounts of prostaglandins measured is still difficult.
Similarly, the relative amounts of PGE to PGE formation in different
areas of the brain reported by Pace—Asciak and Nashet (1976), in which
the labelled endperoxides P002 and PGH2 were added to homogenates of rat
brain, are opposite to the results of Wolfe et al., described
previously. In the studies reported by Pace—Asciak and Nashet,
relatively more PGE was formed by cerebral hemispheres than by whole

brain, and relatively more PGF was formed by cerebellum than by

2alpha
whole brain.
A variety of substances have been shown to stimulate synthesis of

prostaglandins by brain tissue, most notably biogenic amines, including

2O

norepinephrine, dopamine, adrenochrome, L—dopa, and 5—hydr01qrtryptamine.
These substances mainly stimulate PGF2 alpha production, however, not
PGE2 (Ramwell et al. 1966; Wolfe et al. 1976a, 1976b). Analeptics also
stimulate prostaglandin release from cat cerebral cortex (Ramwell and

Shaw 1966) and ADP enhances formation of PGE in rat brain (Abdull and

1
McFarland 1972). Adenosine triphosphate has been shown to inhibit PGE3
production (Abdull and McFarland 1972) or to have no effect on
prostaglandin production (Wolfe et al. 1976a, 1976b).

Quinicrine inhibits PGE2 formation, but not PGF2alpha' Other
substances which inhibit prostaglandin synthesis in brain tissue include
(in order of potency) ketoprofen, indomethacin, prodilic acid,
acetylsalycilic acid, paracetamol, and eicosatetraynoic acid (Wolfe et
al. 1976a, 1976b).

Substances which have no effect on prostaglandin synthesis by brain
tissue in vitro include reduced glutathione, hydroquinone, dibutyryl
cAMP, bradykinin, histamine, morphine, and omission of glucose (Wolfe et
al. 1976a, 1976b). It must be pointed out that these results were
obtained in vitro and may not reflect the situation in vivo where
availability of arachidonic acid is limited by other factors. This
would be especially true for substances which alter prostaglandin
synthesis by action on phosphlipase A activity and arachidonic acid
release rather than by action on cyclo—oxygenase activity.

In summary, it is safe to say that PGD2, PGEZ, PGF and

2alpha’
6—keto—PGF1 alpha (PGI2) are synthesized by central nervous tissue.

Six—keto-PGF production is predominant in cerebral vasculature and

1alpha
choroid plexus. The actual concentrations of prostaglandins in brain

tissue in vivo are probably much lower than the concentrations measured,

 

21
since trauma activates prostaglandin synthesis. Central nervous tissue
does not catabolize prostagland ins; they probably are removed from the

brain by venous blood and cataboli zed in the lungs.

3 . Transport of Exogenous PGE into Central Nervous Tissue

 

In order for exogenously—administered PGE to affect neural
function, it must get out of blood or cerebral spinal fluid and into
brain tissue. Holmes and Horton (1968a) showed that, when tritiated
PGE1 is injected into cerebral spinal fluid or into cerebral blood at
doses high enough to produce central nervous signs in cats and chicks,
very little labelled PGE1 is found in brain or spinal cord. When PGE1
was injected into the lateral ventricles of cats, the largest amount of
radioactivity was found in the liver, and this radioactivity no longer
was associated with PGE1, but probably with a metabolite. The kidneys
and cerebral spinal fluid contained a moderate amount of radioactivity.
The brain contained less than 5% of the injected radioactivity and this
was distributed throughout, although there consistently was more
radioactivity found in the midbrain, cerebellum, medulla, and pons.
When 10 ug of tritiated PGE1 was injected either into a vertebral artery
or into a carotid artery of anesthetized cats, no area of the brain
examined (forebrain, midbrain, cerebellum, or medulla and pons)
contained radioactivity equivalent to as much as 0.1 ug PGE1.
Twenty-five percent of the injected activity was measured in venous
blood in the first three minutes after injection. Since these cats had
displayed central nervous signs (stupor, catatonia) before death, Holmes
and Horton concluded that prostaglandins are active at extremely low

concentrations.

22

The results reported by Holmes and Horton (1968a) are reasonable in
view of present knowledge of prostaglandin transport and mechanisms of
action. Bito (1972) has confirmed that brain tissue does not accumulate
prostaglandins readily. Cell membranes in general are impermeable to
prostaglandins, and uptake into cells is by carrier-mediated transport.
In some tissues this may be passive (facillitated), but in some it is
definitely active (Bito 1972; Bito and Baroody 1974; Bito 1975a, 1975b;
Bito and Baroody 1975; Bito, Davson, and Hollingsworth 1976; Bito,
Davson, and Salvador 1976; Bito, Baroody, and Reitz 1977; Syrota et al.
1982) .

Choroid plexus has a great affinity for prostaglandins and can
accumulate them against a concentration gradient (see references above).
Bito has suggested that the choroid plexus acts as a sponge to absorb
prostaglandins in order to protect the brain from potentially-damaging
high levels of them (Bito 1972, 1975b; Bito et al. 1976). Presumably,
then, the choroid plexus releases the prostaglandins into venous blood,
in which they are returned to the lungs to be metabolized. This also
requires transport of prostaglandins across the vascular endothelial
membrane (Bito and Baroody 1975; Bito et al. 1977; Syrota et al. 1982).
Thus , it is likely that most of the exogenous prostaglandins given into
cerebral spinal fluid or into the cerebral arterial inflow are removed
by cerebral venous blood, are metabolized by the lungs, and are excreted
by the liver and kidneys.

In summary, exogenous prostagland ins must get out of cerebral
capillary blood or cerebral spinal fluid and into brain extracellular
fluid to act on neurons. This may occur in areas of the brain where the

blood-brain-barrier is leaky or in areas of the brain that have the

23

specific prostaglandin carrier. Endogenous prostaglandins may be
synthesized in nerve cell membranes and released directly into the
extracellular space, or they may be synthesized by vascular endothelium
(Gimbrone and Alexander 1975) or formed elements in the blood and enter
the brain as do exogenous prostaglndins.

Once in the extracellular space, prostaglandins could act on
specific membrane receptors. In tissues where the mechanism of
prostaglandin action is known, prostaglandin effects are mediated by
phosphonucleotide cyclase / cyclic nucleotide systems, and activation of
these systems does not require entry of prostaglandins into the cells.
This does not exclude the possibility that prostaglandins also could act
intracellularly in cells which can transport them.

Prostaglandins may get from the extracellular fluid to venous blood
by diffusion, bulk flow, facillitated diffusion, or active transport.
Catabolism of prostaglandins occurs intracellularly in lung, liver, and
kidney (Bito 1975a) . Thus, metabolism of prostaglandins is
compartmentalized so that they are synthesized within the cell membrane,

they act extracellularly, and they are degraded intracellularly.

III. CHAPTEI ONE

PGEZIXJIBMMITATIEECAMIDSINUS
TORAISEAMFRIALPRBSUREINCONSCIOUSSHEEP

INTRODUCTION

Prostaglandin E2 (PGE2) is a potent vasodilator when infused peri—
pherally (MOncada et al. 1980). Hewever, Hull and ChimoSkey (1984) have
shown that, when infused midcervically into the common carotid artery of
conscious dogs, calves, and sheep at a dose of 10 ng/kg/min, PGE2 causes
blood pressure and heart rate to rise. The rise in blood pressure is
mainly due to an increase in total peripheral resistance and is bloCked
by the alphasadrenergic blocking agent phentolamine. There is no chemo-
reflex, but the baroreflex is reset to a higher level. Hull and
ChimoSkey postulated that the pressor effect of PGE2 is mediated by PGE2
acting centrally to stimulate the sympathetic nervous system.

This hypothesis, that PGE2 acts centrally, is supported by work in
rats (Kondo et al. 1979; Hoffman et al. 1981; Takahashi and Bunag 1981b;
Okuno et al. 1982). These investigators independently have shown that
intracerebroventricular (IVT) injections of PGE5 into conscious or
anesthetized rats increases blood pressure. TakahaShi and Bunag showed
that peripheral sympathetic nerve activity is increased, and Okuno et
al. showed that the increase in blood pressure is decreased by
phenoxybenzamine, indicating an alpharadrenergic sympathetic mechanism.

Additionally, Gyang et al. (1973) showed that blood pressure increases

24

25

when PGE is injected into the vertebral artery of anesthetized cats.

1
Note that with these two routes of injection which bypass the carotid
sinus (i.e.: into the vertebral artery or IVT) , PGE2 also has a pressor
effect.

Thus there is strong evidence that either blood-borne PGE2 or PGE2
given into cerebral spinal fluid causes a centrally mediated increase in
blood pressure. However, there has been no work done to determine the
role of the carotid sinus in the pressor response observed when PGE2 is
infused into the common carotid arteries of conscious sheep. This study
was performed to determine the role of a direct action of PGE2 on the

carotid sinus in the pressor response observed when PGE2 is infused into

the common carotid artery of conscious sheep.

METHODS

Six mature female sheep ranging in weight from 48 to 66 kg were used
for these experiments . The sheep were housed individually in the labo—
ratory and allowed to acclimate to their surroundings and laboratory
personnel. This was essential in order to ensure that the sheep were

calm and unstressed during the experiments.

Instrumentation

 

Each sheep was anesthetized with intravenous sodium thiamylal,
intubated, and maintained in a surgical anesthetic plane with halothane
and oxygen. It was placed in dorsal recumbency and prepared for aseptic
surgery. A midline incision, centered over the rami of the mandibles,
was made in the neck. The right or left carotid sinus region (four

sheep right, two sheep left) was exposed by careful dissection to

26

avoid damaging the parotid salivary gland, which was retracted
laterally, and to avoid cutting any of the major nerves in this area
(cranial nerves IX, X, and XII, the sympathetic trunk, the cranial
laryngeal nerve, and various pharyngeal branches).

The carotid sinus region was identified by finding the lingual and
occipital arteries. A fine nerve plexus could be seen in the adventitia
of the carotid artery in this area. A small nerve, thought to be the
carotid sinus nerve, usually could be identified going to this area.
The carotid sinus nerve was cut and the adventitia was stripped from the
carotid artery 3600 over a 3 cm length extending from the external
carotid artery just rostral to the origin of the lingual artery and
caudally past the occipital artery to an area on the common carotid
artery approximately 1 cm beyond where any small nerve fibers could be
seen. This adventitial stripping also was carried up the occipital
artery for approximately 1 cm, where the occipital artery often branched
into 3—5 very small arteries. The surrounding tissues were packed off
with sponges and 10% phenol was painted on the stripped area of the
carotid artery.

The external carotid artery then was dissected as far as possible
until it disappeared over the stylohyoid bone, resulting in a short
length of artery (approximately 1.5 cm) that could be catheterized using
the technique of Herd and Barger (1964). This catheter was non—
occlusive and the tip was positioned beyond the origin of the lingial
artery in order to ensure that it was rostral to the carotid sinus
(Figure 1).

At the caudal end of the incision, the right and left common carotid

arteries were exposed 12—14 cm proximad to the carotid sinuses and

27

non-occlusive catheters were implanted, again using the Herd-Barger
technique (1964). Hydraulic occluders (Rhodes Medical Instruments,
Woodland Hills, CA) were placed around each common carotid artery
upstream to each catheter. An occluder size was chosen such that in the
deflated state there was a loose fit and no pressure was placed on the
arterial wall. When inflated, however, the common carotid artery was
totally occluded. A jugular vein catheter also was implanted in this

area.

 

External
carotid a.

 

Lingual a. Common carotid a.

Cranial Caudal

Figure 1. Anatomy of the carotid sinus region of the sheep and
placement of the external carotid artery catheter.

28

The four catheters were tunneled subcutaneously to a prepared spot
on the neck near the shoulder , where they were brought through the skin
individually (in order to prevent retrograde infection) and tied to a
loop of polyvinyl tubing placed through the skin. The two occluder
lines were allowed to hang straight down and were brought through the
skin on either side of the incision. The incision was closed, leaving a
Penrose drain to prevent excessive swelling by allowing drainage of
fluid. This drain was removed within 72 hours after surgery.
Immediately after surgery each sheep was given 6 x 104 units/kg procaine
penicillin G and then maintained on 3.6 x 104 units/kg/day procaine
penicillin G for five days. The catheters were filled once daily with

1:1000 sodium heparin. The sheep were given one week to recover from

surgery.

Experimental Procedures

 

PGE.) Infusions

 

There were three routes of infusion of PGE2,

10 ng/kg/min: 1) into the common carotid artery past an intact carotid

all at the rate of

sinus, 2) into the common carotid artery past a denervated carotid
sinus, and 3) into the external carotid artery beyond the carotid sinus.
The experimental procedure was the same for each route of PGE2 infusion.
With the sheep standing quietly in its cage, carotid blood pressure was
recorded onto a Gould-Brush chart recorder using Gould Statham pressure
transducers zeroed at the level of the heart. Data also were recorded
onto magnetic tape for storage and playback using a Hewlett—Packard FM
tape recorder. Heparinized saline was infused into each catheter at a

rate of 0.3 ml/min. Control blood pressure and heart rate were obtained

29

in this manner, and then PGE2 was infused, 1O ng/kg/min, at a rate of
0.3 ml/min into the appropriate artery. (PGE2 was made up as a stock
solution of 1 mg/ml in ethanol and stored at -20°C. Just prior to
infusion the proper amount was diluted in normal saline). PGE2 infusion
was continued for 35 minutes. Infusions by each route were repeated
four times randomly on different days. These replicates were averaged

for each sheep. Statistical analysis was performed on the data per

sheep rather than per replicate.

Cardiac Output Determinations

 

Three sheep in which carotid sinus denervation was verified
(described below) were used for cardiac output determinations during
control and during PGE2 infusion into the external carotid artery or
into the common carotid artery past the denervated carotid sinus. These
experiments were performed to make certain that the increases in blood
pressure caused by these two routes of infusion also were due to an
increase in total peripheral resistance, as had been demonstrated
previously by Hull and Chimoskey with PGE2 infusions into the common
carotid artery past an intact carotid sinus (1984).

The experimental procedure for the PGE2 infusions was the same as
described previously, with the addition that cardiac output was
determined several times during control and during PGE2 infusion using
the indocyanine green dye dilution technique. Indocyanine green dye was
injected into the jugular vein while blood was being withdrawn from one
of the carotid arteries through a Gilson model DTL dye tracer. The
values of cardiac output , blood pressure , heart rate , and total

peripheral resistance (blood pressure divided by cardiac output) for 2—3

3O

dye injections during control and during PGE2 infusion were averaged to
produce control and PGE2 values per sheep per day. For the external
carotid artery route of infusion, statistical analysis was performed on
the values from three sheep, three days each, for a total of nine
replicates. For the denervated sinus route of infusion, statistical
analysis was performed on the values from three sheep, two days each,

for a total of six replicates.

 

1 T

T T ' ._
'1') :— on off .1 0| on on
Occlusion Occluolon
cocond L. Common “condo B. Common
Carotid A. Carotid A.

Figure 2. Verification of carotid sinus denervation by unilateral
carotid artery occlusion. Figure 2 shows arterial ressure (mmHg) in
right carotid artery (above) and left carotid artery (p elow). The right
carotid sinus has been denervated. Occlusion of the common carotid
artery proximal to the intact left carotid sinus causes a rise in
systemic blood pressure (measured in the right carotid artery).
Occlusion of the common carotid artery proximal to the denervated right
carotid sinus fails to elicit a carotid sinus baroreflex.

31

Verification of Denervation

 

After all experiments had been performed, each sheep with a
carotid sinus denervation was re—anesthetized with sodiudm thiamylal and
maintained in a very light plane of anesthesia with halothane. While
blood pressure was being recorded in both common carotid arteries, the
occluder on the intact common carotid artery was inflated and the artery
occluded for 60 seconds. The same procedure was repeated on the dener-
vated side. The two sides were compared several times. Denervation was
verified when occlusion on the denervated side failed to cause a rise in
systemic blood pressure while occlusion on the intact side did produce a
reflex rise in systemic blood pressure (Figure 2). It was imperative to
keep anesthesia time short and anesthetic depth light in order consis—
tently to produce a carotid baroreflex on the intact side. This was
because changes in pressure at the carotid sinus region were small due
to backflow of blood from the other carotid artery through the circle of
Willis and/ or from the vertebral artery through its anastomosis with the
occipital artery (Baldwin and Bell 1963a, 1963b, 1963c; McQueen and
Belmonte 1974). Additionally, the response of the carotid sinus to this
small change probably was buffered by the aortic arch receptors and may
have been inhibited by anesthesia. Despite these problems, verification
of denervation was performed in the anesthetized state since blood
pressure tended to be more labile in the conscious state, and transient
increases in pressure due to extrinsic factors (such as noises in the
hall) could not be differentiated from increases in pressure due to
carotid occlusion. Denervation was verified in five of the six sheep (3
right, 2 left). This is reflected in Table 1 which contains only data

from the 5 sheep in which denervation was verified.

32

Statistical Analysis

 

For all variables (blood pressure, heart rate, cardiac output, and
total peripheral resistance), the paired t test was used to canpare

values during control to those during PGE infusion. Since it had been

2
shown previously that PGE2 infusion past an intact carotid sinus caused
all four variables to increase significantly (Hull and Chimoskey 1984),
one—tailed t tables were used to find the value of p. Values of p are
presented in the results; .05 or smaller was considered significant.

In four animals data from control and from PGE2 infusion by each of
the three routes were compared using a blocked Analysis of Variance
(ANOVA) and Student-Newman—Keuls test for multiple comparisons to
determine whether there were significant differences in the blood

pressure or heart rate produced by infusion of PGE2 past an intact

carotid sinus, past a denervated sinus or beyond the carotid sinus.

RESULTS

Infusion of PGE2 at a rate of 10 ng/kg/min into the external carotid
artery of six conscious sheep resulted in an increase in blood pressure
of 17 111an (p<.0005) and an increase in heart rate of 7 bpn (p<.025)
(Table 1). During cardiac output determinations, blood pressure rose
19% from 79 to 94 We (p<.0005), cardiac output (4.86 L/min) did not
change significantly, total peripheral resistance rose 23% from 16.47 to
20.08 mmHg min/L (p<.0005), and heart rate rose 8% from 83 to 90 bpn

(p<.025) (Figure 3A) .

33

Infusion of PGE2 ,

10 ng/kg/min, past the denervated carotid sinus of

five of the six sheep in which denervation was verified resulted in an

increase in blood pressure of 22 umHg (p<.0005) and an increase in heart

rate of 6 bpm (p<.05) (Table 1).

During cardiac output determinations,

blood pressure rose 25% from 80 to 100 mmHg (p<.0005), cardiac output

(4.99 L/min) did not change significantly, total peripheral resistance

increased 29% from 16.84 to 21.69 ang min/L (p<.01), and heart rate

increased 6% from 76 to 80 bpm (p<.025) (Figure 3B).

Data from these experiments for each individual sheep are shown in

Appendix B, Table B—1 .

Table 1 .

Blood Pressure and Heart Rate Responses to Infusion of PGE? 10
he

ng/kg/min, either into the External Carotid Artery Beyond
Carotid Sinus or into the Common Carotid Artery Past a

Denervated Carotid Sinus

 

 

Route of External Carotid Artery Common Carotid Artery
Infusion: (Carotid Sinus Denervated)
(6 sheep/ 23 replicates) (5 sheep /20 replicates

Control PGE2 S . E . D . a Control PGE2 S . E . D .
Blood Pressure H H
(mmHg) 86 103 (1.74) 81 103 (1.96)
Heart Rate * *
(beats/min) 77 84 (1 .93) 79 85 (2. 20)

 

a Standard error of the difference.
* p<.05 re Control

** p<.0005 re Control

A. 50 ‘ * P<.05
" * * P<.01
4O ‘

30‘ *

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MAP CO TPR HR

Figure1 3. A. Hemodynamic changes during external carotid infusion of
ng/kg/min, in three conscious sheep ( replicates). B. Hemo-
dyngmico changes during infusion of PGE2,1O kg/min, past a denervated
carotid sinus in three conscious sheep (6 replicates). The increase in
blood pressure is due to an increase in total peripheral resistance in
both cases. (Asterisks indicate which variables are significantly
different from control 25 minutes after the start of PGE2 infusion.

 

35

>i<

-K- P<.01 RE CONTROL

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

12o '
. * .
Jr * :
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(:2 100 "“1 __;_1
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0 c FGE2 o PGE2 c PGE2

 

 

 

 

 

 

lNTACT DENERVATED EXTERNAL
CAROTID SINUS CAROTID SINUS CAROTID ARTERY

Figure 4. Blocked comparison of blood pressure during control and
during PGE infusion, 10 ng/kg/min, by three routes: into the common
carotid ery past an intact carotid sinus, into the common carotid
artery past a denervated carotid sinus, and into the external. carotid
artery beyond the carotid sinus (4 conscious sheep/16 replicates per
route.

In four sheep in which data were obtained by all three routes of

infusion of PGE including infusion past an intact carotid sinus,

2,
analysis by blocked ANOVA followed by multiple comparisons using the
Student—Newman—Keuls statistical test showed that while blood pressure
during PGE2 infusion differed significantly from control for each route
of infusion, there were no statistically significant differences in
blood pressure among control groups nor among PGE2 groups (Figure 4).

Similarly, there were no significant differences in heart rate among

control or PGE2 groups.

36

DISCUSSION

In cross—circulation. studies in anesthetized dogs, Kaplan et al.
(1969) showed that injection of PGE1 past an intact carotid sinus causes
blood pressure to fall while injection past a denervated carotid sinus
causes blood pressure to rise. They concluded that direct action of
PGE1 on the carotid sinus masks or reverses a central pressor effect.
In contrast to these results, McQueen and Belmonte (1974) more recently
showed that PGE2
caudal to the carotid sinus in anesthetized cats has no direct effect on

(0.4—8.0 ug/kg) injected into the bloodstream just

the firing of carotid sinus baroreceptor nerve fibers.

