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lLlllllllllzlljlllllllllljlllljllllllllllllllfll

LIBRAR Y E -~

Michigan Stat:
fl University

 

This is to certify that the

thesis entitled

The Effect of Sustained Hemorrhagic
Hypotension in Chickens Following
Adrenergic Blockade

presented by

James Mattes Ploucha

has been accepted towards fulfillment
of the requirements for

MASTER OF SCIENCE degreein POULTRY SCIENCE

 

 

a" "
\ o’E/‘CL 71% —‘*'\‘ L's/v
4L 1)
I U

Major professor

Date May 2, 1979

0-7639

 

 

 

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THE EFFECT OF SUSTAINED HEMORRHAGIC HYPOTENSION
IN CHICKENS FOLLOWING ADRENERGIC BLOCKADE

By

James Mattes Ploucha

A THESIS

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

MASTER OF SCIENCE

Department of Poultry Science

1979

ABSTRACT

THE EFFECT OF SUSTAINED HEMORRHAGIC HYPOTENSION
IN THE CHICKEN FOLLOWING ADRENERGIC BLOCKADE

By

James Mattes Ploucha

Research concerning hemorrhagic shock has been extensive in mam-
mals and all studies demonstrate vascular decompensation after large
blood losses, i.e. irreversible shock. Studies designed for other pur-
poses suggest that avian species possess a unique tolerance to the
deleterious effects of hemorrhage, i.e. they do not enter vascular de-
compensation. Many researchers feel that sympatholytic agents may
reduce the mortality of hemorrhagic shock. This study was undertaken
to determine the effects of alpha- and beta-adrenergic blockade on

survival time in the hemorrhaged domestic fowl (Gallus domesticus),

 

and also to evaluate cardiovascular performance and several hemato-
logical parameters, including plasma electrolytes, plasma glucose, and
blood gases and pH. Large deviations from the established mammalian
physiological responses might help to explain why avian species apparent-
ly do not enter vascular decompensation.

Thirty adult Single Comb White Leghorn (SCWL) hens were divided
into five equal groups. The birds were anesthetized with 100 milligrams
of sodium phenobarbital per kilogram of bodyweight. The birds were in
laying condition and weighed between 1.5 and 2.2 kilograms. The caro-
tid artery was cannulated for measuring the arterial blood pressure,
obtaining blood samples, and hemorrhaging. Following a control period,
blood was removed to lower the arterial pressure to 50 mn Hg and blood
was periodically removed to maintain the arterial pressure at 50 mm Hg.

Two groups served as non-hemorrhaged controls. The remaining groups

James Mattes Ploucha

were hemorrhaged. Two of these received a 30 minute drug pretreatment,
one with phenoxybenzamine, an alpha-adrenergic antagonist, and the
other with propranolol, a beta-adrenergic antagonist. The latter was
infused continuously via a cannula in the brachial vein.

The propranolol had a considerable negative chronotrOpic effect
upon the myocardium. Consequently, the survival time was less in these
birds than in the other hemorrhaged groups. Phenoxybenzamine reduced
the total peripheral resistance without positive inotropic effects on
the myocardium. Both of the drugs reduced the mean arterial pressure,
the propranolol having the larger effect.

Hemorrhage reduced the cardiac index and the total peripheral
resistance, whereas mammals Show intense vasoconstriction. Bleeding
volumes were not affected by either drug treatment. Alpha-adrenergic
blockade has a large effect on the bleeding volumes in mammals.

Hematrocrit and hemoglobin levels fell continuously up to the
time of death in the hemorrhaged groups. Mammals experience hemo-
concentration in late shock. Plasma magnesium and plasma sodium levels
did not change in any of the groups. Plasma potassium and plasma glu-
cose increased progressively throughout the duration of the experiment.
Mammals experience severe hypoglycemia in late hemorrhagic shock. All
of the hens became progressively alkalotic throughout the duration of
the experiment. Mammals enter a severe metabolic acidosis. The
phenoxybenzamine reduced the magnitude of the hyperglycemia and hyper-
kalemia and did not increase the survival time.

The results of this study indicate that following hemorrhage the
chicken, unlike the mammal, does not vasoconstrict, enter vascular

decompensation, experience hypoglycemia, or become acidotic. The

James Mattes Ploucha
chicken homeostatic mechanisms act predominantly to maintain blood

volume, not blood pressure.