Hull and Chimoskey (1984) found that midcervical infusion of PGE2
into the common carotid artery shifted the set point of the baroreflex
without changing its sensitivity. This resetting of the baroreflex
could have been an indirect effect of PGE2 acting in the central nervous
system or it could have been due to a local or direct action of PGE2 on
the carotid sinus baroreceptors. Local vasodilation in the carotid
sinus region may cause a decrease in afferent baroreceptor nerve firing,
resulting in an increase in sympathetic nerve activity. Direct action
of PGE2 on the carotid sinus baroreceptors could alter the sensitivity
of the receptors, in which case one might expect the other, non—affected
baroreceptors (ie.: aortic arch and the other carotid sinus) to
dominate and to keep blood and heart rate from_ changing. Another
possibility, however, is that direct action of PGE2 on the carotid sinus
baroreceptors could alter nerve traffic to the nucleus tractus
solitarius, in which case the central nervous system would be affected
indirectly. Thus, direct action of PGE2 on the carotid sinus may cause

the rise in blood pressure observed when PGE2 is infused into the common

37

carotid artery of conscious sheep. However, due to the evidence that
PGE2 does indeed act in the central nervous system (by the vertebral
route or IVT), it is more likely that any direct action of PGE2 on the
carotid sinus is either through augmentation or inhibition of the
centrally-mediated action of PGE2.

In this study, the question of whether the pressor response to
common carotid artery infusion is influenced by a direct action of PGE2
on the carotid sinus baroreceptors was approached in two ways. First,
PGE2 was infused into an artery to the head bypassing the carotid sinus.
The external carotid artery was chosen for this purpose since the adult
sheep has no internal carotid artery and since the vertebral artery of
the sheep only perfuses the caudal medulla (Baldwin and Bell 1963a,
1963b, 1963c) . Thus the external carotid artery is the main source of
blood to the brain of the sheep and infusion of PGE2 into this artery
would be most likely to reach the same areas of the brain as those
supplied by the common carotid artery. Secondly, PGE2 was infused into
the common carotid artery at a site proximal to a denervated carotid
sinus, thereby ensuring that the same areas of brain that had been
studied previously were perfused.

Our results agree with the work of McQueen et al. (1974). Since
there were no significant differences in the changes in blood pressure
or heart rate when PGE2 was infused into an intact common carotid ar—
tery, past a denervated carotid sinus, or beyond the carotid sinus, we
conclude that the carotid sinus has no direct role in the pressor re-
sponse observed when PGE2 is infused into the common carotid artery of
conscious sheep. This conclusion supports the hypothesis that the
cardiovascular responses observed when PGE2 is infused into the arterial

supply to the head are centrally-mediated.

IV. CHAPTETHO

PARASIHPATHETIC m 3m
RESPONSE TO INTRACAROTID PGEZ IN CONSCIOUS SHEEP

INTRODUCTION

Prostaglandin E2 (PGEZ) , a potent peripheral vasodilator (Moncada et
al. 1980) , causes blood pressure and heart rate to increase when infused
into a carotid artery (10 ng/kg/min) in conscious sheep (Breuhaus and
Chimoskey 1983; Hull and Chimoskey 1984) . The increase in blood
pressure during intracarotid (IC) PGE2 infusion is caused by increased
total peripheral resistance (Breuhaus and Chimoskey 1983; Hull and
Chimoskey 1984) and can be prevented by the alpha-adrenoceptor blocking
agent phentolamine in the presence of prOpranolol (Hull and Chimoskey
1984) . Pretreatment with both pr0pranolol and phentolamine does not
abolish the increase in heart rate during IC PGE2 infusion (Hull and
Chimoskey 1984) .

The increase in heart rate which remains during 10 PGE2 infusion
after adrenoceptor blockade may be caused by inhibition of parasympa-
thetic nervous tone to the heart. However, PGE2 may increase heart rate
by increasing cardiac beta-adrenoceptor activity and the dose of propra—
nolol used in the previous study (Hull and Chimoskey 1984) was not
sufficient to block it. Prostaglandin E2 does not produce tachycardia
by direct action on the heart, since 400 ng/kg/min PGE2 infused into the

38

39

right atrium does not alter heart rate (Hull and Chimoskey 1984) . This
latter finding also demonstrates that, at the doses used (10—400
ng/kg/min) , PGE2 is metabolized rapidly by the lungs (Moncada et al.
1980; Hull and Chimoskey 1984) so that it does not enter the systemic
circulation to cause peripheral vasodilation and reflex tachycardia.

In conscious sheep, IC PGE2 increases the set point of the barore—
flex without changing its sensitivity (Hull and Chimoskey 1984) . Intra—
carotid PGEZ,
receptors (Breuhaus and Chimoskey 1983). Centrally-administered PGE2

10 ng/kg/min, has no direct action on carotid sinus baro—

may reset the baroreflex by activating only sympathetic centers in the
brain. However, if IC PGE2 causes both sympathetic activation and
inhibition of parasympathetic tone, PGE2 must act either at multiple
sites in the brain or within the baroreflex pathway itself.

Evidence is presented below that PGE2 has a complex action on
cardiac autonomic regulation. Neither atropine alone nor a larger dose
of propranolol alone can block the increase in heart rate caused by IC
infusion of PGE2 in conscious sheep. However, a combination of atropine
and propranolol does block the heart rate increase . Taken together, the
data presented in this chapter and the information cited above support
the hypothesis that PGE2 acts within the central baroreflex pathway both
to activate the peripheral sympathetic nervous system and to inhibit the
peripheral parasympathetic nervous system, resulting in increased blood
pressure, heart rate, total peripheral resistance, and cardiac output
(Hull and Chimoskey 1984) . If either division of the autonomic nervous
system is blocked peripherally, heart rate still increases during IC
PGE2 infusion by greater contribution to the heart rate response by the

other unblocked division of the autonomic nervous system.

4o

METHODS

Eight mature female sheep, ranging in weight from 50 to 80 kg, were
used for the PGE2 infusions and muscarinic receptor blockade experi—
ments. Seven of these same sheep also were used for the beta-
adrenoceptor blockade experiments and the combined blockade experiments.
The sheep were housed individually in the laboratory and they were
allowed to acclimate to their surroundings and laboratory personnel.

This ensured that the sheep were calm during the experiments.

Instrumentation

 

Each sheep was anesthetized with intravenous sodium thiamylal,
intubated, and maintained in a surgical anesthetic plane with halothane
and oxygen. They were placed in dorsal recumbency and prepared for
aseptic surgery. A midline incision was made in midcervical skin and
subcutaneous tissue. Careful surgical dissection was performed to
expose both carotid arteries without disturbing the vagus nerves. Non-
occlusive polyvinyl catheters were implanted in each carotid artery
according to the technique of Herd and Barger (1964) . One external
jugular vein also was catheterized in the same manner. The catheters
were tunneled subcutaneously to a prepared spot on the neck, where they
were brought through the skin and tied to a loop of polyvinyl tubing
placed through the skin. The incision was closed and the sheep was
allowed to recover from anesthesia. Immediately after surgery each
sheep was given 5x104 units/kg procaine penicillin G intramuscularly and
1.92 gm trimethoprim-sulfa (TribrissinR; Burroughs Wellcome Co.) orally.
They were maintained on the trimethoprim—sulfa for an additional three

days. The sheep were given ten days to two weeks to recover from

 

41
surgery. During this time they became further accustomed to handling
and to the equipment to be used for experiments. The catheters were

filled once daily with 1:1000 sodium heparin.

Experimental Procedures

 

 

PGE2 Infusions

With a sheep standing quietly in its cage, mean and pulsatile
arterial blood pressure were recorded from a carotid artery catheter
onto a Gould—Brush chart recorder using Gould—Statham pressure trans-
ducers placed level with the heart . Data also were recorded onto
magnetic tape for storage and playback using a Hewlett-Packard PM tape
recorder. Heparinized saline was infused into the carotid artery (IC)
at a rate of 0.3 ml/min. Control blood pressure and heart rate were
obtained in this manner and then the heparinized saline was discontinued

and PGE 10 ng/kg/min, was infused IC at the same rate, 0.3 ml/min.

2,
(PGE2 was made up as a stock solution of 1 mg/ml in ethanol and stored
at -20°C. Just prior to infusion the proper amount was diluted in
physiological saline.) PGE2 infusion was continued for 35 minutes.
Infusions of vehicle (the same microliter amount of ethanol diluted in
saline) also were continued for 35 minutes. Blood pressure and heart
rate data were averaged over a two minute period during control and for
two minutes at the beginning of each five minute interval during PGE2 or
vehicle infusion. Infusion of PGE2, 10 ng/kg/min IC, caused blood
pressure and heart rate to increase gradually, beginning approximately
five minutes after the start of infusion. The increases in blood

pressure and heart rate ceased 15—20 minutes later. Steady-state PGE2

values were obtained 25 minutes into the infusions. This experiment was

42

performed only once on any given day and was repeated (replicates) at
least twice. The average number of replicates per sheep was six. There
was no tachyphylaxis to PGE2 infusion in any sheep. The values obtained
from all experiments in any one sheep were averaged to produce mean
values characteristic of that sheep. Statistical analysis was performed

on the data per sheep rather than per replicate.

Muscarinic Receptor Blockade

 

Three series of experiments were performed with atropine. Timed
control experiments were performed by giving atrOpine by itself. In a
second series of experiments atrOpine was given five minutes before the
start of IC PGE2 infusion. Finally, atropine was given 22 minutes after
the start of IC PGE

2 infusion. Muscarinic receptor blockade was pro-
duced with atropine methyl bromide (Signa), 1 mg/kg intravenously (IV).
Atropine methyl bromide was used in these experiments because it does
not cross the blood-brain barrier (Weiner 19&)a) . Effectiveness of the
blockade was determined in the following manner. For each sheep, a
control dose of acetylcholine (ACH) which would stop the heart for 2—5
seconds was found. Among all sheep this dose ranged from .01 to .02
mg/kg IV, but once the correct dose for each sheep was found, it did not
vary from day to day. Five and 15 minutes after administration of
atropine, a dose of ACH two times the control dose produced no change in
heart rate. At 45 minutes after administration of atrOpine, 10 times
the control dose of ACH was given. At no time did this produce a
decrease in heart rate. Further evidence that 1 mg/kg atropine methyl
bromide IV results in adequate muscarinic blockade was obtained in

separate experiments in four of these sheep. Forty minutes after the

i ,_ .h_.7 -lr;1.ri;u,rc)fll ,i._. .1.— w-..

43
initial bolus of atropine, additional boluses of 0.5 mg/kg or 1 mg/kg
atropine methyl bromide IV resulted in no additional increase in heart

rate.

Beta-Adrenoceptor Blockade

 

Three series of experiments were performed with propranolol. In
one series of experiments, pr0pranolol was given by itself to serve as a
time control. In a second series of experiments, propranolol was given
ten minutes before the start of IC PGE2 infusion. In a third series of
experiments , propranolol was given 17 minutes after the start of PGE2
infusion. Beta-adrenoceptor blockade was performed in seven of the same
sheep used above with d,l propranolol (Sigma), 1 mg/kg + .025 mg/kg/min
IV. This is twice the total amount of propranolol given by Hull and
Chimoskey (1984) and is sufficient to reduce the response to a challenge
bolus of isoproterenol to at least one tenth the response produced by
isoproterenol during control. To test the effectiveness of the
blockade, a 5 ug bolus of isoproterenol was given IV during control. At
seven and 15 minutes after the initial bolus of propranolol, 25 ug
isoproterenol IV had no effect on heart rate. At 45 minutes after
propranolol, the heart rate responses to ten times the control dose of

isoproterenol (50 ug) were compared to the control responses.

Combined Blockade

 

In a final series of experiments, IC PGE2 was infused in the
presence of both beta-adrenoceptor and muscarinic receptor blockade.
These experiments were performed in the same seven sheep used in the

beta-adrenoceptor blockade experiments . The same doses of blocking

agents and challenging agents were used. To test the ability of the
combined blockade to prevent the increases in heart rate during IC PGE2
infusion, pr0pranolol was started at time zero and atr0pine was given
five minutes later. Ten minutes after the initial pr0pranolol bolus,
the IC PGE2 infusion was started. Combined blockade also was performed
in the reverse order: PGE2 was started at time zero, pr0pranolol was
given 17 minutes after the start of PGE2 infusion, and atropine was

given 22 minutes after the start of infusion.

Statistical Analysis

 

For each variable (blood pressure and heart rate), control and
experimental values corresponding to 25 minutes of PGE2 infusion were
used. Values from replicates of experiments were averaged for each
sheep and these averaged data were analyzed by blocked ANOVA. Student-
Newman—Keuls test for multiple comparisons was used to determine whether
there were significant differences in control values of blood pressure
or heart rate among experiments or in the blood pressure or heart rate
responses to PGE2 alone, the blocking agent alone, or PGE2 plus blocking
agent. The experiments in which all three drugs were given were
analyzed in two ways. First, the raw data were analyzed as described
above. Second, since the control values for blood pressure and heart
rate in all previous experiments were the same, and since individual
reaponses to each experimental protocol were constant, the data also
were analyzed in terms of the change in blood pressure or the change in
heart rate. This allowed comparison of the changes which actually were
observed when both blocking agents and PGE2 were given together to the
changes which were expected by calculating the sums of the individual

45

changes caused by each drug given separately. A non-parametric test,
the chi—square test, was used to test for significant differences be—
tween the observed results and the expected (calculated) results (Steel
and Torrie 1980) . For all analyses, values of p<.05 were considered to

indicate significant differences.

RESULTS

Control values for blood pressure and heart rate for each individual
sheep did not vary from day to day, and the average control values for
blood pressure and heart rate for all sheep were not significantly
different from one another in any of the experiments. When the same
experiments were repeated in individual sheep, blood pressure and heart
rate responses to the test agents given were highly repeatable.

Infusions of vehicle did not alter blood pressure or heart rate.

Muscarinic Receptor Blockade

 

In eight conscious sheep, atr0pine alone increased heart rate 26 bpn
(p<.05) (Table 2) and 24 bpm (p<.05) (Figure 5) five and thirty minutes
after injection, respectively. Prostaglandin E2 infusion, 10 ng/kg/min
10, increased heart rate 14 bpm (p<.05) (Table 2 and Figure 5). When
atropine was given after PGE2 infusion had raised heart rate, there was
a further 24 bpm increase in heart rate (p<.05), resulting in a total
increase of 38 bpm from control (p<.05) (Table 2). When PGE2 was in-
fused after atropine, heart rate increased a total of 35 bpm (p<.05),
which was significantly different from the increases in heart rate with

atr0pine alone or with PGE2 alone (Figure 5B) .

Atropine alone caused small but significant increases in blood

46

120-

l

 

*O
{.3 *0

l
a
7III"""'l'lI'l’ll'llll’l'flIlI'll,.

  

O
O
1
+1»

1

 

(I)
O
1

l

0‘!
O
1

l

.p
O
1

l

N
O
1

l

 

  

 

 

 

 

 

 

 

 

 

 

 

 

l

 

MEAN ARTERIAL PRESSURE (mm Hg)

ATROPINE
4.
PGE2

ATROPINE 9552

N

O
1

*+

 

HEART RATE (bpm)
E?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O-—

ATROPINE PGE2

ATROPINE
PGE2

Figure 5. Atropine-+ , 8 conscious sheep. Blood pressure and heart
rate in three separate riments (all experiments performed in all
sheep) comparing the responses to 1 mg/kg atropine IV given alone, to H3

kg/min PGE IC given alone, and to atr0pine + PGE given together .
Open bars repgesent control values. Shaded bars reprgsent test values
30 minutes after atropine alone (Atropine), 25 minutes after the start
of PG infusion alone (PGE ), and 30 minutes after atropine and 25
minute after the start of PCB infusion when the two drugs were given
together (Atr0pine + PGE ). a. Blood pressure: *=p<.05 re Control;
o=p<.05 re Atropine. B. Heart rate: *=p<.05 re Control; o=p<.05 re

Atropine; +=p<.05 re PGEZ.

47

pressure five and thirty minutes after injection: 8 mmHg (Table 2) and
4 ang (Figure 5), respectively (p<.05). Infusion of PGE2 increased
blood pressure 22 mmHg (p<.05) (Table 2 and Figure 5). Injection of
atropine during steadyestate PGE2 infusion caused no further increase in
blood pressure (Table 2) . When the PGE2 infusion was started five
minutes after atropine had caused blood pressure to increase, there was
a further significant increase in blood pressure, but the final blood
pressure was no greater than the blood pressure with PGE2 infusion alone
(Figure 5A) . Since the control pressures were not different, the in-
crease in blood pressure caused by PGE2 infusion after atr0pine was not

as great as the increase when PGE2 was given by itself.

Table 2. Muscarinic blockade with atropine methyl bromide (1 mg/kg IV)
during IC PGE2 infusion (10 ng/kg/min) in eight conscious sheep

 

 

Experimental Blood Pressure (mmHg) a Heart Rate (beats/min)
Protocol Control Experimental S .E.D. Control Experimental S.E.D.
PGE2 (25 min) 83 105*0 (1.51) 75 87*° (2.09)
* *
Atropine (5 min) 83 91 (2.93) 76 102 (4.18)
PGE (25 min) + 82 102*0 (1.48) 78 116*°+ (5.50)

Atrgpine (5 min)

 

Standard error of the difference.

significantly different from Control, p<.05
significantly different from Atr0pine alone, p<.05
significantly different from PGE2 alone, p<.05

+0 *W

Data from these experiments for each individual sheep are shown in

Appendix 0, Tables C—1 and C-2.

Beta-Adrenoceptor Blockade

 

In seven conscious sheep, propranolol alone did not change heart
rate (Table 3 and Figure 6). Prostaglandin E2 infusion in these sheep
increased heart rate 15 bpm (p<.05) (Table 3 and Figure 6). When PGE2
had raised heart rate, subsequent administration of pr0pranolol
decreased heart rate 10 bpm (p<.05), but heart rate still was greater
than control (p<.05) (Table 3). When propranolol was given before the
start of PGE2 infusion, PGE2 still increased heart rate 7 bpm (p<.05),
but this increase was less than the increase caused by PGE2 infusion
alone (p<.05) (Figure 6). The effect of propranolol on the heart rate

responses to IC PGE2 was the same regardless of whether pr0pranolol was

given before or after PGE2.

Table 3. Beta-adrenoceptor blockade with propranolol (1 mg/kg + 0.025
mg/kg/min IV) during IC PGE2 infusion (10 ng/kg/min in seven
conscious sheep

 

 

Experimental Blood Pressure (mmHg) a Heart Rate (beats/min)
Protocol Control Emperimental S.E.D. Control Experimental S.E.D.
PGE2 (25 min) 84 105* (1.63) 77 92* (2.19)
Pr0pranolol (10 min) 85 85 (0.34) 76 72 (1.65)
PGE (25 min) + 87 109* (2.90) 78 83*+ (1.86)

Propranolol (10 min)

 

a Standard error of the difference.
* significantly different from Control and Pr0panolol alone, p<.05
+ significantly different from PGE2 alone, p<.05

49

P
m H9)

120 1 t :1
5 100 4 “”11

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

OJ—
PROPRANOLOL PGE2 PROPRANOLOL
PGE2

MEAN ARTERIAL PRESSURE

E3. 12C)-

100: * i"

     

(I)
O
l
(-
*-

 

60+

HEART RATE (bpm)

 

 

 

 

 

 

 

 

 

 

\ ‘
““\“““““““““‘“\‘\

 

I\““\\“\“‘\“\‘\‘\\“\\\\‘
§ \

 

 

 

 

N 4?-
O O O
1 1 1 1 1

PROPRANOLOL PGE2 PROPRANOLOL
PGE2

 

Figure 6. Propranolol + , 7 conscious sheep. Blood pressure and
heart rate in three separate xperiments (all e riments performed in
all sheep) comparing the responses to 1 kg + .025 mykg/min
propranolol IV given alone, to 10 ng/kg/min PG IC given alone, and to
pr0pranolol + PGE given together . Open bars present control values.
Siaded bars reprgsent test values 35 minutes after propranolol alone
(Pr0pranolol) , 25 minutes after the start of PGE infusion alone ( ,
and 55 minutes after propranolol and 25 minutes 2after the start of 32
infusion when the two drugs were given together (PrOpranolol + ).
A. Blood pressure: *=p<.05 re Control and Pr0pranolol. B. Heart ra e:
*=p<.05 re Control and Pr0pranolol; +=p<.05 re PGEZ.

50

In these sheep PGE increased blood pressure 21 mmHg (p<.05).

2
Propranolol had no effect on control blood pressure or on the blood
pressure response to IC PGEZ.

Data from these experiments for each individual sheep are shown in

Appendix C, Tables C—3 and C-4.

        
   
     
        

 

 

 
 

s.

I120~ i ~120
L9 - - 5:
5 80‘ 3' -80 a
a ‘ 53/ ' 70
62 60~ 72/ -6O 3:
CL _‘ :/ _ r'T‘I
g 40« 5; ~40 33?
CE .. :3/ 1- 3
E 20~ % .20 V
< - / t I
E 0- 0
L1_l

E

PROPRANOLOL PROPRANOLOL
+
CONTROL PROPRANOLOL ATROPINE ATROEINE
PGEZ

Fi e 7. Propranolol (1 mg/kg + 0.025 mdkg/min TV) + Atropine (1
1115:; IV) + PGE2 (1O ng/kg/min IC), 7 conscious sleep. Bars represent
values of blood pressure and heart rate during consecutive
administration of substances during one experiment. Note that
administration of propranolol did not change heart rate or blood
pressure. Addition of atropine five minutes later caused a significant
increase in heart rate with no change in blood pressure. Subsequent
infusion of IC PGE caused no further increase in heart rate and raised
blood pressure. = p<.05 re Control and Propranolol. + = p<.05 re
Propranolol + Atropine.

It

51

Combined Blockade

 

In the experiments in which both propranolol and atropine were given
before PGEZ, propranolol by itself did not change heart rate, but heart
rate increased when atropine was added, 17 bpm (p<.05, Figure 7).
Subsequent infusion of IC PGE2 caused no further increase in heart rate.
When all three drugs were given together, heart rate was not
significantly different from heart rate during atropine alone.

The combination of propranolol and atropine did not alter the blood
pressure response to subsequent PGE2 infusion.

Data from these experiments for individual sheep are shown in
Appendix C, Tables C-5 and C-6.

When the changes in blood pressure and heart rate during combined
blockade were compared by chi—square test to the sums of changes caused
by each drug alone (the calculated or expected changes), neither the
observed changes in blood pressure nor the observed changes in heart
rate were as great as the expected changes (p<.05), regardless of
whether the blockers were given before or during PGE2 infusion. Figure
8 compares the observed (experimentally—derived) changes in blood
pressure and heart rate to the expected changes in these variables
(calculated from the sums of exper mentally-derived changes) for the
case in which pr0pranolol and atropine were given during PGE2 infusion.
Note that the observed increase in heart rate when all three drugs were
given (which was shown to be not significantly different from the
increase in heart rate caused by atropine alone) appears to be the same
as the increase in heart rate calculated from the sum of the changes in

heart rate during propranolol alone and atropine alone.

Ila

52

 

 

 

 

 

 

 

A. A
E 2
E 70: OBSERVED CALCULATED :70 g
E 50: ~50 r”
.1 :1: : E
g 30. ‘30 :
Lg 403 :-10 ’3‘
E -30 '_30 I
o S
B. A O
a 70: OBSERVED CALCULATED [70 i
a 50.. [-62%
a: .

Z 10‘ :1:
LIJ c1 :3
:2: “0: - a
a -304 g

PGE PlOP ATRO Pg:

PROP

+

ATRO

Fi e 8. (10 ng/kg/min IC) 4» Propranolol (1 Ig/kg + 0.025

min IV) AtrOpine (1 mg/kg IV), 7 conscious sheep. A.
Canparison of changes in blood pressure . Note that the observed change
in blood pressure at 25 minutes PGE infusion + 10 minutes propranolol +
5 minutes atrOpine (PGE+PROP+ATRO) Ts significantly less than the change
in blood pressure calculated (PGE+PROP+ATRO calculated) from the sum of‘
the observed changes in blood pressure during PGE alone (PGE) ,
propranolol alone (PROP), and atropine alone (ATRO . "PROP+ATRO
calculated" is the sum of PROP + ATRO. *=p<.05 re (PGE+PROP+ATRO
calculated). B. Comparison of changes in heart rate. Note that the
observed change in the heart rate at 25 minutes PGE infusion + 10
minutes propranolol + 5 minutes atropine (PGE+PROP+ATRO observed) is
significantly less than the change in heart rate calculated
(PGE+PROP+ATRO calculated) from the sum of observed changes in heart
rate during PGE2 alone (PGE) , propranolol alone (PROP), and atropine
alone (ATRO) . "PROP+ATRO calculated" is the sum of PROP + ATRO.
*=p< .05 re PGE+PROP+ATRO calculated.

53

Table 4 demonstrates that isoproterenol, 5 ug, raised heart rate 65—
72 beats per minute in all experimental series prior to administration
of propranolol. Following propranolol, isoproterenol, 50 ug, raised
heart rate only 6—10 beats per minute, whether propranolol was given

alone, before PGE2, after PGE2, or before PGE2 and atropine.

Table 4. Heart rate increases to test boluses of isoproterenol, 5 ug
before and 50 ug after experimental agents, during four
experimental protocols in seven conscious sheep

 

Heart Rate Increase (beats per minute)
Is0pr0terenol, 5 ug Isoproterenol, 50 ug

 

Experimental Before Propranolol After Propranolol
Protocol (mean i S.E.M. )a (mean i S.E.M.)
Pr0pranolol, alone 68 i 2.74 9 i 1. 24
PGEZ, then Propranolol 68 i 4.54 6 _4_-_ 1.29
Propranolol, then PGE2 72 i 5.38 9 i 2.46
Propranolol, then 65 i 7.69 10 i 1.77

Atropine , then PGE2

 

a Standard error of the mean.

DISCUSSION

This laboratory has shown that the increase in heart rate observed
during IC administration of PGE2,
calves, is not abolished with alpha— and beta—adrenoceptor blockade

1O ng/kg/min to conscious sheep and

(Hull and Chimoskey 1984) . The current study was performed to determine
if inhibition of parasympathetic tone to the heart is partially

responsible for the increase in heart rate. If IC PGE2 acts only

54

at areas in the brain that control peripheral sympathetic nervous system
activity, it might be expected that either the peripheral para-
sympathetic nervous system plays no role at all in the response to IC
PGEZ, or that the parasympathetic nervous system actually is activated
by the baroreflex to Oppose the increases in blood pressure and heart
rate caused by PGE2 driving the peripheral sympathetic nervous system.
Alternatively, IC PGE2 may act at multiple sites in the brain or within
the baroreflex pathway itself, both to activate the peripheral sympa-
thetic nervous system and to inhibit resting parasympathetic tone.

In the first series of experiments presented here, muscarinic recep—
tor blockade did not affect the heart rate response to IC PGEZ. These
data, taken alone, suggest that in sheep inhibition of the parasympa—
thetic nervous system does not contribute to the increase in heart rate
observed when 10 ng/kg/min PGE2 is given IC, nor does activation of the
parasympathetic nervous system inhibit the PGEZ-induced increase in
heart rate.

Inhibition of cardiac parasympathetic tone may account for a small
part of the increase in blood pressure caused by IC PGE2 infusion, since
the increase in blood pressure during 10 PGE2 infusion in the presence
of atropine is less than the increase in blood pressure when PGE2 is
infused alone. If inhibition of parasympathetic tone to the heart
contributes to the increase in blood pressure during IC PGE2 infusion,
PGE2 must increase cardiac output. Cardiac output was shown to increase
during IC PGE2 infusion in a previous study by this laboratory (Hull and
Chimoskey 1984) .

Since the atropine experiments failed to show that the increase in

heart rate caused by IC PGE2 is caused by parasympathetic inhibition, we

55

re—examined the effect of beta-adrenoceptor blockade on this increase.
Since propranolol is a competitive antagonist (Weiner 1980b), it is
possible that the inability of Hull and Chimoskey to prevent the
PGEZ-induced tachycardia was caused by insufficient beta-adrenoceptor
blockade. In the present study, a larger dose of propranolol
significantly reduced, but still did not abolish, the increase in heart

rate caused by 10 PGE infusion. Although small, this remaining

2
increase in heart rate during beta-adrenoceptor blockade is significant,
especially since the parasympathetic nervous system's ability to lower
heart rate in response to the PGEZ-induced 20+ mmHg increase in blood
pressure is unopposed. In these experiments it is unlikely that PGE2
overcame the propranolol blockade, since the degree of blockade (as
determined by heart rate responses to isoproterenol) was not different
when propranolol was given alone or with PGE2 (Table 4) . Furthermore,
the degree of blockade produced by pr0pranolol was the same , regardless
of whether the PGEZ-induced tachycardia was prevented or not . Thus ,
only part of the increase in heart rate during PGE2 infusion is caused
by beta—adrenoceptor activation alone.

Recently, a nonadrenergic—nonmuscarinic mechanism of increased heart
rate following increases in afterload has been reported (Evans et al.
1984; Rigel et al. 1984). To determine whether this mechanism contri-
butes to the change in heart rate during IC PGE2 infusion, combined
blockade with both propranolol and atropine was performed prior to IC
PGE2 infusion. In these experiments PGE2 did not raise heart rate
beyond the level obtained during propranolol and atropine.

Since combined beta-adrenergic and muscarinic blockades abolish the

PGE2-induced tachycardia, and since alpha—adrenergic blockade prevents

56

the PGEZ-induced increase in blood pressure (Hull and Chimoskey 1984) ,
the increases in blood pressure and heart rate during IC PGE2 infusion,
10 ng/kg/min in conscious sheep, are caused primarily by peripheral
sympathetic activation and secondarily by parasympathetic inhibition.
Furthermore, it appears that during blockade of one division of the
autonomic nervous system, 10 PGE2 increases heart rate by means of the
other , unblocked , division of the autonomic nervous system. Thus , the
contribution of the sympathetic nervous system to the heart rate re-
sponse to IC PGE2 is increased during muscarinic receptor blockade,
thereby compensating for the loss of ability to increase heart rate by
withdrawing parasympathetic tone. Likewise, during beta-adrenoceptor
blockade, the contribution of parasympathetic withdrawal to the PGEZ-
induced tachycardia is increased, perhaps even changing from a merely
permissive role to a causative role.

The interpretation described above of the data in this chapter
implies that there is a set point and control system for heart rate,
just as there is for blood pressure. It is known that the baroreceptors
are sensitive to pulse strength as well as to mean arterial pressure
(Angell James and Daly 1970); they may also be sensitive to frequency.
If 10 PGE2 increases the heart rate "set point" , then one might expect
the central nervous system to attempt to raise heart rate by whatever
means is available to it.

The central pressor and tachycardic effects of PGE2 also have been
studied in rats (Hoffman and Schmid 1979; Kondo et al. 1979; Hoffman et
al. 1981; Takahashi and Bunag1981b; Okuno et al. 1982). Our results in
sheep are in agreement with the results of Hoffman and Schmid in rats

(1979) , who also found evidence for both sympathetic activation and

 

57

parasympathetic inhibition in response to centrally—administered PGEZ.

It has been shown previously that PGE 10 ng/kg/min IC in conscious

2,
sheep, causes a shift of the baroreflex to a higher Operating level
without a change in sensitivity (Hull and Chimoskey 1984). Intracarotid
PGE2 does not produce its central cardiovascular effects by acting
directly on the carotid sinus baroreceptors (McQueen and Belmonte 1974;
Breuhaus and Chimoskey 1983) . These observations (McQueen and Belmonte
1974; Breuhaus and Chimoskey 1983; Hull and Chimoskey 1984) , together
with the data from this study, support the hypothesis that 10 PGE2 acts
within the central baroreflex pathway to cause both activation of the
peripheral sympathetic nervous system and inhibition of peripheral

parasympathetic nervous tone .

V. WTHREE

WWW HEEDD AID
REPONSE TO CENTRAL AND MAI. INFUSIONS 0F ANGIO'EBTSIN II

INTRODUCTION

A central pressor effect of angiotensin II (AII), separate from its
direct effect on vascular smooth muscle, first was demonstrated by
Bickerton and Buckley in a cross—circulation preparation in which blood—
borne AII, infused into the head of a vascularly—isolated, neurally-
intact recipient, increased blood pressure in the recipient's trunk
(Bickerton and Buckley 1961 ). Since that study, a separate brain
renin-angiotensin system has been documented (Fischer—Ferraro et al. ,
1971; Ganten et al., 1971; Phillips et al., 1979) and many
investigations of the central actions of All have been performed (for
reviews see Buckley and Ferrario 1977 and 1981; Phillips 1978 and 1980).
In addition to its pressor effect, centrally—administered All has been
shown to cause antidiuresis, urinary sodium excretion, release of
certain anterior pituitary hormones including ACTH and vasopressin, and
drinking behavior in conscious animals. Since it is not possible or
practical to use cross—circulation techniques in conscious chronically-
instrumented animals, and since circulating All is a direct
vasoconstrictor, central actions of All in conscious animals have been
studied by comparing responses to intracarotid (IC) vs intravenous (IV)
infusions of All and by infusion of AII into cerebral spinal fluid.

58

59

The purpose of the present study was threefold. First, this paper
describes a method of chronic catheterization of the cerebral ventricles
of sheep which is more convenient and easier to maintain than
conventional methods (Pappenheimer et al. 1962; Akerlund et al. 1973) .
Second, the presence or absence of drinking responses to intracerebro—
ventricular (IVT) infusions of All in sheep was used to verify proper
ventricular catheter placement and patency. Drinking and pressor
responses to IVT AII were compared to responses to IC or IV AII. Third,
a dose of All was determined which, when given IVT to conscious sheep,
increases blood pressure 20-25 mmHg. Other hemodynamic responses,
plasma sodium, osmolal and vaSOpressin concentrations, and packed cell

volumes also were measured during IVT infusion of AII.

METHODS

Eighteen mature female sheep ranging in weight from 45 to 80 kg were
used in this study. The sheep were housed in individual pens and they
were allowed to acclimate to their surroundings and to laboratory
personnel before surgical preparation. Because sheep are flock animals,
at least two sheep were kept in each room. These conditions ensured
that the sheep were calm and unstressed during the experiments.

Ventricular cannulas were placed in the first five sheep according
to the technique of Pappenheimer et al (1962). Because of problems with
maintenance of the catheters that developed in these sheep, the
technique was modified, and the fifth sheep and thirteen additional
sheep were catheterized as described below. Problems encountered with
the metal ventricular cannulas will be presented in the results , but

data from these sheep were not included in this presentation.

6O

Instrumentation

 

Twelve sheep were anesthetized with intravenous sodium thiamylal,
intubated, and maintained in a surgical plane of anesthesia with
halothane and oxygen. They were placed in dorsal recumbency and their
necks were prepared for aseptic surgery. Through a midline incision,
both carotid arteries were exposed without disturbing the vagus nerves,
and non-occlusive polyvinyl catheters made from TygonR tubing (Fischer
Scientific) were implanted in each artery (Herd and Barger, 1964). One
external jugular vein also was catheterized in the same manner. The
catheters were tunneled subcutaneously to a prepared spot near the
shoulder, brOught through the skin individually, and tied to a loop of
polyvinyl tubing.

In order to measure cardiac output and to calculate total peripheral
resistance, the last of the twelfth sheep also had an electromagietic
flowmeter (Zepeda; Seattle WA) placed on its main pulmonary artery in a
subsequent surgery (described next). Two additional sheep (making a
total of fourteen sheep) were anesthetized as described above, placed on
a respirator, and a left lateral thoracotomy was performed to implant an
electromagnetic flowmeter on the main pulmonary artery and non-occlusive
catheters in the right atrium and aortic arch. The thoracotomy was
closed and the left common carotid artery was catheterized
non-occlusively through a separate incision placed directly over the
artery. All catheters and the flowmeter wires were tunneled
subcutaneously to a prepared spot at the shoulder, as described

previously.

61

All fourteen sheep then were placed in eternal recumbency with their
heads supported on sandbags and taped in a position such that the
parietal surface of the skull was horizontal (Pappenheimer et al. 1962).
This positioning put the axis of the brainstem in the horizontal plane
and the tip of the ventricular catheter , which was perpendicular to the
horizontal axis, followed a trajectory that was inclined 25° rostrally
fran the coronal zero described by McKenzie and Smith (1973) (Figure 9).

 

 

 

Figure 9. Position of ventricular catheter in lateral cerebral
ventricle.

62

The surgical site was prepared for aseptic surgery and a
caudad-directed curvilinear incision was made in the skin over the
junction of the parietal and frontal bones (the coronal suture). The
rostral flap of skin was dissected off the surface of the parietal bone
and reflected forward (Figure 10A) . Hemorrhage was controlled by
ligation. To ensure viability at the end of the procedure, the skin
flap was covered with a sponge moistened with physiologic saline. The
periosteum of the skull was incised with a scalpel along the midline and
reflected laterally to the side of catheter placement (7 left, 7 right).

A trephine with a 5 mm diameter was used to create a hole in the
skull. Pappenheimer et al (1962) used the coronal suture as a landmark
for cannula placement in the goat. According to May (1970) , the coronal
suture of the sheep remains distinct throughout life but is very
irregular. Because the coronal suture was not always easy to find in
these mature sheep without performing more extensive surgical dissection
(and trauma), its position was identified as follows.

The most rostral boundary of attachment of the muscles of the neck
to the skull could be identified easily. Imaginary lines were drawn
from the midline of this boundary rostrally through the center of each
cornual process. The trephine was placed to one side of the midline and
moved forward from the muscle boundary until it was centered between the
midline and the oblique imaginary line with approximately 2 mm ease on
each side (Figure 10B). By following this procedure, the coronal suture
was located and formed the rostral border of the trephine hole in nine
sheep. In five sheep the trephine hole was placed directly over the
suture.

 

Figure 10. Placement
of the ventricular
catheter. A. s =
skin, co = coronal
suture , m 2 muscle,
and cp = cornual
process. B. Trephine
hole centered between
the midline and an
oblique imaginary
line passing through
the cornual process .

63

 

Figure 10 (cont'd.).
C. Trephine hole made
and screws placed.
D. Catheter (with
infusion line
attached) ready to be
advanced into brain.

64

 

 

65

Before the trephine hole was completed, three smaller holes were
drilled around it and filled with stainless steel machine screws. The
trephine hole was completed and the plug of bone removed (Figure 100).
If bleeding occurred, a corner of a gauze sponge was placed in the hole
temporarily until the bleeding stopped.

The ventricular catheter then was prepared for placement. This
catheter had been made previously from .053" I.D./ .085" 0.D. polyvinyl
tubing cut 2 mm longer than the shaft of a 16 gauge 5-1/4 inch
AngiocathR stylet (The Deseret 00., Sandy, Utah). Three tabs of a
larger size tubing had been placed 4 cm, 6.5 cm, and 10 cm from the
bottom of the catheter, in the same way that tabs are placed on
Herd—Barger catheters (Herd and Barger 1964) . The AngiocathR stylet was
slipped into the ventricular catheter and tied at the top with a piece
of silk (Figure 10D). A sterile infusion line filled with artificial
cerebral spinal fluid (CSF) (Table 5) was connected to the stylet, and
pressure in the stylet-ventricular catheter was measured with a
Gould-Statham pressure transducer onto a Gould-Brush chart recorder.

The catheter was held by a micromanipulator and positioned over the
trephine hole so that it was perpendicular to the parietal surface of
the skull. A .059 ml/min infusion of artificial csr was started through
the stylet, and the catheter and stylet were advanced into the brain.
Pressure was allowed to rise . The stylet and catheter then were
advanced slowly until pressure dropped abruptly to ventricular pressure.
The depth in millimeters from the brain surface was noted. The stylet
and catheter then were advanced further until pressure again started to
rise, at which point they were withdrawn until pressure dropped back to

ventricular pressure, and this depth from the brain surface was noted.

66

The catheter was left at the deepest level possible where ventricular
pressure was steady. This was important, since catheters left at the
depth where pressure first drOpped abruptly were more likely eventually

to be sealed by the ventricular lining and become non-functional.

Table 5. Composition of Artificial Cerebral Spinal Fluid (CSF)

 

 

meL
NaCl 125.25
KCl 3.10
CaCl2 2H20 1.20
MgCl2 ’ 6H20 1.00
NaHCO3 24.50
NaH2PO4 2H2O 0.50
Dextrose* 2.53
Dissolved in distilled water gassed with 5% C02
Total Na Concentration 150.30
Total Cl Concentration 138.80

L 7 .40
* CSF sterilized as individual 100 ml bottles. Dextrose was added
to each 100 ml bottle as it was used.

After the correct depth of catheter placement was determined, a silk
suture was tied from the lowest tab on the catheter to one of the
screws. The catheter was cemented in place with dental acrylic worked
into the hole in the Skull and around the screws. The dental acrylic
used was a cold—cure type (Hygenic Corp., Akron, Ohio) which bonded to
the polyvinyl tubing but did not dissolve it. Once the dental acrylic
had begun to set, the silk suture around the top of the catheter was cut
carefully and the stylet was removed. The infusion line was reconnected
directly to the catheter and pressure continued to be monitored to make

certain that removal of the stylet had not inadvertantly disturbed the

 

67

catheter placement.

The second tab on the catheter was sutured to subcutaneous tissue to
provide strain relief. The rest of the catheter was brought through the
skin caudad to the incision, and the third tab on the catheter was
sutured loosely to the skin. An obturator was placed in the end of the
catheter. The curved flap of Skin was brought back over the mound of
dental acrylic and the skin incision was closed in two layers with
interrupted sutures. The flap ensured that the skin incision was not
directly over the point of entry of the catheter into the brain, and the
curvature of the incision aided wound healing by minimizing strain on
the suture line caused by the mound of dental acrylic.

Immediately after surgery each sheep was given 3 x 106 units of a
combination of procaine and benzathine penicillin. G (HeL-Bi—PenTM,
Raver—Lockhart) intramuscularly and 1.92 gm trimethoprimesulfa
(TribrissinR, Burroughs wellcome Co.) orally. They were maintained on
the trimethoprimesulfa for an additional three days. Flunixin meglumine
(BanamineR, Shering, Inc), a non-steroidal anti-inflammatony drug, was
given IV, 1 ngykg, for the first two days after surgery. This was done
in an attempt to eliminate or decrease any adhesions or scar tissue in
the ventricles resulting from placement of the ventricular catheter .
The sheep were given ten days to two weeks to recover from surgery.
During this time they became further accustomed to handling and to the
equipment to be used for experiments. The carotid and jugular catheters
were filled daily with 1:1000 sodium heparin. The ventricular catheters

were left sealed except during IVT experiments.

Experimental Procedures

 

All experiments were performed with each sheep standing quietly in
its cage. One of the carotid artery catheters was connected to a
Gould—Statham pressure transducer placed at heart level, and arterial
pressure was recorded continuously onto magnetic tape (Hewlett-Packard)
and displayed in both pulsatile and mean form on a Gould—Brush
oscillograph. Mean arterial pressure data were obtained by averaging
blood pressure every 30 seconds over a two-minute period, beginning at
the designated time. For example, arterial pressure data five minutes
after the start of any infusion represent the average of values taken
every 30 seconds, from five minutes to seven minutes after the start of
the infusion. Heart rate data were obtained by counting the number of
peaks in the pulsatile blood pressure trace over this same two minute
period. During control periods, heparinized saline, .3 ml/min, was
infused into the carotid artery catheter and, if appropriate, into the
venous catheter. Subsequent peripheral drug infusions were substituted
for the heparinized saline infusions and given at the same volume flow
rate.

In the sheep with electromagnetic flowmeters on their main pulmonary
arteries, right atrial pressure and cardiac output also were recorded.
Total peripheral resistance was calculated from (arterial pressure -
right atrial pressure) divided by cardiac output. Stroke volume was
calculated from cardiac output divided by heart rate. Each sheep's
flowmeter was calibrated in situ with the indocyanine green dye dilution
technique.

During IVT infusion experiments, ventricular pressure also was

monitored and recorded as described above. Care was taken when handling

69

the ventricular catheter to prevent infections. Special packs
containing syringes, sponges , gloves, stopcocks, needle hub adaptors, an
infusion line (PE 20), and a line to the transducer (PE 240) were gas
sterilized with ethylene oxide. The infusion line was filled with
sterile artificial CSF and connected by a stopcock to the catheter end
of the transducer line. The transducer line was flushed retrograde with
sterile CSF before being connected to the transducer.

The ventricular catheter was wiped with ethanol and then a clean
sponge was placed under the catheter and secured by hooking it over the
crownpiece of the sheep's halter. This kept the catheter tip up out of
the sheep's wool. The obturater was removed and placed in a beaker of
ethanol for the duration of the experiment . The free end of the
ventricular catheter was immersed in an iodine solution (BetadineR, The
Purdue Frederick 00. , Norwalk, Conn.) for one minute. The tip then was
rinsed in two sequential ethanol dips, rinsed with artificial CSF, and
attached to the transducer-infusion line via a sterile 16—gauge needle
hub adaptor.

In those sheep in which CSF flowed freely from the catheter, the
iodine and ethanol dips were performed while the obturator was still in
the catheter. This was done to prevent either solution from being drawn
up the catheter into the ventricle, in the event that the sheep lowered
its head.