ACKNOWLEDGEMENTS

I wish to express my appreciation to my major professor, Dr. R. K.
Ringer, for his assistance in the preparation of this manuscript. I
also thank Drs. H. C. Zindel, J. B. Scott, D. Polin, J. L. Gill, and
R. J. Ballander for their assistance with various aspects of this study.
Further appreciative acknowledgement is expressed to Mr. D. V. Stoffs,
for his expert technical assistance, and Mrs. B. Trosko and Mrs. D.
Richmond, for their critical review of this manuscript.
1 also thank Mr. Thomas and Mrs. Leotta M. Ploucha, my parents, and
Dr. Anthony J. and Geraldine A. Miltich, my father and mother-in-law, who
have contributed in so many ways to my studies and my life. I
Finally I thank my wife, Lael, and my daughter, Courtney, for their

unrelenting love and encouragement.

ii

TABLE OF CONTENTS

Eggg_
LIST OF TABLES .......................... v
LIST OF FIGURES ......................... vii
LIST OF ABBREVIATIONS ...................... ix
INTRODUCTION ........................... 1
LITERATURE REVIEW ........................ 3
I. Circulatory Homeostasis .................. 3
A The Shock Phenomenon .................. 3
B. Alpha-adrenergic Blockade ............... 9
C. Beta-adrenergic Blockade ................ 15
D Normovolemic Anemia .................. l8
E Other Factors in Shock ................. 20
II. Respiratory Homeostasis .................. 22
A. Avian Blood Gases and pH ................ 22
B. Intrapulmonary Receptors in Aves ............ 23
C. Intracardiac Receptors in Aves ............. 25
D. Other Receptors in Aves ................ 25
E. Hypoxia in Aves .................... 27
OBJECTIVES ................ - ........... 29
MATERIALS AND METHODS ...................... 30
1. Experimental Stock ................... 30
II. Anesthesia ....................... 30
III. Surgical Procedure ................... 30
IV. Drug Administration .................. 3l
V. Shock Protocol ..................... 32
VI. Blood Pressure Measurement ............... 33

iii

Pag '

VII. Heart Rate Measurement ................. 33
VIII. Blood Chemistry Analysis ............... 34
IX. Cardiac Output Determinations ............. 35

X. Statistical Analysis .................. 36
RESULTS ............................. 38

Experiment I: The changes in heart rate, mean arterial

blood pressure, cardiac index, total peripheral resistance,
stroke volume, and stroke work induced by a thirty min-

ute pretreatment with propranolol or phenoxybenzamine in

adult Single Comb White Leghorn hens ............ 38

Experiment IIA: The changes in heart rate,

cardiac index, stroke volume, stroke work, bleeding

volumes, survival times, mortalities, and the occurrence

of arrhythmias and lesions in adult Single Comb White

Leghorn hens subjected to sustained hemorrhagic hypo-

tension without pretreatment and following pretreatment

with propranolol or phenoxybenzamine ............. 43

Experiment 118: The changes in hematocrit, hemoglobin,

plasma potassium, plasma magnesium, plasma sodium, and

plasma glucose concentrations in adult Single Comb White

Leghorn hens subject to sustained hemorrhagic hypo-

tension without pretreatment and following pretreatment

with propranolol or phenoxybenzamine ............ 53

Experiment IIC: The alterations in arterial pH, ar-
terial blood gases, and the incidence of respiratory
arrest, in adult Single Comb White Leghorn hens subjected
to sustained hemorrhagic hypotension without pretreat-
ment and following pretreatment with propranolol or

-phenoxybenzamine ...................... 66
Experiment 110: Group #5 data and overall mean initial
values (n=30) ........................ 73
DISCUSSION ............................ 76
SUMMARY AND CONCLUSIONS ..................... 94
LITERATURE CITED ......................... 96
APENDICES ............................ l06

iv

Table
lA.

18.

LIST OF TABLES

The effect of a thirty minute pretreatment with propranolol
or phenoxybenzamine on cardiac index, mean arterial blood
pressure, heart rate, and total peripheral resistance in

adult Single Comb White Leghorn hens ............