During control periods, artificial CSF was infused IVT at a volume
flow rate of .039 ml/min. This infusion rate did not alter ventricular
pressure and was tolerated easily by the sheep. Subsequent IVT AII
infusions were performed by diluting the appr0priate amount of AII in

artificial CSF and giving it at the same volume flow rate as during

70

control. Intracerebroventricular experiments were repeated a minimum of
four days apart and often seven days apart. Experiments in which AII
was given IV or IC were perf0rmed in between IVT experiments.
Angiotensin II (Sigma) was prepared as a stock solution of 10 mg/ml
in artificial CSF and stored refrigerated. Just prior to use the proper
amount was diluted in artificial CSF for IVT experiments or in saline
fer 10 or IV infusions. In pilot experiments, IVT infusions of All at
10—50 ng/kg/min all caused drinking. The dose of IVT AII used in this
study (50 ng/kg/min) was selected because it caused blood pressure to
increase approximately 20 mmHg. vehicle control experhments also were
performed. During some experiments, blood was drawn to measure packed
cell volume and plasma vasopressin, osmolal, and sodium concentrations.
Angiotensin II also was given at a rate of 10 ng/kg/min 10 or IV to
eight sheep and at 50 ng/kg/min IV to six of these same sheep. The
sheep were not allowed to drink during any of the All infusions, since
drinking ‘behavior increases the magnitude of' the ;pressor response
(Hoffman et al. 1977; Fitzsimons and Kucharczyk; 1978) . Presence or
absence of drinking behavior was assessed at the end of the infusions.
In experiments in which blood samples were taken, blood was
withdrawn during control and 35 minutes after the start of All infusion
from a carotid artery catheter through a length of polyethylene tubing
which was long enough to extend out of the cage, and which held three
milliliters of fluid. This allowed the investigator to take blood
samples without standing up or approaching (and possibly disturbing) the
sheep. Blood was withdrawn into iced syringes. The first ten ml of
blood withdrawn were discarded, and then, using a new syringe, the

sample was drawn and transferred into iced EDTA tubes. After the blood

71

sample was taken, the volume of blood withdrawn was replaced with
physiologic saline. In these experiments, packed cell volume was
determined in microhematocrit tubes after the experiment was over. The
rest of the blood was centrifuged immediately at 40 F for five minutes
at 2700 rpm. Plasma sodium concentration was determined by flame
photometry (Instrumentation Laboratory, Model #943) and plasma
osmolality was determined by the freezing point depression technique
(Micro—Osmette; Fischer Scientific). Plasma also was stored at -80°C

until analyzed for vasopressin concentration, described below.

Vasopressin Assay

 

Plasma vasopressin concentrations were determined by Dr. Roger
Grekin at the University of Michigan, Ann Arbor. The assay has been
described previously (Pierce et al., 1984). Briefly, vasOpressin was
extracted from 2 ml plasma samples with octadecylsilane cartridges
(Sep—pak C18; Waters Associates, Milford, MA) and assayed in triplicate.
Synthetic arginine vasopressin (Sigma, 350 IU/ng) was used for the
standard curve. There was no detectable cross—reactivity with oxytocin,
vasotocin, or ACTH, and no interference from unknown plasma factors.

The assay could detect as little as 1.25 pg/ml vasopressin.

Statistical Analysis

 

Where possible, experiments were repeated in each sheep, and data
for each sheep represent the average of several experiments of each
type. In the results section, "n" identifies the number of sheep used
in the experiment, and "r" represents the total number of replicates of

the experiment that were performed in those sheep. Statistics were

72

performed on the data per sheep (n—1 degrees of freedom) and not on the
data per replicate.

Mean arterial pressure, heart rate, right atrial pressure, cardiac
output, total peripheral resistance, stroke volume, packed cell volume,
plasma sodium, and plasma osmolality were compared by the paired t-test
or by blocked analysis of variance (ANOVA), as was appropriate.
Multiple comparisons were made by Student-Newman—Keuls test . Some
plasma vas0pressin concentrations were less than the sensitivity of the
assay (1.25 pgfiml). These samples were assigned values of 0.63 pgjml
(halfway between 0 or 1.25 pg/ml) and nonparametric statistics (Signed
Ranks Test) were used to determine whether IVT AII infusion altered
plasma. vasopressin. concentration. 'Values of jp<.05 ‘were considered

significant .

Verification of Catheter Placement

 

All ventricular catheters were injected or infused postmortem with
crystal violet or methylene blue dye. The brains were removed from the
skulls and cut in half midsagitally. The point of entry and the

position of the tip of each catheter was recorded.

RESULTS

Problems encountered with stainless steel ventricular guide cannulae
included neurologic deficits, inflammation/ infection, and poor
durability. Two sheep were sacrificed less than one month after cannula
placement when they developed severe head tilts (with the ear on the
side of cannula placement held higher than the Opposite ear) and loss of

equilibrium. In one of these sheep the deficit was preceded by a fever,

 

suggesting that the source of inflammation was infection. In the second
sheep, no fever was detected. Antibiotic and non-steroidal
anti—inflammatory therapy returned body temperature to normal in the
first sheep, but the neurologic deficits progressed in both sheep. The
third sheep was found dead the day after surgery. Although it appeared
that the cause of death was enterotoxemia, complications from placement
of the ventricular cannula carmot be ruled out. The cannula was
malpositioned in the fourth sheep, and the fifth sheep had a seizure one
week after catheter placement and sheared off the hub of the cannula.
The problems described above did not occur in the sheep which
received polyvinyl ventricular catheters (see Appendix D for individual
sheep history). All polyvinyl ventricular catheters entered the
posterior part of the lateral ventricle 2 to 18 mm rostral to the
splenium of the corpus callosum except one (discussed under "angiotensin
II infusions ") . One sheep would not eat after surgery. A second
developed septicemia, apparently from its carotid artery catheter.
These two sheep were sacrificed immediately, before sufficient data
could be obtained to include them in the results. In the remaining
twelve sheep, the ventricular catheters remained functional an average
of 6i1 month, ranging from two months to greater than one year. During
this time the sheep remained healthy and peripheral arterial or venous
infusions could be performed daily. Intracerebroventricular experiments
usually were performed weekly, although it was possible to perform IVT
experiments as often as every five days for extended periods of time
with no problems encountered. It also was possible to repeat an IVT
experiment within two days, but this could not be done habitually

without causing slight changes in control blood pressure and increasing

 

74

the risk of infection. The relatively low control values of blood
pressure and heart rate and the stability of these values from day to
day for each sheep indicate that the presence of the ventricular
catheters and the experiments performed were neither painful nor

stressful for these animals and that the sheep were handled humanely.

Angiotensin II Infusions

 

Thirteen of the fourteen sheep which received polyvinyl cerebro—
ventricular catheters drank water immediately when they were released
after experiments in which All was given IVT. The sheep were released
15—30 minutes after the All infusion had been turned off. One sheep
drank only rarely after IVT AII, and when she did drink it was only a
few sips and usually was delayed 5—10 minutes after being released. At
postmortem this sheep's catheter stopped 3 mm above the ventricle. Data
from IVT experiments in this sheep have not been included.

Intracerebroventricular infusions of AII, 50 ng/kg/min in eleven
conscious sheep (42 replicates), caused a gradual rise in arterial
pressure. Thirty minutes after the start of infusion, blood pressure had
increased from 86 i 1.6 mmHg to 109 i 2.5 mmHg (p<.01, Figure 11).
Blood pressure began to fall as early as 15 minutes after the end of
infusion, but did not return to control levels until 30—60 minutes
afterwards. Heart rate (78 i 3 bpm) did not change. In the three sheep
with electromagnetic flowmeters (six replicates), right atrial pressure
increased from 1.1 i .8 1:11ng to 3.7 i 1.1 mmHg (p<.05) cardiac output
(6.5 i .2 L/min) did not change, total peripheral resistance increased
from 13.14 i .86 to 16.34 i .70 mmHg min/L (p<.01), and stroke volume
increased from 86 i 2 to 88 :1 ml (p<.05).

 

75

 

 

’6?

I

E 115-

E. .

a 105-

D

U) a

£3

a: 95"

D.

2 .

a? 85-

LL]

2 .

Z 75 I I I r I I I
:5 0 5 10 15 20 .25 30
2

TIME (minutes)

Fi e11. Time course of blood pressure response to IVT AII, 50
ngfiré/min, in eleven conscious sheep. All points differ from control
and each other (p<.05) except 15 and 20 minutes, which do not differ
from each other, and 25 and 30 minutes, which do not differ from each
other.

Values of packed cell volume , plasma sodium concentration, plasma
osmolality, and plasma vasOpressin concentration before and 35 minutes
after the start of IVT AII infusion are shown in Table 6. Only plasma
vasopressin (which increased) changed significantly (p<.01). Although
statistics were performed on all the AVP data (see Methods), the value
of vasopressin reported during IVT AII infusion is the average value in
the five sheep in which it was greater than the sensitivity of the
assay. Although plasma osmolality decreased in seven of the nine sheep,
the change was not significant (p=.07).

Intracerebroventricular infusions of vehicle (CSF) for the same

duration as IVT AII had no effect on any variable reported above.

76

Table 6. Effect of Intracerebroventricular (IVT) Infusion of
Angiotensin II (All), 50 ng/kg/min, on Packed Cell Volume,
Plasma Sodium Concentration, Plasma Osmolality, and Plasma
Vasopressin Concentration in Conscious Sheep

 

 

Control IVT AII
n (mean + SE11) (mean + SIM)
Packed Cell Volume (96) 9 27 i 1 27 i 1
Plasma [Na+] (mEq/L) 9 145.5 : .9 145.0 3: .9
Plasma Osmolality (mOsm/L) 9 309 i 3 306 i 3
Plasma Vasopressin (pg/ml) 8 < 1.25* (p<.05) 5.90 i 1.62

 

* All control values were less than the sensitivity of the assay.

Table 7 summarizes the arterial pressure, heart rate, and drinking
responses of eight conscious sheep to IC and IV AII, 10 ng/kg/min, and
of six sheep to IV All, 50 ng/kg/min. The hemodynamic data shown were
taken during control and 25 minutes after the start of infusion. With
peripheral AII infusions, blood pressure rose quickly (within five
minutes), leveled off for the duration of the infusion, and fell rapidly
after the infusion was turned off. (Blood pressure began to decrease
within minutes after the end of infusion and had returned to control by
15-30 minutes afterwards.) Drinking responses were recorded after the
sheep were released, approximately ten minutes after the end of the All
infusions. The "(IO-IV) AII, 1O ng/kg/min" values for changes in
arterial pressure and changes in heart rate were calculated to estimate
the central component of the responses to peripherally-infused AII at 10
ng/kg/min (Fink et al. 1980). No statistics were performed on these

values .

 

77

Table 7. Blood Pressure, Heart Rate, and Drinki Responses to
Peripheral Infusions of Angiotensin II All)

 

 

Angiotensin II Blood Pressure Heart rate Drinking
(BP, mmHg) (HR, beats/min) Behavior
Route Dose Control Change Control Change After Release
ng/kg/min) meanism in BP mean-ism in HR #yes #no
*
ICAII 10 8611 +30 77:4 -4 3 5
(n=8,r=11) * *
IV All 10 85 + 1 +25 85 + 2 -13 2 6
(n=8,r=10) - —
(IO-IV) AII 10 + 5 + 9
(11:8) -x- *
IV All 50 86 i 2 +47 80 i 5 —1O 1 5
(n=6,r=6)

 

* p<.01 re Control

Blood pressure and heart rate data for individual sheep from all

experiments presented in this chapter can be found in Appendix E.

DISCUSSION

 

The use of flexible polyvinyl tubing catheters instead of rigid
stainless steel cannulae for catheterization of the lateral cerebral
ventricles of sheep has certain advantages. The catheters appear to
cause less inflammation, since longer implantation times are possible
without development of neurologic abnormalities. This may be due to the
difference in material, but it may reflect a difference in the ease of
adequately cleaning the free end of the catheter or cannula before
infusions. The free ends of the polyvinyl catheters can be lifted up

78

out of the wool and dipped in disinfectant solutions, whereas this is
not possible with the rigid hubs of the steel cannulae. Additionally,
the further these hubs stick out from the skull (making them easier to
clean) the more susceptible they are to being brOken off (sheep #11).

One disadvantage of the polyvinyl catheters is that patency is not
easily re—established if they become plugged. In these sheep, patency
was maintened longer when the tips of the catheters were left against
the tissue at the bottom of the ventricle rather than just barely into
the ventricle, since this prevented the ventricular lining from sealing
over the catheter. However , leaving the catheter tip against tissue
prevents CSF from flowing freely back up the catheter. This makes it
necessary to assess proper catheter placement by some other method, and
excludes the use of this technique fer CSF sampling. The technique can
be used f0r infusions, however, since infusion fluid will flow back up
around the catheter into the ventricular space (Hoffman and Phillips,
1976b). Regular weekly infusions into the catheters probably helps to
maintain catheter patency.

Drinking behavior in response to IVT infusion of All can be used in
conscious sheep as a premortem indication that the ventricular catheter
is patent and is indeed in a cerebral ventricle, despite failure of CSF
to flow from the catheter. Phillips (1978) reported that IVT AII is
1000X more potent than IV All in its ability to cause drinking. In the
present study, only one of six sheep drank after 50 ng/kg/min AII IV,
whereas 13 of 14 sheep drank after 50 ng/kg/min AII IVT. The only sheep
that did not drink in response to IVT All had a catheter that ended
several millimeters above the lateral ventricle. When sheep were

sacrificed either because they had lost a drinking response to IVT AII

79

that they had had earlier or because ventricular infusion pressure was
becoming elevated, postmortem examination revealed that their catheters
were becoming or had become plugged.

Previous studies with peripherally or centrallyeadministered AII in
sheep and goats have focused on AII's effects on drinking'behavior and
the hormones of body fluid balance (Andersson and westbye 1970;
Andersson et al. 1972; Andersson 1977; Blair4west et al. 1980; Nicholle
et al. 1983). The peripheral rate of All infusion used in the present
study produces circulating levels of AII well within the physiologic
range of AII in sheep (Blair—West et al. 1980). The IVT rate of All
infusion was higher than that used in other studies, but was necessany
to produce the desired change in blood pressure. This is in agreement
with work in conscious rats in which IV AII also produced greater
pressor responses than IVT AII (Hoffman and Phillips 1976b).

The results of AII infusions reported here in conscious sheep are
comparable to those reported by Bonjour and Malvin (1970) in conscious
dogs, and by Mouw et al. (1971) in pentobarbital—anesthetized dogs. In
the latter study IV and IC infusions of AII, 10~17 ng/kg/min, produced
similar changes in blood pressure to those reported here, and smaller
doses of IVT AII (.01-5 ng/kg/min) increased plasma, vasopressin
concentration without changing blood pressure, plasma, sodium
concentration, plasma osmolality, or packed cell volume. The present
study provides new evidence that the IVT AII-induced increase in blood
pressure in conscious sheep is caused by an increase in total peripheral
resistance. Cardiac output and heart rate do not change, and right

atrial pressure and stroke volume are increased slightly.

 

VI. CHAPTEFOUR

0(1le OF INTRACAROIID VS.
INTRACEREBROVENTRICULAR INFUSIGIIS OF PEEZ

INTRODUCTION

Prostaglandins of the E series (PGEs) are potent vasodilators when
injected peripherally (Moncada et al. 198)), but as early as 1969 Kaplan
et al. demonstrated a centrally-mediated vasopressor effect of PGE1 in
anesthetized dog. Further studies of the central pressor effects of
PGE8 have been performed because of their potential role in central
arterial pressure regulation (Skarnes et al. 1981; Takahashi and Bunag
1981b) . Prostaglandin E2 is synthesized by brain neural and vascular
tissue (Hagen et al. 1979; Abdel-Halim et al. 1980;) and does not
circulate, since it is metabolized rapidly by the lungs (Ferreira and
Vane 1970; Piper et al. 1970; Moncada et al. 1980;). Thus PGE2 could
act locally in the brain to increase blood pressure without leaking into
the periphery to decrease blood pressure. Since the studies of Kaplan
et al. (1969) , PGEs have been shown to increase blood pressure when
infused into the cerebral blood supply or into cerebral spinal fluid of
a variety of species. (Hoffman and Schmid 1979; Kondo et al. 1979;
Hoffman et al. 1981; Skarnes et al. 1981; Takahashi and Bunag 1981b;
Feurstein et al. 1982; Okuno et al. 1982; Breuhaus and Chimoskey 1983;
Hull and Chimoskey 1984).

81

The present study was performed (1) to determine if PGE2 increases
arterial pressure when infused into the lateral cerebral ventricles
(IVT) of conscious sheep and, if so, then (2) to compare dose response
curves of IC vs IVT PGEZ, (3) to note any hemodynamic or behavioral
differences between the responses to IC and IVT PGEZ, and (4) to measure
plasma catecholamines, vasopressin, sodium, and osmolality and packed
cell volume before and during IC and IVT PGE2 infusion in conscious
sheep. Evidence is presented that PGE2 is much less effective at
raising arterial pressure when given IVT than when given IC. Despite
this , certain plasma changes and behavioral effects, which are not
observed when PGE2 is given IC, occur when PGE2 is given IVT.

Prostaglandin E2 administered into cerebral spinal fluid may not act at

the same sites in the brain as does blood—borne PGE2.

METHODS

Fifteen mature female sheep ranging in weight from 45 to 80 kg were
used in this study. The sheep were housed in individual pens and they
were allowed to acclimate to their surroundings and to laboratory
personnel before instrumentation. Because sheep are flock animals, at
least two sheep we re kept in each room. These conditions ensured that

the sheep were calm and unstressed during the experiments.

Instrumentation

 

The methods used to place vascular and cerebral ventricular

catheters in sheep were described in Chapter 3.

82

Experimental Procedures

 

All experiments were performed with each sheep standing quietly in
its cage. One of the carotid artery catheters was connected to a
Gould—Statham pressure transducer placed at heart level , and arterial
pressure was recorded continuously onto magnetic tape (Hewlett-Packard)
and displayed in both pulsatile and mean form on a Gould-Brush
oscillograph. During control periods, heparinized saline, .3 ml/min,
was infused into the carotid artery catheter and, if appropriate, into
the venous catheter . Subsequent peripheral drug infusions were
substituted for the heparinized saline infusions and given at the same
volume flow rate .

During IVT infusion experiments, ventricular pressure also was
monitored and recorded as described above. Care was taken when handling
the ventricular catheters to prevent infections . The ventricular
catheter tips were disinfected as described in Chapter 3. During
control periods, sterile artificial CSF was infused IVT at a volume flow
rate of .039 ml/min. This infusion rate did not alter ventricular
pressure and was tolerated easily by the sheep. Subsequent IVT PGE2 or
vehicle infusions were performed by diluting the appropriate amount of
PGE2 in artificial CSF and giving it at the same volume flow rate as
during control. Intracerebroventricular experiments were repeated a
minimum of four days apart and often seven days apart. There was no set
order in which experiments were performed, although very high doses of
IVT PGE2 never were given before the individual sheep's sensitivity to
IVT PGE2 was tested using lower doses of PGE2. Also, because
prostaglandins are known mediators of inflammation, doses of PGE2

greater than 100 ng/kg/min were not repeated in consecutive IVT

II»

83

experiments. .Experiments in. which PGE2 was given. IC or IV 'were
performed on days when IVT experiments were not performed.

In experiments in which blood samples were taken, blood was
withdrawn during control and 35 minutes after the start of PGE2 or
vehicle infusion from a carotid artery catheter through a length of
polyethylene tubing which was long enough to extend out of the cage and
which held three milliliters of fluid. This allowed the investigator to
take blood samples without standing up or approaching (and possibly
disturbing) the sheep. Blood was withdrawn into iced syringes. The
first ten ml of blood withdrawn were discarded, and then, using a new
syringe, the sample was drawn and transferred into iced EDTA tubes.
After the blood sample was taken, the volume of blood withdrawn was
replaced with physiologic saline. In these experiments, packed cell
volume (PCV) was determined in microhematocrit tubes after the
experiment was over. The rest of the blood was centrifuged immediately
at 40 F for five minutes at 2700 rpm. Plasma sodium concentration was
determined by flame photometry (Instrumentation Laboratory, Model 11943)
and osmolality was determined by freezing point depression technique
(Micro—Osmette). Plasma also was stored at -80°C until it was analyzed

for catecholamines and vasopressin, described below.

 

Intracarotid PGE2 Infusions: Intracarotid PGE2 infusions were
performed as reported—E'eviously (Breuhaus and Chimoskey 1983) . For 10
infusions, PGE2 (Upjohn) was made up as a stock solution of 1 mg/ml in
ethanol and stored at -20° 0. Just prior to use, the proper amount was
diluted in physiologic saline at room temperature. Infusions of vehicle

were performed by adding the same microliter amount of ethanol (without

84

PGE2) as was used for PGE2 infusions at 10 ng/kg/min. Twelve sheep were

used to generate IC PGE2 dose response curves. All sheep received IC
PGE2 at a rate of 10 ng/kg/min. Seven of these sheep received an
infusion of vehicle (0 ng/kg/min), eight received 5 and 30 ng/kg/min,
six received 60 ng/kg/min and eight received 100 ng/kg/min. Blood
samples for PCV, plasma osmolality, and plasma concentrations of sodium,
vasopressin, and circulating catecholamines were collected during
exeriments in which PGE2 was given IC at 10 ng/kg/min and at 100
ng/kg/min. Body temperature was recorded in seven sheep before and

during IC infusion of PGE , 10 ng/kg/min.

Intracerebroventricular PGE2 Infusions: Twelve sheep received IVT

 

PGE in doses ranging from O ng/kg/min (vehicle) to 1000 ng/kg/min.

2
Infusions of vehicle were performed by adding the same microliter amount
of ethanol (without PGE2) as was used for infusions of PGE2 at 300
ng/kg/min. Seven sheep received infusions of vehicle; nine received 10,
30, and 100 ng/kg/min; all twelve received 300 ng/kg/min; four received
750 ng/kg/min; and three received 1000 ng/kg/min IVT PGE2. Only sheep
that showed little or no change in blood pressure at 300 ng/kg/min IVT
PGE2 received higher doses. For IVT infusions, PGE2 was made up in
stock solutions of 10 mg/ml ethanol and 100 mg/ml ethanol to minimize
the amount of ethanol infused into the ventricles, especially at the
higher doses of PGE2. The stock solutions were stored at -20° C, and
just prior to use the proper amount of PGE2 was diluted in artificial
CSF at room temperature. An attempt was made to find an IVT PGE2

infusion rate which caused a similar increase in blood pressure as did

10 ng/kg/min IC PGE2 in each sheep. This was not always possible.

85

Blood samples for PCV, plasma sodium, plasma osmolality, vasopressin,
and circulating catecholamines were taken during infusion of PGE2 at 300
ng/kg/min IVT. Body temperature was recorded in seven sheep before and
during IVT PGE2 infusions.

Intravenous PGE2 Infusions: Intravenous (IV) PGE2, 1O ng/kg/min,

 

 

was infused in six sheep. This was done to determine whether the serous
nasal discharge observed during IO and IVT PGE2 was caused by a central

action of PGE2 on the lung, and will be explained in the discussion.

Adrenergic Blockade of IVT PGE2: In four sheep, the ability of

 

adrenoceptor blockade to prevent the IVT PGE2-induced increase in blood
pressure was tested. Alpha-adrenoceptor blockade was obtained with
phentolamine, 1 mg/kg IV given five minutes before the start of IVT PGE2
infusion. Because phentolamine alone causes pronounced tachycardia in
conscious sheep (Hull and Chimoskey 1984) , the beta-adrenoceptor blocker
propranolol, 1 rug/kg IV, was given ten minutes before administration of
phentolamine . Adequacy of phentolamine blockade (tested with
phenylephrine) and of propranolol blockade (tested with isoproterenol)
will be described in the results. Separate experiments were performed
in the same four sheep to determine the hemodynamic effects of

adrenoceptor blockade alone.

Catecholamine Assay

 

Plasma was acidified by the addition of 2N perchloric acid
containing 100 mg/100 ml EGTA (1 part acid to 9 parts plasma). Plasma

concentrations of norepinephrine and epinephrine were measured by a

Eli,

86
modification of a radioenzymatic assay described by Umezu and Moore
(1979). The assay was run on three separate occasions. Sensitivity of

the assay was 125 pg/ml on two occasions and 250 pg/ml on one occasion.

Vasopressin Assay

 

Plasma vasopressin concentrations were determined for us by Dr.
Roger Grekin at the University of Michigan, Ann Arbor. The assay has
been described previously (Pierce et al. 1984). Briefly, vasopressin
was extracted from 2 ml plasma samples with octadecylsilane cartridges
(Sep—pak C18; Waters Associates, Milford, MA) and assayed in triplicate.
Synthetic arginine vasopressin (Signa, 350 IU/ng) was used for the
standard curve. There was no detectable cross-reactivity with oxytocin,
vasotocin, or ACTH, and no interference from unknown plasma factors.

The assay could detect as little as 1.25 pg/ml vasopressin.

Statistical Analysis

 

Where possible, experiments were repeated in each sheep, and data
for each sheep represent the average of several experiments of each
type. Statistical analyses were performed on the data per sheep and not
on the data per replicate .

Mean arterial pressure, heart rate, PCV, plasma sodium
concentration , plasma osmolality, plasma catecholamines , and body
temperature data were compared by the paired t-test or by blocked
analysis of variance (ANOVA) , as was appropriate. Multiple comparisons
were made by Student—Newman—-Keuls test . One-way ANOVA was used to
compare blood pressure and heart rate responses to different doses of

PGE2, since not every animal received every dose of PGE2. Some plasma

87

samples had less vasopressin than was detectable by the assay (1.25
pg/ml) . These samples were assigned values of 0.63 pg/ml (halfway
between 0 and 1.25 pg/ml) and a nonparametric test, the Signed Ranks
test, was used to determine whether PGE2 altered plasma vasopressin
concentration. Values of p<.05 were considered significant. Results
are expressed as means i standard errors of the mean (SE11) , or for

paired samples, means and the standard error of the difference (SED) .

Verification of Ventricular Catheter Placement

 

Proper positioning of the ventricular catheters and free access of
infusions to CSF were tested before death by observations of the
presence or absence of drinking responses to infusions of angiotensin
II, 50 ng/kg/min IVT (Chapter 3; Hoffman and Phillips 1976b). If, after
a period of time, a sheep lost its drinking response to IVT angiotensin,
no data were used from IVT experiments performed after the last IVT
angiotensin experiment in which the sheep drank.

All ventricular catheters were injected or infused postmortem with
crystal violet or methylene blue dye. The brains were removed from the
skulls and cut in half midsagitally. The point of entry and the
position of the tip of each catheter in the lateral cerebral ventricle

was verified .

RESULTS

Of the fifteen sheep used in the study, fourteen were prepared
surgically with polyvinyl ventricular catheters as described above. One
sheep did not drink in response to IVT angiotensin II and, at postmortem

examination, the tip of its ventricular catheter was several millimeters

above the lateral ventricle. Data from IVT experiments in this sheep

were not included.

Intracarotid PGE.) Infusions

 

Figure 12 shows blood pressure and heart rate responses to
increasing doses of IC PGE2 in twelve conscious sheep. (Not every sheep
received every dose of PGE2, as indicated in "Methods".) The
dose—response curve for IC PGE2 is limited; doses greater than 30
ng/kg/min cause no greater steady-state increases in blood pressure than
the increases caused by 30 ng/kg/min, although blood pressure does rise
more quickly at higher doses.

Intracarotid PGE2 caused a small increase in body temperature which
was significant (p<.01, Table 8) because it increased in every sheep.
Packed cell volume and plasma sodium, osmolality, vasopressin,
epinephrine, and norepinephrine did not change during IC PGE , 1O

ng/kg/min. Intracarotid PGE

2, 100 ng/kg/min, did increase PCV (p<.01,

Table 8).

Intracarotid PGE2 infusions produced a serous nasal discharge and
sneezing at all doses in all animals. At higher doses (60 and 100
ng/kg/min IC), PGE2 caused slight shivering in some sheep. Often the
sheep urinated and defecated as IC PGE2 began to increase blood pressure
and heart rate. However, the sheep showed no signs of discomfort,
alarm, or excitement during IC PGE2 infusion and often chewed their
cuds. They showed no tendency to drink after PGE

2
straight to their feed buckets when released after an experiment.

, but usually went

Intracarotid infusions of vehicle had no effect on any reported

above.

 

BLOOD PRESSURE (mmHg)

 

 

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o
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I I I 1 I

10 15 20 25 30
TIME (minutes)

. =10. 4 =30. - =100 ng/kg/min

On
UM

Figure 12. Intracarotid PGE at three doses. A. Time course of blood
pressure responses. B. T e course of heart rate responses. Blood
pressure and heart rate are significantly greatgr than control at all
time points for all doses. At 10 ng/kg/min,PG reaches steady state
15 minutes after the start of infusion. At 30 né/kg/ reaches
steady state at 10 minutes. At 100 ng/kg/min, PGE reaches steady state
at 5 minutes. (Control values for blood pressure 5nd heart rate are not
significantly different from each other).

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91

Intracerebroventricular PGE2 Infusions

 

The blood pressure responses of eleven sheep to doses of IVT PGE2
from 10 to 300 ng/kg/min are shown in Figure 13. Blood pressure did not
increase in four of the eleven sheep, and larger doses of PGE2 in these
sheep (to 1000 ng/kg/min) also were ineffective. Greater doses of IVT
PGE2 were not used because of the occurrence of side effects or toxicity
described below. The increases in arterial pressure in response to IVT
PGE2 were not as great as the increases in blood pressure in response to
IC PGE2 (One-way ANOVA, p<.05, Figure 14).

Heart rate did not change at any dose of IVT PGE2. In seven of
these sheep in which body temperature was measured during IVT PGE2
infusions, there were no significant changes in body temperature at any
dose 35 minutes after the start of infusion. Intracerebroventricular
PGE2 significantly increased PCV (p<.01), decreased plasma sodium
concentration (p<.01), decreased plasma osmolality (p<.05), and
increased plasma vasopressin concentration (p<.05) . Plasma
concentrations of circulating catecholamines did not change (Table 8).

Intracerebroventricular PGE2 caused shivering in all sheep at some
dose. In general, shivering began at doses of PGE2 that did not
increase blood pressure as much as did 10 ng/kg/min IC PGE2 (an IC dose
at which no shivering occurs). In fact, one sheep began to shiver and
showed other side effects of IVT PGE2 at 30 ng/kg/min, yet never had
much of a hemodynamic response to IVT PGE2, even at 1000 ng/kg/min.

A serous nasal discharge and sneezing also occurred during IVT PGE2
infusions in all sheep, regardless of whether or not they responded
hemodynamically. Other effects of IVT PGE2 which were not observed

during IC PGE2 infusion were: pupillary constriction in eight of eleven

92

100-

A

\
\
1

'\

on
O
1

BLOOD PRESSURE (mmHg)

 

 

I I I

O 5 1'1) 1'5 20 25 30
TIME (minutes)

. :10, 1 =30, . =100, o =3OO ng/kg/min

Figure 13. Time course of blood pressure responses to IVT P at four
doses in eleven conscious sheep. For all doses, all time ints are
significantly greater than control but are not different from each
other. Steady state was reached 5 minutes after the start of infusion.
(Cont§ol values for all doses are not significantly different from each
other .

rt.

 

93

 

 

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1 10 100 1000
PGE2 (ng/kg/min)

CHANGE IN HEART RATE (bpm)

 

 

Figure 14. Comparison of lo dose response data for intracarotid REE
(solid circles) and IVT PGE open circles). Intracarotid PG produce
significantly greater press r and tachycardic res nses than I PGEI at
all doses. (IVT PGE does not change heart rate . A. Changes in b ood
pressure. B. Change; in heart rate.

94

sheep, a dry hacking cough near the end of the 35 minute infusion period

or after the PGE infusion had been turned off in all sheep (but not

2
during every experiment), and vocalization in seven out of eleven sheep.
The frequency of vocalization and the length of time of infusion before
the onset of vocalization appeared to be dose-related. Those sheep that
vocalized showed no signs of pain or discomfort. Vocalization did not
result in further increases in blood pressure in those sheep that had a
pressor response to IVT PGE2, and it did not produce increases in blood
pressure or heart rate in sheep that otherwise had no pressor response
to IVT PGE2.

Intracerebroventricular infusions of vehicle had no effect on any
variable reported above.

Blood pressure and heart rate data for individual sheep from these

experiments can be found in Appendix F.

Intravenous PGE2 Infusions

 

During IV PGE2 infusion, 10 ng/kg/min in six conscious sheep, there
were no changes in blood pressure or heart rate. No nasal discharge

occurred. There was no visible effect of IV PGE2 at this dose.

Adrenergic Blockade of IVT PGE2

 

Adequacy of propranolol blockade was tested with isoproterenol.
Before propranolol, 5 ug isoproterenol increased heart rate 67 i 7 bpm.
Sixty minutes after administration of propranolol, 25 ug isoproterenol
increased heart rate 12 :1; 6 bpm. Adequacy of phentolamine blockade was
tested with phenylephrine. Before phentolamine, 100 ug phenylephrine
increased blood pressure 25 i 4 111an and decreased heart rate 20 i 5

it-

95

 

E

E 20

E

a

3 10

(f)

(f)

LLJ

D:

0- O

Q

C)

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—10

2

Lil

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C.)

PROPRANOLOL PROPRANOLOL
+ PGE2 +
PHENTOLAMINE PHENTOLAMINE

+

PGE2

Figure 15. Changes in blood pressure during three experimental
protocols in the same four sheep. The first bar shows the change in
blood pressure 45 minutes after the administration of propranolol and 35
minutes after the administration of phentolamine. The second bar shows
the pressor response of these sheep to IVT PGE , 300 ng/kg/min, 30
minutes after the start of infusion. The last ar shows that when
propranolol and pentolamine are given prior to PGE infusion, there is
no change in blood pressure 30 minutes after the start of PGE infusion.
Asterisks indicate that blood pressure is significantly different from
control, p<.05.

96

bpm. Sixty minutes after phentolamine, 1000 ug phenylephrine increased
blood pressure 26 i 5 mmHg and decreased heart rate 27 i 5 bpm.

The combination of propranolol and phentolamine had no effect on
heart rate, but decreased blood pressure (Figure 15). Propranolol and
phentolamine prevented the PGE2-induced increase in blood pressure

(Figure 15).

DISCUSSION

This laboratory (Breuhaus and Chimoskey 1983, 1984a, 1984b; Hull and
Chimoskey 1984) has shown that intracarotid (IC) infusions of PGE , 1O
ng/kg/min, increase arterial pressure and heart rate in conscious sheep,
dogs and calves. The increase in arterial pressure is caused by an
increase in total peripheral resistance mediated by increased
alpha-adrenergic vasomotor tone, although there also is a slight, but
significant, increase in cardiac output. Blood flow in certain regional
vascular beds, measured in calves, is decreased; renal, cranial
mesenteric, and iliac resistances are increased. The increase in heart
rate is caused by both cardiac beta-adrenergic activation and by
inhibition of resting cardiac parasympathetic tone. The baroreflex was
studied over a limited range and found to be reset without a change in
sensitivity. The resetting of the baroreflex probably was central in
origin, since the effect of IC PGE2 on blood pressure is not mediated by
direct action at the carotid sinus baroreceptors (Breuhaus and Chimoskey
1985) and since 10 PGE2 has no direct action on carotid sinus nerve
firing (McQueen and Belmonte 1974) .

In the present study, IVT PGE was less potent than IC PGE in terms

2 2
of its hemodynamic effects . Intracarotid PGE2 increased blood pressure

97

and heart rate in all sheep, whereas IVT PGE2 had no effect on blood
pressure or heart rate in four sheep and increased heart rate in only
four of the seven sheep in which it did have a pressor effect. One
possible explanation for this is that PGE2 is removed rapidly from CSF
by the choroid plexus and transported to venous blood (Bito 1972, 1975a,
1975b) . Once there, PGE2 would circulate to the lungs and be
metabolized (Ferreira et al. 1967; Piper et al. 1970; Moncada et al.
1980) . This laboratory has shown previously that 400 ng/kg/min PGE2
causes no change in blood pressure or heart rate when infused into the
right atria of conscious calves (Hull and Chimoskey 1984).

Despite its less potent hemodynamic effects, IVT PGE2 produced some
changes that are not observed during 10 PGE2 infusion, including
behavioral changes and changes in plasma sodium concentration, plasma
osmolality, and plasma vas0pressin concentration. In this study, the
largest increases in plasma vasopressin concentration were observed in
sheep that had no pressor response to IVT PGE2. This observation and
the fact that adrenergic blockade completely prevented the PGE2-induced
pressor response, confirm that vasopressin release does not contribute
to the pressor response to PGE2 in conscious sheep. Increased plasma
levels of vasopressin may account for the decreases in plasma osmolality
and plasma sodium concentration, however, since vasopressin causes
antidiuresis and natriuresis (Chan and Sawyer 1962) . Urinary water and
sodium excretion were not measured in this study, but were found to be
increased by IVT PGE2 in conscious goats (Andersson and Ieksell 1975;
Ieksell 1976) . Since there was no evidence of loss of plasma volume
during IVT PGE2 infusion (plasma osmolality was decreased instead of

increased), the increase in packed cell volume probably was due to

98

splenic contraction rather than due to hemoconcentration.

Prostaglandin E2 was infused IV to determine whether it would cause
a serous nasal discharge, an. effect seen during, IC and IVT PGE2
infusions. This was done to determine whether the nasal discharge was
due to facial vasodilation, was a central effect of PGE2, or whether it
might be caused by action of PGE2 returning in the venous system to the
lungs to increase pulmonary secretions. Since IV PGE2 did not produce a
nasal discharge (thus eliminating the last possibility) and since IVT
PGE2 did produce a serous nasal discharge (thus eliminating the first
possibility) it appears that this effect of IC and IVT PGE2 is
centrallyamediated.

we conclude that PGE2 given IVT may not reach the same sites in the
brain. as IC PGE2, or it may' reach the same sites in different
concentrations. Since PGE2 is synthesized by brain vascular tissue
(Hagen et al. 1979; Abdel—Halim et al. 1980), it is likely that
endogenous PGE2 gains entry to cardiovascular centers in the brain from
the blood route rather than from CSF. This does not exclude the
possibility that PGE2 could participate in cardiovascular regulation

through synthesis by neural tissue in discrete areas of the brain.

VII. CHAPTEFIVE

WWIUMSINHANDHEEZAMINDW
TOINCREASEBIIDDPRISSUREINCONSCIOIBEEP

INTRODUCTION

It is welléknown that prostaglandin synthesis and renin release are
interrelated in the kidney. Prostaglandins cause renin release from the
kidney (Larsson et al. 1974; Spokas et al. 1982; Freeman et al. 1984)
and chronic infusions of PGE2 into the renal arteries of conscious dogs
increase plasma renin activity (Hockel and Cowley 1979) . Conversely,
angiotensin II (AII) stimulates prostaglandin ‘release from. several
tissues (McGiff et al. 1970; Gimbrone and Alexander 1975; Terragnc 1981;
Nasjletti and Malik 1982).

Central effects of PGE2 and AII are similar: both raise arterial
pressure by increasing sympathetic nerve activity and causing
vasopressin release (Hoffman and Phillips 1976; Takahashi and Bunag
1981b; Unger et al. 1981), both cause antidiuresis and natriuresis, and
the actions of both are potentiated by hypertonic saline (Andersson and
Leksell 1975; Leksell 1976; Kapsha and Severs 1981). It has been shown
that Chronic intrarenal infusions of PGE2 in conscious dogs produce mild
hypertension which can be reversed by angiotensin receptor or
convertingeenzyme 'blockade (Hockel and Cowley’ 1980). It ‘has 'been

suggested that intracerebroventricular (IVT) AII causes vasopressin

99

100

release by stimulating PGE2 synthesis in the brain (Share 1979).

This study was performed to determine whether PGE and AII act in

2
series in the brain to increase blood pressure. Specifically, we tested
whether central AII-receptor blockade prevents the pressor effect of
centrally-administered PGE2 or whether prostaglandin synthesis blockade

prevents the pressor response to centrally-administered AII.
METHODS

Instrumentation

 

Nine mature female sheep (50-80 kg) underwent sterile surgery for
placement of vascular catheters and cerebral ventricular catheters as

described in Chapter 3.

Experimental Procedures

 

All experiments were performed with each sheep standing quietly in
its cage. One of the carotid artery catheters was connected to a
Gould-Statham pressure transducer placed at heart level, and arterial
pressure was recorded continuously onto magnetic tape (Hewlett—Packard)
and displayed in both pulsatile and mean form on a Gould-Brush

osc illograph .

Intracarotid PGE2 Infusions: Intracarotid PGE2 infusions were

 

performed as reported previously (Breuhaus and Chimoskey 1985). During
control periods, heparinized saline, .3 ml/min, was infused into the
carotid artery catheter. Prostaglandin E2 (Upjohn) was made up as a
stock solution of 1 mg/ml in ethanol and stored at 40%. Just prior to

101

use, enough PGE2 for a 35 minute infusion was diluted in physiologic
saline at room temperature and substituted for the heparinized saline.

Intracerebroventricular Infusions: During IVT infusion experiments,

 

ventricular pressure also was recorded. Care was taken when handling
the ventricular catheters to prevent infections . The ventricular
catheter tips were disinfected and attached to a sterile
transducer/ infusion line via a sterile 16-gauge needle hub adaptor.
During control periods, sterile artificial CSF was infused IVT at a
volume flow rate of .039 ml/min. This infusion rate did not alter
ventricular pressure and was tolerated easily by the sheep. Subsequent
IVT drug infusions were performed by diluting the proper amount of drug
in artificial CSF and giving it at the same volume flow rate as during
control. (For IVT experiments, stock solutions of drug were made up as
follows: PGE2, 100 mg/ml in ethanol stored at -20°C; AII (Sigma), 10
mg/ml in artificial CSF stored at 4°C; and [Sar1Thr8] AII (Sarthran,
Sigma), 20 mg/ml in artificial CSF stored at 40C.)

AII-Receptor Blockade of Central PGE2: The first series of

 

experiments tested whether central administration of PGE2 in conscious
sheep exerts its pressor effect through activation of the brain
renin—angiotensin system. Each sheep's hemodynamic response to
intracarotid (IC) PGE2, 1O ng/kg/min, and to IVT PGE2, 300 ng/kg/min,
infused for 35 minutes (Chapter 4; Chimoskey and Breuhaus 1985) was

102

first characterized. Next, a dose of AII was found which, when given
IVT for 35 minutes, increased blood pressure as much as PGE2. This dose
was found to be 50 ng/kg/min (Chapter 3). A dose of sarthran was then
found which, when also given IVT, prevented the IVT AII pressor
response. In pilot studies this dose was found to be 1000 ng/kg/min.
Sarthran was started 15 minutes before and continued during the IVT AII
infusion. Finally, the ability of 1000 ng/kg/min IVT sarthran to
prevent the pressor responses to 10 ng/kg/min IC PGE2 or to 300
ng/kg/min IVT PGE2 was tested. Again, the sarthran infusion was started
15 minutes before and continued during the PGE2 infusions. Sarthran was
given by itself for 50 minutes to determine whether it had any effect on

resting blood pressure. Intracarotid and IVT vehicle infusions also

were performed .

Prostaglandin Synthesis Blockade of Central AII: The second series

 

of experiments tested whether prostaglandin synthesis blockade prevents
the pressor response to IVT AII in conscious sheep. Prostaglandin
synthesis blockade was obtained with two different types of
cyclo—oxygenase inhibitors and two different protocols. In the first
protocol, six sheep received indomethacin, 4 mg/kg, dissolved in sesame
oil and given subcutaneously two hours before IVT AII infusion. This
dose and method of administration of indomethacin have been shown to

prevent PGE2 synthesis and the central pressor and hyperthermic

103

responses to endotoxin in conscious sheep. The blockade is effective
within one hour of administration of indomethacin and lasts at least
four hours (Skarnes et al. 1981). In this study IVT AII was given well
within the known effective period of indomethacin (IVT AII was infused
from 120 to 155 minutes after injection of indomethacin). Additionally,
the efficacy of indomethacin blockade was tested in five sheep by
determining whether it would prevent the pressor response to IC
arachidonic acid (see below).

In the second protocol, five sheep were given flunixin meglumine, 3
mg/kg intravenously, thirty minutes before IVT AII infusion. This dose
is 5X the recommended clinical dose, which is supposed to prevent
prostaglandin synthesis for 24 hours. Efficacy of prostaglandin
synthesis blockade was tested in five sheep in the following way. A
dose of arachidonic acid was found which increased blood pressure. On
different days, either indomethacin was given subcutaneously two hours
before 10 arachidonic acid infusion or flunixin meglumine was given one

half hour before IC arachidonic acid infusion.

Statistical Analysis

 

Blood pressure and heart rate data were compared by the paired t
test or by blocked analysis of variance (ANOVA), as was apprOpriate.
Multiple comparisons were performed with Student—Newman—Keuls test.
Values of p<.05 are considered significant. Results are expressed as

means i standard error of the mean (SIM).

II.b

104

Verification of Ventricular Catheter Placement

Proper positioning of the ventricular catheters and free access of
infusions to CSF were tested before death by observations of the
presence or absence of drinking responses to infusions of angiotensin
II, 50 ng/kg/min IVT (Chapter 3; Hoffman and Phillips 1976b). If, after
a period of time, a sheep lost its drinking response to IVT angiotensin,
no data were used from IVT experiments performed after the last IVT
angiotensin experiment in which the sheep did drink.

All ventricular catheters were injected or infused postmortem with
crystal violet or methylene blue dye. The brains were removed from the
skulls and cut in half midsagitally. The point of entry and the
position of the tip of each catheter in the lateral cerebral ventricle

was verified.

RESULTS

One sheep did not drink after IVT All and at postmortem this sheep's
ventricular catheter had become sealed. Data from this sheep were not
included. All other sheep drank after IVT All and their ventricular

catheters were positioned properly in their lateral cerebral ventricles.

AII—Receptor Blockade of Central PGE2: Intracerebroventricular AII,

 

50 ng/kg/min, caused a gradual rise in blood pressure which reached
steady state 15 minutes after the start of infusion (Figure 16). At 50
minutes, blood pressure had increased from 87 i 1.4 to 110 -_1-_ 2.7 mmHg

(p<.01). Heart rate did not change. Intracerebroventricular sarthran,

105

10(1) ng/kg/min, had no effect on blood pressure or heart rate, but
prevented the IVT AII pressor response (Figure 16) and the IVT AII
drinking response.

 

 

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Figure 16. Blood pressure responses to IVT AII, 50 ng/kg/min from O to
30 minutes; to IVT sarthran, 1000 ng/kg/min from --15 to 50 minutes; and
to the combination of IVT sarthran started at -15 minutes and IVT AII
started at 0 minutes. During IVT AII infusion alone, blood pressure is
greater than control (p<.05) after time 0, and reaches steady state at
20 minutes (ie . pressures at times 20-50 minutes are not different fran
each other). IVT sarthran and IVT sarthran + IVT AII do not alter blood
pressure and are not different from each other.

106

In addition to a gradual increase in blood pressure (Figure 17), IC
PGE2, 1O ng/kg/min, caused an increase in heart rate. Steady state was
reached at 15 minutes . At 30 minutes, blood pressure and heart rate had
increased from 85 i 1.6 to 106 i 2.0 mmHg (p<.01) and from 80 i 3.0 to
95 i 2.8 bpn (p<.01). Intracarotid infusion of vehicle (the same
microliter amount of ethanol diluted in saline) had. no effect on blood
pressure or heart rate. Sarthran, 1000 ng/kg/min IVT, did not alter the

pressor (Figure 17) or tachycardic responses to IC PGE2.

 

 

 

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Figure 17. Blood pressure responses to IC PG , 1O ng/kg/min from O to
50 minutes; and to the combination of IVT arthran, 1000 ng/kg/min
started at -15 minutes, and IC PGE started at 0 minutes. There is no
difference in the blood pressure résponses to IC PGE with or without
sarthran pretreatment. For IC PGE infusion alone, glood pressure is
greater than control (p< .05) fron? 5-30 minutes after the start of
infusion, and reaches steady state at 15 minutes (ie. pressures at times
15-50 minutes are not different from each other). For sarthran + IC
PGE , blood pressure is greater than control (p<.05) from 5—50 minutes
aftgr the start of infusion, and reaches steady state at 10 minutes.

107

Intracerebroventricular PGE2, 300 ng/kg/min, increased blood
pressure from 86 j; 2.0 to 100 i 5.9 mmHg (p<.01) 50 minutes after the
start of infusion (Figure 18). Heart rate did not change.
Intracerebroventricular infusion of vehicle (the same microliter amount
of ethanol diluted in artificial CSF) had no effect on blood pressure or
heart rate. Sarthran, 1000 ng/kg/min IVT, did not alter the pressor
response to IVT PGE2 (Figure 18).

 

 

 

 

 

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Figure 18. Blood pressure responses to IVT PGE2, 300 ng/kg/min from O
to 50 minutes, and to the combination of IVT sarthran, 1000 ng/kg/min
started at --15 minutes, and IVT PGE started at 0 minutes. There is no
difference in the blood pressure regsponses to IVT PGE with or without
sarthran pretreatment. For IVT PGE alone, blood prgssure is greater
than control at all time points (1%.05) and reaches steady state 15
minutes after the start of infusion (ie. pressures from 15-30 minutes
are not differint from each other). For sarthran + IVT PGE2, blood
pressure is greater than control (p<.05) at 10-50 minutes.

 

 

 

CHANGE IN BLOOD PRESSURE (mmHg)

FLUNleN
INDOMETHACIN MEGLUMINE
+ +

IVT All IVT All IVT All

Figure 19. Com ison of changes in blood pressure caused by IVT All
alone, 50 n kg/min; caused by IVT AII after pretreatment with
indanethacin, 4 mg/kg subcutaneously; and caused by IVT AII after
pretreatment with flunixin meglumine, 3 mg/kg intravenously. All
increases in blood pressure differ significantly from control (*=p<.05),
but are not different from each other.

Prostaglandin Syniflsis Blockade of Cgtral AII: The results of
prostaglandin synthesis blockade with either indomethacin or flunixin
meglumine were the same; neither blocker prevented the IVT AII pressor
responses (Figure 19), and both appeared to potentiate the IVT AII

drinking responses (subjective observation).

109

In five sheep IC instions of arachidonic acid, 4 mg/min for 20
minutes, increased blood pressure 15 mmHg (p<.01). Intracarotid
infusion of the vehicle for arachidonic acid (0.1 M NaHCO3 in 10%
ethanol) did not alter blood pressure. After indomethacin or flunixin
meglumine, IC arachidonic acid also did not alter blood pressure.

Blood pressure and heart rate data for individual sheep from

experiments presented in this chapter can be found in Appendix G.

DISCUSSION

Potentiation. of the central AII pressor response with. centrally;
administered PGE has been described (Gyang et al. 1973; Ieksell 1976).
Although the hypothesis that central AII acts through brain PGE2
synthesis is attractive and is supported by circumstantial evidence
(Share 1979), the present study does not support it, since indomethacin
and flunixin meglumine did not prevent the IVT AII pressor response.
These results in conscious sheep are in agreement with those of Phillips
who found that meclofenamate increased the drinking response to IVT All
in conscious rats, but had no effect on the pressor response to IVT AII
(Phillips 1980).

This is the only study in which central AII-receptor blockade has
been performed with central administration of PGE2. The results

indicate that PGE does not exert its central effects solely by

2
stimulation of the brain renin—angiotensin system.

In conclusion, this study provides evidence that central All and
PGE2 do not act in series (i.e. production or administration of one does

not exert its effects solely through synthesis or release of the other).

It is possible that central administration of one substance increases

 

 

110

blood pressure partially through synthesis or release of the other and
partially through other mechanism(s) , and that during prostaglandin
synthesis blockade or AII—receptor blockade the increase in blood
pressure is maintained by greater contribution from these other
mechanism(s) . Whether these other mechanism(s) exist and what they may
be is unknown, but this type of compensatory switching of mechanisms was
observed in Chapter 2. This study also does not exclude the possibility
that All and PGE2 have similar central actions because they share common
central pathways. If this is true, however, it is likely that PGE2 and
All activate these pathways independently of each other .

 

VIII. MARY AND CONCLUSHE

In summary, although PGE2 is a potent vasodilator in the periphery,
(Moncada et al. 1980), central administration of PGE2 causes blood
pressure to rise. In sheep, the increase in blood pressure is mediated
totally by peripheral adrenergic activation (Chapter 4; Hull and
Chimoskey 1984) -

Intracarotid (IC) administration of PGE2 also causes heart rate to
increase due to a combination of cardiac beta-adrenergic activation and
cardiac parasympathetic inhibition (Chapter 2). Intracarotid PGE2 has
no effect on plasma renin activity or partial pressure of 002 (Hull and
Chimoskey 1984) , or on plasma osmolality or plasma concentrations of
sodium, epinephrine, norepinephrine, or vasopressin (Chapter 4) . At
higher doses, IC PGE2 increases packed cell volume (PCV) , probably by
splenic contraction.

In conscious Sheep, IC PGE2 is more potent 'than. intracerebro—
ventricular (IVT) PGE2 (Chapter 4). This difference cannot be explained
by direct action of IC PGE2 on carotid sinus baroreceptors (Chapter 1).
It may be caused by PGE2 reaching different sites in the brain by the
two routes or by PGE2 reaching the same sites in different
concentrations, especially since IVT PGE2 produces certain changes such
as pupillary constriction, vocalization, and coughing which are not

observed during IC PGE2 administration.

111

 

112

Intracerebroventricular PGE2 increases PCV and decreases plasma
osmolality and sodium concentration in conscious sheep. The decreases
in plasma sodium concentration and plasma osmolality may be the result
of increased urinary sodium excretion and decreased free water clearance
(Andersson and Ieksell 1975) caused by increased vasopressin secretion.
Although IVT PGE.2 does increase plasma vasopressin concentration in
sheep, this increase is not partially responsible for PGEZ's pressor
effect in sheep (as it is in rats: Takahashi and Bunag 1981b, Okuno et
al. 1982), since adrenergic blockade alone prevents the PGE2 pressor
response. Also unlike IVT PGEZ's effects in conscious rats (Hoffman and
Schmid 1979; Feurstein et a1. 1982; Okuno et al. 1982), IVT PGE2 does
not increase plasma catecholamines or heart rate in conscious sheep.

Intracerebroventricular administration of angiotensin II (AII) has
cardiovascular effects similar to those of IVT PGE2. Interactions
between the renin—angiotensin system and PGE2 have been described
(McGiff et al. 1970; Larsson et al. 1974; Gimbrone and Alexander 1975;
Terragno 1981; Nasjletti and Malik 1982; Spokas et al. 1982; Freeman et
al. 1984). The original hypothesis of Chapter 5 was that central PGE2
increases blood pressure by stimulating release or synthesis of central
AII. Instead, the experiments presented in Chapter 5 show that PGE2 and
AII can act independently of each other in the brain; AII receptor
blockade has no effect on the pressor responses to IC or IVT PGE2, and
prostaglandin synthesis blockers have no effect on the pressor response
to IVT AII.

Thus, the central mechanism of the PGE2 pressor response remains to
be elucidated. More recent studies support an interaction of PGE2 with

central catecholaminergic pathways (Breuhaus and Chimoskey 1984a) . In

113

these studies in conscious sheep, the central alpha2 agonist clonidine
both prevented and reversed the central pressor and tachycardic effects
of PGE2. Since PGE2 inhibits catecholamine release in a variety of
tissues including brain (Bergstrdm et al. 1975; Frame and Hedqvist 1975;
Malik 1978; Hillier and Templeton 198)), central administration of PGE2
may increase blood pressure by inhibiting catecholamine release in areas
of the brain where catecholamines are tonically vasodepressor, such as
the A—1 region of the ventrolateral medulla or the A—2 or C-2 regions of
the dorsomedial medulla (Brody et al. 1984).

In conclusion, PGE2 may play an important role in arterial pressure
regulation. Prostaglandin E2 is synthesized by brain tissue (Wolfe and
Coceani 1969; Wolfe et al. 1976), brain blood vessels (Hagen et al.
1979; Abdel—Halim et al. 198)), and endothelial cells (Gimbrone and
Alexander 1975). Because PGE2 is pressor when administered centrally,
increased central PGE2 production would result in increased blood
pressure. The effect of increased central PGE2 production would be
self-regulating, however, since PGE2 is a potent vasodilator in the
periphery. Any PGrE2 released from the brain into cerebral venous blood
would be removed rapidly from the blood with passage through the lungs
(Ferreira and Vane 1967; Piper et al. 1970; Moncada et al. 1980). As
long as the rate of PGE2 release did not exceed the ability of the lungs
to remove it in a single pass, blood pressure would remain elevated.
Further central PGE2 production, however, would result in leakage of
PGE2 into the periphery. The resultant direct vasodilation would
counter PGEZ'S central pressor effect.

There is evidence that endogenous prostaglandins play a role in

central arterial pressure regulation. The rise in blood pressure

114

associated with IVT administration of bradykinin can be inhibited by
inhibition of prostaglandin synthesis with indomethacin and enhanced by
administration of the prostaglandin precursor arachidonic acid
(Takahashi and Bunag 1981a) . Similarly, the increases in blood pressure
caused by endotoxin (Skarnes et al. 1981) or IVT carbachol (Phillips
1980) are prevented by prior treatment with prostaglandin synthesis
inhibitors. Prostaglandin E2 may well be the prostaglandin involved in

these pathways since central administration of PGF has little

2alpha
(Hoffman et al. 1981) or no (Kondo et al. 1979) effect on blood pressure
and IVT PGI2 causes blood pressure to decrease (Kondo et al. 1979).
Finally, if central PGE2 plays a role in normal arterial pressure
regulation, it is possible that any metabolic change resulting in an
increase in PGE2 in brain tissue could lead to hypertension. The
possibility of a role for PGE2 in the genesis of hypertension is
supported by evidence that increases in blood pressure and sympathetic
nerve activity due to IVT injections of PGE2 are greater in
spontaneously hypertensive rats (Takahashi and Bunag 1981 b) and certain
strains of genetically hypertensive rats demonstrate a decreased ability

to catabolize prostaglandins before they become hypertensive

(Pace-Asciak 1976; Limas and Limas 1977).

 

APPEIDICIES

 

Sheep ID #

1
2

12
13

 

APPENDIX A

IDENTIFICATION OF MAJOR CCIHHIDfllflliS ("THE GIRLS")

Name
Betty Bot
Zorro

Possum

Minnie Pearl

Peachy

Ethyl

Gertrude
Lucy
Bertha
Agnes

Dana

Roz

Violet

Contributions
Chapter 1: carotid sinus
Chapter 1: carotid sinus

Chapter 1: carotid sinus, Intravertebral
pilots, IVT pilots

Chapter 1: carotid sinus

Chapter 1: carotid sinus, Intravertebral
pilots, IVT pilots

Chapter 1: carotid sinus, Intravertebral
pilots, IVT pilots

Intravertebral pilots

Intravertebral pilots

Intravertebral pilots

IVT pilots

Chapter 2: parasympathetic; Chapter 5: IVT
AII; Chapter 4: IVT PGE2, catecholamines,
vasopressin; Chapter 5: IVT sarthran;
Clonidine studies; Naloxone studies
Chapter 2: parasympathetic; IVT pilots
Chapter 2: parasympathetic; Chapter 5: IVT
AII; Chapter 4: IVT PGE2, catecholamines,

vasopressin; Chapter 5: IVT sarthran;
Clonidine studies

115

20
21

22

25

24

25

Daisy

Smokey

Silver

Susie

Pinky

Blondie

Emma

Charlotte

Trudy

Helga

Sadie

Maude

116

Chapter 2: parasympathetic; Chapter 5: IVT
AII; Chapter 4: IVT PGE2, catecholamines,
vasopressin; Chapter 5: IVT sarthran

IVT pilots; IC PGE2 catecholamines and
vasopressin

Chapter 2: parasympathetic; Chapter 5: IVT
AII; Chapter 4: IVT PGE2, catecholamines and
vasopressin

IVT pilots; IC PGE2 catecholamines and
vasopressin

Chapter 2: parasympathetic; Chapter 5: IVT

AII; Chapter 4: IVT PGE2, catecholamines,

vasopressin; Chapter 5: IVT sarthran; .
Carbachol pilots; Clonidine, naloxone, and

stress studies

Chapters 2, 3, 4, and 5; Clonidine studies
Chapters 2, 3. 4, and 5; Clonidine studies
Chapters 5 and 4; Clonidine, naloxone, and
stress studies; Hypertonic saline and

carbachol pilots

Chapters 5, 4, and 5; Clonidine, naloxone, and
stress, and hypertonic saline studies

Chapters 5, 4, and 5; Clonidine, naloxone,
stress, hypertonic saline, yohimbine, and
studies

Chapters 5 and 4; Clonidine, naloxone, stress,
hypertonic saline, yohimbine, and ANP studies

Chapters 5 and 4; Clonidine, naloxone, stress,
hypertonic saline, yohimbine, and ANP studies

APPHIDIX B

117

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sp mpCospaomxm opopmmom mouse Soap moogm Hoswp>flucH soap spam opom ppoom can opdmmopm wooam .elo oHpoe

122

on Hospsoo on mo.va

.plo oapoa CH on mammaoso aseppmppopm

.Apopm<v HoaocMHQOpm p0 msaop Hopppsp ogp coppo mopSst ms >H HosopopOpmomp

m5 om 0p oncommop mp opdp peso: qp omqoSO oSp op coaomsoo Aopopomv Hoppsoo mappsw >H Homoeopocmoop
ws m 0p oncomwop up opoc peso: SH omcogo osp mo uomwopmxo owoxooap Hoaocopmocm Mo hoosvoc< o

;2*

 

 

 

pm.m >0.N pp.m mm.m mm.w mm.m ww.w mw.p 2am
oop ms em as mop mm mop em ¢p+ pp + zoos
* .x. * *
mpp NS mm mm mpp mop mpp ow om+ ow + ON
mm we mm we mop so oop ow op+ so + mp
mop mm pm mm ppp mop mm pm w + ms + mp
mpp om oop pm mm mm so we mm+ mm + mp
mm we pm we mop mop mop om ¢p+ so + sp
mm me om we so om mm ow mp+ me + mp
om ow mop ow mop mop ppp mm m + wpp+ pp
osHQOpp< Hoaoqopmopm Asps mpv Hosp ocH90pp< Hoaococmoam papa mpv Hosp copp< opopom % QH
Hoaoqocmocm + mmwm [coo Hoaocopmocm + mmwm Icoo Mooam moosm
mesa News Nmea News memos
Aopdsps\wpoopv opom ppoom Ammssv oHSmmopm wooam

ssa\os\ms op “News
Mo sepmSMSH quOpoooppsH mappsm so>pw ocoz wa\ws pv osHQOpp< can Asps\wM\ws mmo. + spa\wx\ws
pv Hoaoqopm0cm msoso>occh sepcz mp moogm Hossp>pssH Hosp opoa opom ppoom ego oHSmmopm sooam .mlo oppoe

125

8v Hospsoo on mode *

.plo oapoe sp no mpmhaoso HoOHpmppopm p
.ppopp<v Hoaoompmopm mo mSHop Hopppcp opp sopwo wopchs ms >H Hosocopocmowp

m5 Om op oncommop mp opop peso: 9p omqono onp op ooHMQSOO Aopopomv Hoppsoo mapp5o >H Hosopoposmomp

ws m op oncommop cp

opmc paoon sp omcoso ogp on womwopmxo oodMooap Hoposopmocm Mo hoo56o©< o

 

 

 

m>.¢ mm.m m¢.¢ mo.m m®.m ©©.N mo.m mo.m 2mm
om mm me me p mop mm mm em op+ mm+ coos
* * *
mm om S mm .8 p so no em 0 p+ 8+ om
mop mop we so mpp om mm mm mp+ ¢m+ mp
sop em as mo Npp mm pm N@ m + 05+ mp
ppp mpp mm mop so pr we no wp+ mm+ mp
we we mm pm mo p mm mm mm s p+ 5+ e p
em mm 3 out mm mm mm mm m + sm+ m p
em me me as m p p mm om om m + 3+ p p
mmwm osHQOpp< pops mv Hosp mmcm ospmocp< Amps mv Hopp sopp< opopom % QH
ospm0pp< + LopQOAm Isoo oQHQOpp< + Loam0pm Isoo Mooam moosw
HoaococQOpm Hoaococmocm HoaosopQOpm Hoaoqopmopm opmoe
popssps\mpoopv opom ppoom Ammssv oHSMmopm wooam
:ps\mM\ws op .QOpoSMsH mmwm
opposooocpsH Mo ppopm opp opomom so>pw opoz wa\wspv ospm0pp< woo Asps\wx\ws mmo. + mx\ws
pv HoaooopQOLm ozoso>ocpoH SOpgs op moogm Hosop>pocH Soap opmm opnm paoom ego oHSmmopm oooam .mlo oppoe

Il‘,

APPHIDIX D

 

.pxop ooo .pIQ oHcoa ho COHposonxo soapsSM pow .o>pHo Happo mooso I ***

 

 

 

.Qpp aopospoo osp po>o mspaooo on: mapapa poHSOpspso> I w
app sopoapoo who Hooo 0p mapsspwop oos ozxoag upososo I o
ooxooao poo sepppoom opp so osow pos om: paoapsomxo osp oaooa :IIIr < I p
oopospoppo p09 was hHao hpowpso mo ospp opp po cospoppo moo I o
*** m on ooh ospsos s ooh oa mN
*** m III III mapsos +m ooh 0s vN
*** m III III ospsoa +Np ooh on mm
as mp q ooh oa ospaos h ooh ospaos h I ooh NN
oas Np q o: ooh ospaos w ooh ospaos N I ooh pm
as op H on ooh oppaos m ooh on ON
oas op m o: ooh ogpaoa o ooh onpaos N I ooh mp
oas pp H ooh o: spaoa p on Moos p I ooh mp
as N A o: o: oMoos m.N ooh 0: hp
oas m m on ooh oppsos N ooh 0: mp
as op A o co spaos p ooh on mp
mm as m A ca 0: oapsoa m ooh Moos p I ooh ep
1. as m m oa oa ospaos h ooh 0: mp
as m m ooh oa ospaos op ooh 0: pp
IIIIIIII os ooh ohms 0 III Mooh Np
as N H a o: soos p III mooh pp
I o os ohms 0 III oooh op
as N m ooh oa oMoos N III oooh m
ooh on apaos p pooh ooh m

ass 8393s possess 2.2 aosQH sow

sepppoom upwoaopdoz mo oooq sopoSpoo aha waoponpoo
sopospoo ouopoaomlcopospoo osph ho popp< sons Soah %.Q.H
sopsospooa Narcissus sop soosom fiosop oars hose sou Em mooso
paoaooopm sopospoo soHSOHLpsoh mo soHpoOppppoh .pIm oppoe

APPMDIX D

VERIFICATIGIOFCAWPIACMTANDHMBIEBW

The problems which we encountered in developing this preparation are
presented here in the hope that this will prevent others who wish to use
this technique from experiencing similar difficulties. Table D—1
summarizes the success or failure of catheter placement for each sheep
used in this study. All catheters entered the posterior part of the
lateral ventricle. The numbers given under "Postmortem Catheter
Position" indicate the distance in millimeters that the catheter was
found rostral to the splenium of the corpus collosum. The letter "L"
indicates that the catheter was to the left of midline; "R" means it was
to the right. "Length of Time Catheter Functional" indicates the time
from surgery until either 1) elective euthanasia was performed (if no
problems developed with the catheter), 2) there was a change in catheter
function (loss of drinking response to IVT All or loss of ability to
infuse into the catheter indicated by elevated ventricular pressure), or
5) the animal demonstrated some neurologic abnormality (such as a head
tilt). The table also indicates which sheep were sacrificed because of
problems with their ventricular catheters, and whether these were
related to development of neurologic signs or to loss of catheter
patency.

Sheep #‘s 5, 5, 10, and 12 received stainless steel guide cannulas
according to the technique of Pappenheimer et al. (1962). Pilot data

125

 

126

only were obtained from sheep #5 before she developed a head tilt and
gradually lost control of her equilibrium. Sheep #5 had an extensor
paralysis in ‘her right hind limb immediately after surgery “which
improved over the next few days. She began to develop a head tilt after
the first IVT experiment. Several days later she had a fever, her head
tilt was worse, and problems 'with. her right hind limb returned.
Euthanasia with an overdose of pentobarbital was performed in both of
these sheep. In the sheep that developed head tilts, the tilt was such
that the car on the side of the catheter placement was higher than the
other car. Sheep #10 was found dead in her cage the morning after
surgery. We are uncertain whether this death was related to her
ventricular cannula or caused by a gastrointestinal upset. The evening
before she had recovered well from anesthesia, had no neural deficits,
and was eating and drinking. She had had an unusually big appetite
immediately after surgemy and, in retrospect, she may have upset the
bacterial balance in her rumen and developed enterotoxemia, but no
postmortem was performed. Sheep #12's cannula apparently was
malpositioned since control IVT infusions could not be performed without
causing, discomfbrt and raising "blood pressure. Therefore, no IVT
experiments were perfbrmed in this Sheep. Sheep #11 sheared off the hub
of her ventricular guide cannula one week after surgeny. No IVT
experiments had been performed. She then was the first Sheep to receive
1a polyvinyl catheter using the technique described in Chapter 5. This
catheter was placed in the lateral cerebral ventricle opposite to the
stainless steel cannula. At postmortem, both cannula and catheter had
entered their respective ventricles at approximately'the same position.

No data from sheep using the stainless steel cannulas were presented.

127

Of the fourteen sheep that received polyvinyl ventricular catheters,
data from two of these sheep (#'s 15 and 17) were not presented. Sheep
#15 developed diarrhea and would not eat after surgery. The diarrhea
cleared up, but she never regained an appetite. She appeared normal and
was healthy in every other way, but she could not be enticed to eat.
Different feeds, appetite stimulants, IV dextrose infusions, feeding a
slurry made from pellets by stomach tube, and transfaunation
(transferring fresh rumen contents from a normal donor to the sheep by
stomach tube) were tried. Remarkably, she maintained her weight for
approximately one month. Euthanasia was performed when she continued
not to eat and her health began to deteriorate. At postmortem it was
found that the catheter had entered the posterior part of the left
lateral ventricle 10 mm in front of the splenium of the corpus callosum.
However, it had passed through the ventricle, and the tip rested in the
menigeal space between the cerebellum and the dorsum of the brainstem,
just over the rostral colliculi. During IVT infusions, fluid apparently
had leaked backwards along the catheter to the lateral ventricle, since
this sheep drank in response to IVT AII, 5O ng/kg/min. The State of
Michigan Animal Health Diagnostic laboratory performed a complete gross
postmortem examination on this sheep but could not determine a cause for
the lack of appetite.

Sheep #17 developed a uveitis in her right eye and a septicemia.
These responded initially to local and systemic antibiotic
(chloramphenicol) therapy, but the uveitis returned after discontinuing
treatment. This problem more likely was related to embolization from
this sheep's right carotid artery catheter (which was flapped and did

not withdraw) than to a problem associated with her ventricular catheter

 

128

(which was on her left side). This sheep did drink in response to IVT
AII, but four weeks elapsed from the time of her last IVT experiment
until euthanasia. At postmortem it was found that the ventricular
catheter originally had just entered the posterior left lateral
ventricle 2 run in front of the splenium of the corpus callosum, but the
catheter was not very deep and the ventricular lining had sealed over
the tip of the catheter, leaving only a dimple to indicate that the
catheter had ever been through the lining. No dye had entered the
ventricles.

Sheep #11 developed disequilibrium and a head tilt approximately one
year after surgery and had to be sacrificed. Her catheter was still
patent at postmortem examination.

The ends of the ventricular catheters of sheep #‘s 15 and 14 were
dangling freely in the ventricular space. The choroid plexus had
wrapped itself around the tips , but dye had flowed freely throughout the
ventricles.

Euthanasia was performed on sheep #16 because an elevated
ventricular pressure during IVT experiments suggested that the
ventricular catheter was getting plugged. This was confirmed at
postmortem; there was only a small. spot of dye coming into the ventricle
at the end of the catheter. The ventricular lining was in the process
of sealing over the catheter.

Cerebrospinal fluid flowed freely from sheep #18's ventricular
catheter the first time she had an IVT experiment. However, later that
week the catheter pin was discovered to be missing. After that, CSF no
longer flowed freely from the catheter. This sheep did not drink after
her first IVT AII experiment, although she sniffed at the water. During

129

that experiment ventricular pressure was approximately 14 mmHg, so
certainly the catheter was not plugged at that time. Ventricular
pressure gradually increased during subsequent IVT experiments,
suggesting that the catheter either was sealing off or choroid plexus
was beginning to plug the tip. This sheep continued to have an
inconsistent drinking response to IVT All. When she did drink it
usually was delayed. At postmortem it was found that the tip of the
catheter was several millimeters above the dorsal lining of the
ventricle. The lack of drinking response to IVT All in this sheep
suggested that infusions were not reaching the third cerebral ventricle.
Alternatively, it may be that centrally— administered AII did not elicit
a drinking response in this sheep, since she did not drink in response
to 10 All either. However, the very definite pressor response to IVT
PGE2 obtained repeatedly in this sheep had to be a central effect. This
sheep did, however, require higher doses of PGE2 IVT than did other
sheep that responded to IVT PGE2, and she had relatively few side
effects associated with the infusions (although she did have one—the
dry hacking cough). It is possible, therefore, that PGE2 and AII gained
access to the cerebral ventricles in this sheep by diffusion through the
thin layer of cerebrum that lay between the ventricles and the catheter
tip. It may also be true that these data demonstrate that the central
site of action of PGE2 is different from the central site where AII
elicits its drinking response. In other sheep whose catheters had
sealed off, a definite change in function was observed, i.e., loss of
drinking behavior in response to IVT AII. Since this sheep did not
drink in response to IVT AII even when it was likely that her catheter

was still patent, it was impossible to use this as a guide to know when

 

130

her catheter had sealed off. She never lost her ability to respond to
IVT PGE2. Because we cannot be certain that All and/or PGE2 reached
this sheep's CSF, we did not include her data in any of the results
presented in the body of the thesis.

Sheep #19's ventricular catheter was showing signs of becoming
sealed when she ruptured a carotid artery at the site of catheter-
ization, and had to be put to sleep.

As indicated in Table D-1, sheep #20's ventricular catheter became
non-functional after five months . This was apparent antemortem when the
sheep stopped drinking in response to IVT All and began to object to
ventricular infusions by shaking her head. This sheep died after
surgery to replace a jugular catheter . At postmortem she was extremely
bloated, and gas and a dark red serous fluid exuded from the cut surface
of her cervical muscles. A presumptive diagnosis of Clostridial
enterotoxemia was made. Also at postmortem it was found that the dorsal
ventricular lining indeed had sealed over the end of the ventricular
catheter, but, again, a scar was present in the lining indicating that
at one time the catheter had penetrated into the ventricle. No IVT data
were used from this sheep after she stopped drinking in response to IVT
AII.

Sheep #21 was sacrificed when her catheter showed signs of becoming
plugged.

Sheep #22 suddenly became unable to stand and showed signs of
increased intracranial pressure seven months after catheter placement.
At postmortem her catheter was still patent.

At the time of this writing, sheep #'s 25, 24, and 25 are still
alive and healthy. All three drink in response to IVT AII.

 

APPEDIX E

.oopssps on po mm H oopdaps mN po
mm .oopasps 0N po mm H oopscpa mp po
Ammv oazooosh cooam .Hocpaoo op m0.¥m

*

mph mooso oosaoap poc oc saw was .mm coo: o
Hospsoo u o p
mooso pom oopoOHHmop Mo pocasz n pmopv o

 

151

 

 

h.m o.¢ o.¢ w.m N.m p.m m.N m.N s.N m.N m.N s.N w.N m.p 2mm
m.Np N.mp p.mp m.pp h.op m.op m.® m.w w.h h.h m.h o.w m.m N.m mm
hh wh hh hh mh hh ow *mop *mop *wop *mop *Nop *hm mm osoos
ow ph hm mm mm Oh sh mop mop sop pop pop mm mm s mN
Nh mh sh mh pm sh mh MNp MNp wpp opp opp ppp pm N sN
mh oh oh oh oh hh hh oop oop mm >0 mm pm cm 0 MN
om sop Nop mm mm No hm mpp hpp opp mpp opp hop hm m NN
mm mm mm mm mm so so mop pop oop oop mm mm pm m pN
Op om mm mm No so om spp ppp mop mop pop hm hm m ON
ho mm mm mm mm mm mm opp mpp ppp Npp mop mop 00 m mp
om mm mm Co No mm mm mpp mop mop Nop mm pm mm m mp
Nw on on pm mm mm pm 5 mm so so mm mh mh p m p
he mm Oh hm Nh mm oh sop Nop Nop mm hm Nm pm p vp
oh oh eh mh Nh oh oh mop mop mop pop mm mm on m mp
mh pm no No Nw Nw pm wpp mpp mpp opp hop Nop mm m pp

spa on mo ON 5 8 m o Es om. mm ON E 2 m so was: aooso

paps\opoopv opom psoom Ammasv ossoooph soon
Sesame om .HH saosopoaoé
aoHSOHLpso>osposoooppsH op moosm Hossp>pooH po ooosoaoom opom psoom saw oHSooopm sooam .plm oppoe

152

 

poopsoo on mo.va *
sopospsp so psspo oep soaps oopsspa om spas Hp sposopoaosa n spa s

 

 

 

m.p m.p Oh.o mw.o mN.o ON.o p.p w.o w.m m.m m.w s.m awn
h.N N.m pN.p we.p es.o sm.o m.p ¢.p m.w w.m o.pp m.m mm
*mw mm *sm.op sp.mp Q¢.© hs.® *h.m p.p eh oh *mop ow sooz
hm em em.mp mm.Np pm.m mN.© w.m h.N mm mh sop em mN
pm om Ethp Shin: sho hNo m.m Nd sh Oh pr no em
ow em so.mp ow.pp mm.m hw.© o.N m.o pm No pop Nw MN
HH< pospaoo HH< Hospsoo HH< Hospsoo HH< Hospaoo HH< Hospsoo opp< Hospcoo
as Essossv lasso sass Ass: sass
oasao> oesopopoom pampso oHSooosm opom opSooosm hmH
ososeo assosaasoa osassso posses pesos soopm aooso
aspoe shops
ooppopp<
haosoaasm cpoz spoSB ao osopoaSQph oppoawoaospoopm apps moosm op .apaxwxxwa Om .HH
apoaop0pma< aoH50pspao>osposooospsH 0p moocm Hossp>posH ho oooaohoom opaoShooaom .NIm oppoh

 

poapsoo on mo.va *

 

153

 

es 2. as p.p he as R s; so
Mh mh Mh hh *opp *opp *ppp om Coos
mm mm mm mh pmp Nmp mNp Nw sN
pm 00 mm pm smp wmp omp om ON
oh Mh Nh mm hpp mpp Npp om mp
mm em mm mm mpp ppp mop mm mp
0h Nh oh mo hm pm No No op
mo mo pm mo mop mop mop mo sp
Mh sh hh Nw mop mop mm pm mp
mm so ow mm NNp NNp hpp om pp
ass mN ops mp cps m Hospsoo apsmN spa mp spa m Hospaoo moogm
papa\opoopv opom paoom Awmasv opsooopm wooam

 

spsxosxos op .HH sposopoaoss sppossosspsp
0p mooom Hosop>prH mo ooosomoom opom psoom coo opfiooopm wooam .MIm opcoe

 

134

 

 

 

 

oopsaps m op mo.vh +
H828 on mode *
m.p e.p m.N m.N h.N h.N p.N m.p Sam
Nh ph mh mm + Opp + wOp NOp mm zoos
* * * * * *
mm mm sh mm NNp ONp Opp em sN
so No hm Oh Npp hOp mop mm ON
sh mh mm NO mop sop pOp pm mp
so mm hm Oh mop sop pOp em mp
Mh Oh mh mm mm no No Nw Op
Oh ph Mh Nm mop mop em NO ep
Nh Oh Nh wh mpp Npp mOp mm mp
pm hh Nw mm Opp ppp mop 0% pp
spa mN spa mp cps m Hospsoo cps mN spa mp spa m Hospsoo moosm
Aspsaeoofi opom pesos homes osmoosa sees
sps\wsxwa Op .HH aposopopws< ozoso>oppah
op hoocm Hozsp>posH po oooaomoom opom psoom woo osSooosh sooam .slm oppoh

135

 

spa m on mO.vg + Hospaoo op mO.vg *

 

 

 

v.m N.m N.s p.m O.h w.O 0.0 m.N 2mm
N.mp h.Np mwOp O.Np hump 0.0p Onhp h.m Om
+*Oh *hm *SO Ow *mmp *mmp *wNp Om zoos
as me we mm mmp smp mmp pm ON
hm mm mm Nw mNp pmp ONp em mp
mm NO mm mm wmp omp wNp Nw mp
sh sh mm mm OOp sop pOp Ow Op
pm Oh mh Om pmp Omp sz Nw mp
mo hh 0h ow map Nsp omp so pp
spa mN spa mp Spa m Hospaoo cps mN cps mp spa m Hoppsoo moosm
papa\opoopv opom psoom Ammasv opfiooosm OOOdm
cpsst\ws Om .HH spoaop0pws< ozosohospap
Op moosm HoSOH>psaH mo oooaomoom opom paoom was oppooopm oooam .mlm oacoe

APPEWDIX F

 

156

 

Hoppsoo n O p
V o

mooso pom oopooppmop p0 popasz n Amos

 

 

 

m.o p.o o.o s.o p.o o.o s.o o.o m.p m.p o.o o.p o.p m.p zoo
oo oo po co pm pm oo oo oo ho oo oo oo oo zoos
oo oo mo so mo oop oo mo pm No mo No No po p pm
mm mm mm mm mm co mo so oo oo ho ho oo ho p cm
mo mo po oh so No oh oo oo oo oo oo mo oo p op
sh mh 0o oh oh hh hh oo oo mo no No mm mm p op
no no oo oo so po so mo mo so ho oo oo mo p sp
oo mo mo so mo po so om co oo mm mm oo oo p mp
oo oo oo ho oo oh oo oo oo oo ho po oo ho p pp
sea om om om op op o o spa on om om op op o no Isaaosv aooso
Aspa\opoopv opom paoom Awmasv opSooopm OOOdm
Aopopso>v spsposxos o
.Nmmm UHPOHNOOpPE Op mmmflm §©H>HUCH .HO mmmsoommmm mummm demm .98 mpSmmmpm OOOHmp. . plop mHnrNB

157

 

"poop p ppomv Hocpaoo op pO.vm

 

 

 

Apoppaoo op wopoaaoo opos opoo ass Om osp hfiso *
Hospaoo n O p
mooso pom oopoOHHmop po copssz n Asopv o
o.s m.m s.s O.m h.s m.s s.s o.N m.N o.N m.N o.N p.N o.p saw
*mO 0O sO NO mO mm sm sOp sOp pOp OOp mO mO hm coos
mm Oh hw Ow mm hh Oh mO mO pO OO Ow sw pm p pN
NOp OO OO NO ON sm Oh sOp mOp NOp pOp OOp mO Ow p ON
ON Ow Oh Oh Ow mh Mh mpp Npp Npp OOp mop OO sw N Op
mO OO pO ON ON pm OO OOp Npp NOp OO wO sO mm p mp
mop mop mop mOp mOp hO OO mOp mop sOp NOp OOp OO MO p sp
ho oo oo ho ho mo oo mo mo so no po mo No p mp
sz sz Opp Opp mpp ppp wO mpp Opp Npp Opp OOp mOp mO p pp
so No mo oh pm pm oh oo mo so no Oo oo oo p o
spa on om om op 2 o spa om om om mp 9 o no sao: aooso
Aaps\opoopv opom p.p.oom Ammssv ossooopm woosm
spsfishs m
.wam OppopooocpsH op moosm passp>posh ho oooaomoom opom paoom was oadooopm oooam .NIh oppop

 

158

.oopssps mp po sosooop opop ppoo: coo opSooopm cooap pop opOpo homopm

.HOppaoo op mo.vh

*
Heppaoo n O p
gooso poh oopOOHHgop Mo popasz u pmopv o

 

 

 

s.N N.N M.N m.N M.N s.N M.N O.p O.p O.p O.p O.p O.p N.p saw
*sO *sO *MO *NO *OO *hO OO mop mop *mOp *MOp *pOp *mO sO soos
pO OO hO mO OO MO Oh OOp hOp mOp sop NOp OO pO O ON
Opp OOp Opp Opp hOp OOp MO OO OO hO OO NO NO OO O sN
OO OO OO OO mO pO hh MOp Mop MOp pOp OO pO hh Op MN
hO OO mO OO NO OO mO sop sOp mOp sop NOp mO sO O NN
OO OO hO mO pO OO NO hO OO OO hO OO pO MO O pN
OO sO sO pO pO hh OO hpp hpp hpp spp Opp pOp OO O ON
sO mO MO OO MO pO Mh mop hOp OOp OOp OOp hO hO m Op
OO pO pO OO OO hO Oh hOp hOp mop sOp pOp OO NO O Op
mO mO MO OO OO sO pO OOp hOp OOp OOp pOp mO OO m sp
OO hO OO hO hO hO sO NOp NOp OOp OO OO pO NO O Mp
OOp OOp mop sOp MOp OO NO OOp hOp hOp OOp sop OOp pO O pp
MO mO sO mO MO OO Oh sOp sOp MOp NOp pOp mO OO O O
spa cm om Om op op m o spa co ow om op op m on shoopp aoosm

Apps\opoo£ opom psoom Awmasv opsooopm @0on
.m Hashasapop

mph OppopoooppoH op hooom HosOp>psaH so oooaoaoom opom ppoom cam opdooopm sooam

.m:p oppsp

.oopacps Op po oopooop opop ppoo: Oso opSooopm OOOHQ pop opopo homopm

.HOpp:0O op mO.vm

*
HOpppOO n O n
V o

mooso pom oopooHHmop mo popasz u poop

 

 

 

 

m.M m.M s.M m.M O.s s.s O.s M.N h.N h.N O.N N.M O.N O.p ZOO
*sOp OOp *mOp sop *NOp Oo OO *ppp *Npp *Mpp *Mpp *ppp *mOp OO pooa
oo Oo mo so OO MO Oh Mop pOp pOp NOp Oo po pO p pN
pOp OOp mOp MOp ho NO Oh opp pr pr ONp pr ppp hO p ON
oo OOp Oo ho Oo Oo Oh opp opp NNp NNp pr Npp hO p op
mo OOp pOp OOp Opp po Oh OOp spp Opp spp OOp MOp OO p Op
hOp OOp oop OOp OOp Oo OO hOp OOp Opp Opp OOp mop OO p sp
spp hpp Opp opp sz Opp hOp sop sop OOp mop mop mop mO p Mp
pr MNp opp hpp Opp ppp OOp Opp pr ONp MNp ONp Mpp NO p pp
NO so Mo Mo po OO pO hop OOp Opp mop Mop oo Oo p o
spa OM ON ON mp Op O O ops OM ON ON mp Op m 2O opmopv moosm

Apps\opoopv opom ppoom Ammasv opdooopm @005
.m Easels: on

MOO OppopoooppoH op mooom Hossp>posp ho ooosomoom opom ppoom Ono opdooopm cocam

.slh oanoe

140

AHOppsoo op oopogsoo opos spas spa OM opp hHso "poop p ppomv

poppsoo op oo.vp *

HOppaoO n O p
mooSo pom oopoOpHQop ho popadz n Ahopv o

 

 

 

N.h 0.0 M.h p.h 0.0 N.h 0.0 h.N O.M O.M O.s O.M O.M O.N EEO
*OOp MOp OOp pOp oo so MO *Mpp Mpp spp mpp spp Npp OO zoos
Opp OOp OOp NOp Oo OO po Npp Opp OOp OOp Opp OOp NO p NN
Oo Mo mo OO Oo pO sh MOp pOp pOp pOp OOp OO NO p pN
spp spp spp spp spp Npp MOp pr ONp ONp mNp mNp ONp OO p ON
Opp NOp OOp sop Oo Oo NO hpp Opp NNp sz MNp pr hO p Op
OMp mNp OMp NNp pr Opp mop mpp Opp Opp pr opp hpp so p pp
Oh hh hh sh h sh NO OOp ppp ppp Opp OOp OOp OO p o
spa Om mo om op op m o ass on om om op op m as sppopv aooso
psps\opoopv opom ppoom Ammasv opdooopm scoop
apsfifiws OO
.wah OppopoooppsH op hooom passp>posp po oooaomoom opom ppoom was opSooopm sooam .th ospop

 

141

.spa Op po opop ppoos pop poopssps m pm cocooop opSooopm oooap pop opopo hooopm

.Hoppsoo op mO.vm

*
poppsoo u o a
v O

gooso pom oopoOpHgop po popasz u poop

 

 

 

N.h o.o o.o o.o o.o o.o m.h o.m o.m o.m O.N m.m o.m o.p zoo
ppp Npp ppp oop oop sop so spp opp spp spp opp Npp no shoe
* * * * * * * * * * *
pop sop pop so no so mo opp opp opp pop opp ppp po p om
omp cop pop oop sop oop opp opp oop oop ppp ppp mop mo p so
oo pop mop mop oop so so Npp pop opp opp hpp opp op p om
opp ppp ppp opp opp oop oop mop oop opp hop hop sop po p mm
oo oop mop oo ho mo mo mop Nop pop sop oop mo mo p pm
opp opp spp opp spp Npp so omp omp ONp omp omp oop oo p om
mo mo No oo mo po mo opp ppp opp ppp mop omp mo p op
mop mop mop mop mop cop oop ppp opp omp opp opp opp mo p pp
spa om om cm op op o o spa oo oo co op op m as sflaopp sooso
Asps\opoopv opom ppoom Ammasv opSooopm woosm
m Seasons 08
. sop sppopsosppsp op pooso posepspssp po woosoaoom opsm spasm sea opSaoopo soopm .OIp opssp

142

HoppaOO n O p
moopo pom ooHOOHHQop po popasz u Amopv o

 

 

 

m.s h.s O.m p.h p.m m.s N.s h.N p.m O.M p.m N.M N.h O.N zoo
oo ho so so oo ho ho oo oo oo Oo oo Oo ho poops
hO hO mO mo NO hO Oh so oo oo OOp oo NOp oo p ON
mo oh oh Oh oo po po oo Oo oo Oo Oo oo mo p o p
Oo oo oo hh oo Oo no no so no so mo mo so p op
so po po po oh oh No hh mh Oh Oh oh oh oh p Op
oo 5 p mo p pop oo ho so mo so so no po so oo p s p
sop pop OOp pOp mop mop sop oo oo oo oo oo ho ho p op
No mo so Oo Oo No no ho ho mo ho Oo Mo so p pp

spa on mo ON op Op m 0 Es Om om Om mp Op o no oapopp poops

paps\opOopv opom ppoom Ammasv opsooopm OOOHm
N Afloat: Heaps: O
.ImOm pOHSOpppSo>OppopOOOppsH Op gooom HOOOp>pOOH po oooaomoom opom ppoom was opSooopm OOOHO .hIh oHQOE

poppo peso sopp paopopwpo pop pap .aps m was HOppsOO op mO.vm *
Ops mooso ouSHOap pos Os ZOO was .OO .ssoz o

HOppaOO n O p

mooso pom oopsOHHQop po ponasz u pmopv s

 

 

 

M.O s.O s.O O.m M.O p.O 0.0 p.p p.p O.p s.p O.p m.p p.p 2mm
Oo Oo OO OO oO OO OO *hO *OO *hO *OO *OO sO MO ossos
Oo OOp Oo so ho OOp mOp MO NO NO OO OO Oh Oh p MN
Oo ho OO ho Oo so ho OO OO OO OO OO OO NO p NN
oo OOp oo Oo oo oo ho OO Oo hO Oo hO OO sO p pN
pm Nm Nm mm Mm Nm Mm OO hO OO po oO OO OO p ON
nw OO Oh Oh Oh Oh Oh hh OO hO po sO mO pO NO p op
1. hs Om Om Om os os Om NO po OO po OO hO OO p Op
No No OO oO OO OO OO MO sO OO sO OO MO OO p sp
ppp OOp Npp Opp Npp OOp NOp mO MO OO sO mO mO pO p Mp
NO so OO OO OO po Oo No OO Oo OO OO OO hO p pp
spa Om om Om mp Op m o 9E On om Om op Op o no was: poops
Asps\opsopv opsm ppsom Ammasv opzooopm OOOHO
.m Hoops: Op

MOO psHSOprso>OppopoosppsH Op hoogm HsSOp>pOOH ho ooosomoom opsm ppsom was opaooopm OOOHO .Olh oapse

 

opspom oapp pospo Has ps op5ooopm OOOHQ ass was HOppOOO op mO.vm +
poppo :oso aOpp paopopppo pop pap .sps m was HoppaOO op mo.v@ *
ops sooeo oaaosp pos os s.s as do “so: o
p
.O

 

 

HOppOOO n O

gooso pom oopsOpHgop p0 popasz n pmopv
O.s s.m p.m m.s s.s O.s O.m h.N s.N O.p O.p s.p s.p m.p 2mm
Oh Oh Oh hh hh Oh Oh +*mo *No *po *po *Oo OO OO oasos
Oh ph ph Nh ph sh OO pOp OO OO OO hO mO NO p MN
Oo OOp NOp so po OO po so Oo so No Mo pO mO p NN
Nh OO ph Oh Oh hO Oh mO sO sO sO MO sO mO p pN
OO OO OO MO sO mO OO OOp MOp ho Oo so so OO p ON
OO sh ph Oh OO Oh Mh oo ho ho Oo so OO OO p op
OO NO NO NO pO MO mO Oh Oh NO Oh OO Oh Oh p Op
sh Nh sh sh sh Mh mh hO mO hO OO hO sO NO N sp
oo OOp pOp oo OOp MOp hOp Oo mo Oo mo po No so p Mp
Oh Oh Nh OO sh Oh Oh OO OO hO OO Oo mO MO p pp

spa Om mm Om mp Op m O spa Om mm Om mp Op m so s39: poops

Aspaapsofl opsm pesos

Ammasv opsooopm OOOHO

 

.N

Sesame Om

MOO psHSOpppso>oppopoospch Op hooOO HsSOp>pOOH ho ooosomoom opsm ppsom cos opdooopm OOOHO

.Olm oanse

 

145

.oopsaps mp ps ooposop opspo hosopO

.HOppaOO op mO.a

*
ops pooso osSposp pos os zoo ass .Oo .ssoz o
poppsoO n O n

.w

mooao pom oopsOHHmop MO popasz u Amopv

 

 

O.m h.s m.s p.s s.s o.m o.m O.m o.m O.m p.m o.N h.N p.p zoo
po oo Oo oo Oo oh hh *mo *mo *po *mo *oo *oo mo ossos
NO mm om hm om pO om No oh mh mo Oh hh Oh p mN
ho oo Oo Oo oo po oh mOp mOp pOp oo Oo mo mo m mm
hh hh Oh oh oh Oh sh mo po oo po so po Oo p po
oh oo Oo mo oo oo hh pOp mOp OOp oo oo ho mo p ON
mo oh po po oh so Oo pOp oo mo so po mo mo p op
mh hh hh oh mh mh oh so po No so oo oo oh p op
mh Nh oO oO OO hO mO Oo mo mo OOp mo oo so p sp
Oo mo mo mo mo so mo No so No po mo Oo Oo p mp
so oo so po Oo po oo hOp sOp sOp oo ho so ho p pp
spa Om mm Om mp Op spa Om mm Om mp Op so sppopp aooso

Asps\opsopv opsm ppsom

AOOasv opsooopm OOOHm

 

sps\oa\os OOp .mpOp
psHSOprSo>OppopoosppsH op mooOO HsSOp>pOaH ho ooosomoom opsm ppsom was opaooOpm OOOHO

.OpIm oHQsE

.oopaaps m ps Oososop opspo hosopO .pospo soso aOpp psopopppo pos pap .Hoppaoo op mO.vm *
Ops mooso oOSHosp poa OO Sam was .OO .asoz o

HoppsOO n O p

hooso pom oopsOpHmop so popasz n pmopv s

 

 

s.M O.N O.N O.N N.N O.p M.N p.M p.M p.M s.N M.N O.N M.p 9mm
Oh Oh hh hh hh Oh mh *so *Mo *No *Mo * po * po mO oesoa
mm hm OO NO NO mO sO hO OO mO mo OO Oo mO p mN
mO ph ph NO Oh Oh mO Oo OO hO hO OO No OO p sN
NO OO Oh Oh Oh OO Oh mO sO MO mO pO NO hh s MN
Oo Oo po oO MO mO OO NOp pOp Oo Oo Mo Mo OO N NN
sO sO OO OO mO OO mO Oh Oh OO mO MO mO OO p pN
,0 OO Oh Oh OO Oh OO mO hOp OOp hOp sOp sOp OOp Oo M ON
”w pO hh mh sh Oh mh mh Opp OOp mOp MOp pOp Oo OO M op
mo OO po oO OO OO NO mO mO sO OO hO OO sO p Op
Oh sh Oh Oh Oh Mh Oh OO O mO OO O NO O p Op
Oh Nh Oh OO ph Oh Mh MO No No po OO hO mO N s p
sh sh mh sh hh hh Oh mO OO mO hO OO hO mO N Mp
OO OO po hO hO MO OO sOp mOp OOp sOp NOp oo po M pp
spa OM mN ON mp Op m O spa OM mN ON mp Op m 9O sphopv moosm
Asps\opsopv opsm ppsom Ampsasv ossooopm OOOHO

 

Sessions OOm NOON
psHSOprao>oppopoosppsH Op moogm HsSOp>pOaH mo oooaomoom opsm ppsom was opsooopm OOOHO .ppIh oacse

 

 

147

poppsoO n O Q
d

mooSo pom oopsOHHmop po popasz n Amopv

 

 

hh Oh oh No Oo No po Oo oo ho ho oo No so p pN
ph oO oO Oh ph ph mh Oo oo oo mo Oo Oo Oo N op
OO oo mo OO mo Oo mo ho OOp Oo mo ho ho mo p sp
po NOp ho OOp pOp mOp oo mo po ho ho mo Oo Oo p mp
spa Om mN ON mp Op m O spa Om mN ON mp Op m o.O sppopp pooso

psps\opsopp opsm pesos

AOOasV opsooopm OOOHm

 

sp .sps\oa\os Omh .N

oooom posoH Op Osoaoom pop Opm pssp moosm
mOp psHOOpppso>oppopoosppOH op ooosomoom opsm ppsom ops opsooopm OOOHO

.NpIm oHQsB

HOppOOO n O O.
mooppo pom oopsoflmop .po popssz u pmopv s

 

 

O No po ho Oo mo No mo po oh O No oo oh p N
Nh mh ph oO ph Oh OO 8 p O p O p ho ho mo No s o p
Nh sh oh hh Oh NO mo oO oO ho hO oO OO ho N m p
spa Om mN ON mp Op m O sap Om mN ON mp Op m OpO s39: poops
Asps\opsoo.v opsm ppsom Ammasv ossooopm OOOHO

 

SH .QHS\WM\®C 000p .N

oooom poprH op anmoom pop 3Q psppp moopm
MOO psdepppaoppOppopoosppOH Op ooosomoom opsm pssom was opsooopm OOOHO

. M pIh oHQse

APPEVDIX G

 

 

149

 

 

 

O.M O.M s.N N.N m.N O.M O.N N.s O.N O.p EEO
OO pO OO OO OO OO OO pO pO OO ssos
0O OO OO OO mO OO MO pO MO pO MN
po No Mo No Oo OO OO OO OO Oo NN
NOp ppp pOp hO OOp Nop NOp spp pop so ON
OO pO MO 0O OO Oh. Oh. pO mO po op
Mo so OO NO NO No Oo No No No sp
OO mO OO mO NO OO hO OO hO OO Mp
Mo po Mo MO so Oo OO so pOp Oo pp
cps OM mN ON mp op m 0 ml OpI O moosm

 

sps\OM\Os OOOp .sspsppsm psHSOprso>OppopoosppaH
Op hooOO HsSOp>pOpH po ooosomoom opdooopm OOOHO

pOmasv opdoooph OOOHO

.pIO oflflse

p.h M.h 0.0 h.O 0.0 h.O N.O O.m M.m O.m 2mm

 

 

OO OO Oh Oh oh oh oh OO OO OO zoos
oh OO oO OO Oh hO oO OO OO mO mN
Oh Oh ph hh Oh hh mo mo mo OO NN
hO ph hO sO mO Oh sO oO Oh ph ON
OO OO OO pO mO hm NO mO hO mO op
ph sh Oh Oh Oh Oh OO Oh oh NO sp
NOp mOp so No mo mo po OO mo mo mp
Opp oOp Npp Opp ppp Opp OOp hOp hOp OOp pp
spa Om ON ON mp Op O O m- Op- O oooso

 

papa\opsoo.v opsm ppsom

sps\oop\os 88 Steam
HOHSOpppaoppoppoponppaH Op gooppm HsspOppppOsH p0 ooosomoom opsm peso: .NIO odpse

151

 

poppsoO op mO.vp * sposoppo ppsp po so sopospsp pps o
opopppsoh opsp oosppps op ppOpo pps p
oOpsaps mpI ps ooppspo aOposppsp Sopsppsm s

m.N O.N N.M O.N h.M p.M N.M M.M O.N O.N spam

 

*NO pO *NO pO *No OO OO OO OO hO :sos
NO pO hh Oh Oh mh Mh ph Mh Mh MN
hO hO OO OO mO OO mO sO mO MO NN
OOp MOp so so oo mo mo OO mo Mo ON
NO OO OO OO po OO OO OO OO OO Op
NO No OO so OO pO po mo pO No sp
OO OO Oo OO so OO OO mO OO OO Mp
pOp OOp sOp NOp OOp OOp OOp so OO po pp

 

Ln

ass OM mN ON m p O p

v

oO pm: Op- oO pooso

 

AOmasv opdooopm OOOHO

sps\os\os Om .pp sposopoposs
p.phh + .EEOME: OOOp .ppOpppppsO 2..th psdepppppoppOpOpOponppaH
Op moosm gasps/posh po oooaomoom opfiooopm OOOHO .MIO oapsp

152

sposoppo spas po so sopospsp pps o

opopppso> opsp ooSpppO op ppOpo pps p

oopdpps mpI ps ooppspo :OpoSpap aspsppsm s

 

 

O.M O.M p.s p.M N.s O.s O.s M.s O.s O.s 2mm
Oh Oh Oh hh Oh hh hh Oh Oh Oh ssos
ph Nh ph sh Mh Nh ph sh Mh hh MN
OO OO hO hO OO po No so oo so NN
pO sO Oh Oh NO Oh hh OO mO OO ON
ph sh Oh Nh sh Mh sh sh sh Oh op
OO OO hO Oh sO mO OO OO mO OO sp
hO Oh OO Oh OO OO hO hO mO OO Mp
Oo mo ho OO so pO Oo Mo OO OO pp
cps OM mN ON mp Op m oO me OpI sO hoosm

 

sps\op\os Om .pp sposopOposs php + .sps\oa\os OOOp .soasppoo pphpp

psps\opoopp opso ppooo

psHSOpppao>oppopoosppsH op mooOO HsSOp>pOsH po ooosomoom opsm ppsom

.sIO oanse

sspsppsm Ops HoppaOO op mO.vm + soppspo aOpoSpsp NOOO p
Hoppsoo op mO.vh * oopsspa mpI ps ooppspo OOpospsp Ospsppsm s

s.p m.p h.p o.p o.p O.p s.N o.N h.N p.N 2am
+sOp +sOp +mOp +NOp +NOp Oo *po *po *po hO esos

*

 

153

NOp OOp OOp OO Oo so MO Oh Oh Oh MN
mop sop Mop Oo OOp Oo oO OO pO hO NN
hop Opp Mpp Npp ppp NOp ho hO Oo OO ON
OOp NOp sop NOp OO OO OO Oo MO hO Op
OOp OOp OOp mop OOp mop pop sop pOp MO sp
OO Mop mOp NOp OOp ho po OO OO hO Mp
pop NOp OOp OOp pop so hO OO CO CO pp

 

m

spa Om mN ON mp Op pO mI OpI oO pooso

 

Ammasv opSooopm OOOHO

sps\os\os Op .NpOp
OppOpsosppaH + .apa\OM\Oa OOOp .aspsppsO psHSOpppso>OppopoosppaH
op mooom HOSOH>HOOH po oooaomoom op5ooopm OOOHO .mIO oapse

154

soppopo sopospsp NOON p

oopssps mpI ps ooppspo aOpoSpsp OspsppsO s

 

 

m.h m.h O.O O.m m.m p.m O.s O.m N.s Nm so
mo so NO mo pO Oo mh mh mh hh soos
OO Oo mo No oh Oh ph ph Oh OO mN
hO mO oO mh Nh sh Nh Oh po oh NN
OO pO OO pO pO OO mm Nm sm Om ON
OO mo so No Oh sh OO hO Oh OO op
OO Nh Oh Oo Oh po po so mo ho sp
Oo Oo ho Oo mo mo OO OO mo OO mp
spp Opp Opp OOp mOp oo No oo so mo pp
ass Om mN ON mp Op 9 m- Op: oO pooso

 

sps\os\os Op .NOOp Oppopoooppsp + .sps\oa\os OOOp .sossppoo

Aspa\opsopv opsm ppsom

psHSOpppao>OppopoosppaH Op moosm HOSOp>pOaH po ooosomoom opsm ppsom

.OJO oapse

155

aspappsO was HOppsOO op mO.vm * spOsoppo asap so sOpospsp NOOO O
oHOprso> Opsp oospppo Op ppspo NOOO p
oopsaps mpI ps woppspo sOpospap aspsppsO O

OO h.m O.m h.s s.m h.s hm m.m o.N O.N so
NOp pop oo *Oo *mo No No Oo O OO soos

 

 

ho so mo Oo po oh pO oh Oo mh mN
Oo Oo so Oo ho mo ho mo oo oo NN
pr hpp opp oOp Npp OOp OOp NOp oo so ON
hOp hOp mOp so mo No Oo oO Oo mo op
so mo so so mo ho Oo Oo mo mo mp
mpp spp Opp Opp Opp sOp OOp ho mo oo pp
spa Om mN ON mp Op m oO pm- OpI oO pooso

 

Ammasv opSooopm ooodm

sps\oa\os OOm .NOON ppp + .sps\oa\os OOOp .sopsppoo possopppso>
IOppoponppsH Op moogm HOSOp>pOsH po ooosomoom opdooopm OOOHO .hIO oapse

156

 

spanppo asap :0 sOpo5psp NOOO o

oHOpppso> Opap oospppo Op ppspo NMOO Q

oopssps mpI ps ooppspo sOpospsp asppppsO s

 

 

s.m p.m o.p N.N h.N p.m s.s s.m N.m m.N zoo
Oh Oh Oh Oh hh Oh Oh mh mh sh ssoa
OO mo oh po oh OO hh po hh OO mN
mo mo oh No mo ho Oo oo hO oh NN
ph mh sh Nh sh Nh ph Nh Oh Nh ON
mO OO hO OO mO mO hO OO mO hO op
hh Oh hh mh oh hh Oh mh Oh hh mp
sO so oh mh Oh sh hO OO OO hO pp
spa Om mN ON mp Op m oO pm- OpI oO pooso

 

sasasspsm Nsspsp+.sa<a<sssop.sasrsopssp

Asps\opsopv opsm ppsom

psHSOpppso>OppopoosppsH op mooOO HsSOp>pOsH po oooaomoom Opsm ppsom

.wO osap

 

 

 

 

 

sap m op mO.vp +
poppsoO op mO.vp *
Hoppppoou O s
M.m s.s m.M s.M M.s O.N s.s N.N O.N O.N M.N O.M o.N O.N sham
hh mh mh sh ph sh Mh +*Opp +*Opp +*Npp +*Opp +*hOp *NOp OO asos
NO OO O NO hh Oh Nh spp Mpp ppp ppp OOp mOp OO NN
OO MO Oh Oh sh hh hh MNp ONp spp ppp Opp pOp OO ON
Om om NO sO Om mO OO Mpp Npp OOp OOp Mop ho hO op
Oh Mh Nh OO sO ph Oh mpp Opp OOp MOp OO ho po Mp
OO O Oh Oh O OO hO MNp MNp opp Opp Opp Npp Oo pp
spa OM mN ON mp Op O spa OM mN ON mp Op m sO hoosm
Aspa\opso£ opsm ppsom Amman: opdooopm OOOHO
Oshawa s .aposgposOOsH ozoossfipopsm pophs. opdom 03h .apshsphw: Om .HH aposopOHOpps
psdpoprsoppOppoponppsH op hooam dongs/Hosp .po oooppogoom opsm ppsom was o.pdooOpm OOOHO .OIO oHOpsp

158

.oopssps mp ps wososop opspo hOOOpO

.HOppsOO op mO.vm *

HOprOO HO O

 

 

O.s o.s O.m O.s m.s m.m O.h N.m s.O o.O m.h N.h o.O s.m zoo
Oo Oh hh oh Oh oh No OOp mOp pOp pOp ho ho ho soos
.x. * * * .x.
OO mO NO mO mO sO pO Oo mo po po hh Oh OO mN
so so NO so mo oo mo Npp Opp oOp Npp oOp oOp Oo NN
mo NO NO po OO Oh ho sOp mOp mOp OOp ho mo mo pN
mo No No so mo mo mo opp spp Opp Npp mOp mOp so pp
spa Om mN ON mp Op m O spa Om mN ON mp Op m oO pooso

Asps\opsopv opsm ppsom

Ammasv oppooopm OOOHO

 

OM a .ospaSHOoz aprOSHh oSOso>sppaH pop p50 p o .aps M a . aposopOp
M ps O pom OO Om Hp

psHSOpppao>OppopoosppsH Op mooOO HOSOp>pOOH po ooosomoom opsm ppsom was opSOOOpm OOOHO

.OpIO oanse

LISTOFREFERFNCE

 

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endogenous prost andin biosynthesis by brain homogenates.
Prostaglandins 17(5 : 411—418, 1979.

Abdel-Halim, M.S., I. Lunden, G. Cseh, and E. Kngggrd. Prostaglandin
profiles in nervous tissue and blood vessels of the brain of various
animals. Prostaglandins 19(2): 249—258, 1980.

Abdull, Y.H. and E. McFarland. Control of prostaglandin biosynthesis in
rat brain homogenates by adenine nucleotides. Biochem Pharmacol 21:
2841- 2847, 1972.

Akerlund, L.E., I. Andersson, and K. Olsson. A cannula system for
frequent infusions into the CSF of the cerebral ventricles of the
goat. Physiology and Behavior 10: 161-162, 1973.

Andersson, B. Regulation of body fluids. Ann Rev Physiol 59: 185—200,
1977.

Andersson, B., L. Eriksson, O. Fernandez, C.G. Kolmodin, and R. Oltner.
Centrally—mediated effects of sodium and angiotensin II on arterial
blood pressure and fluid balance. Acta Physiol Scand 85: 598-407,
1972.

Andersson, B. and LG. Ieksell. Effects of fluid balance of
intraventricular infusions of prostaglandin E1. Acta Physiol Scand
95: 286- 288, 1975.

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