The effect of a thirty minute pretreatment with propranolol
or phenoxybenzamine on cardiac output, stroke volume, and
stroke work in adult Single Comb White Leghorn hens

The effect of sustained hypovolemic hypotension on heart
rate at the time of the final three samples in adult Single
Comb White Leghorn hens without pretreatment and following

pretreatment with propranolol or phenoxybenzamine ......

The effect of sustained hypovolemic hypotension on cardiac
index, stroke volume, and stroke work in adult Single Comb

White Leghorn hens .....................

The effect of sustained hypovolemic hypotension of total
peripheral resistance in adult Single Comb White Leghorn

hens ............................

The changes in initial bleeding volume (IBV), secondary
bleeding volume (SBV), maximal bleeding volume (MBV),
SBV/IBV ratio, survival time, and mortality in adult Single
Comb White Leghorn hens subjected to sustained hypovo-
lemic hypotension without pretreatment and following pre-

treatment with propranolol or phenoxybenzamine .......

The effect of sustained hypovolemic hypotension on hema-
tocrit at the time of the final three samples in adult
Single Comb White Leghorn hens without pretreatment and

following pretreatment with propranolol or phenoxybenzamine.

The effect of sustained hypovolemic hypotension on hemo-
globin at the time of the final three samples in adult
Single Comb White Leghorn hens without pretreatment and

following pretreatment with propranolol or phenoxybenzamine.

The effect of sustained hypovolemic hypotension on the
initial and final plasma concentrations of potassium,
magnesium, sodium, and glucose, as well as the mean time

to the final sample, in adult Single Comb White Leghorn
hens without pretreatment and following pretreatment with
propranolol or phenoxybenzamine ..............

The effect of sustained hypovolemic hypotension on ar—
terial pH. p02, and pCOz in adult Single Comb White Leg-
horn hens without pretreatment and following pretreatment

\with propranolol or phenoxybenzamine ............

V

Page

39

42

44

49

52

54

55

59

7O

Table
l0.

ll.

12.

Page

The change in cardiac index, heart rate, mean arterial

pressure, and hematocrit in adult Single Comb White Leg-

horn hens after 225 minutes with and without replacement

of sampling blood losses of 2.5 ml/hr ............ 74

The mean values for cardiac index, heart rate, mean ar-

terial blood pressure, hematocrit, hemoglobin, and plasma
glucose, potassium, magnesium, and sodium concentrations

in thirty adult Single Comb White Leghorn hens ....... 75

The initial bleeding volume (IBV), secondary bleeding

volume (SBV), maximal bleeding volume (MBV), SBV/IBV

ratio, and initial blood volume data from research in

the literature on the dog and chicken during hypovolemic
hypotension ......................... 80

vi

LIST OF FIGURES

Figure Page

l. The effect of a thirty minute pretreatment with pro-
pranolol or phenoxybenzamine on the cardiac index, mean
arterial blood pressure, heart rate, and total peri-
pheral resistance of adult Single Comb White Leghorn hens. 41

2A. The change in heart rate at the time of the final three
samples in adult Single Comb White Leghorn hens subject-
ed to sustained hypovolemic hypotension .......... 46

28. The change in heart rate'at the time of the final three
samples in adult Single Comb White Leghorn hens subject-
ed to sustained hypovolemic hypotension and pretreated
with propranolol or phenoxybenzamine ........... 46

3A. The change in cardiac index in adult Single Comb White
Leghorn hens subjects to sustained hypovolemic hypoten-
sion ........................... SI

38. The change in stroke volume in adult Single Comb White
Leghorn hens subjected to sustained hypovolemic hypoten-
sion ........................... 51

4A. The change in hematocrit at the time of the final three
samples in adult Single Comb White Leghorn hens sub-
jected to sustained hypovolemic hypotension ....... 57

4B. The change in hematocrit at the time of the final three
samples in adult Single Comb White Leghorn hens sub-
jected to sustained hypovolemic hypotension and pre-
treated with porpranolol or phenoxybenzamine ....... 57

5A. The change in hemoglobin at the time of the final three
samples in adult Single Comb White Leghorn hens sub-
jected to sustained hypovolemic hypotension ....... 61

58. The change in hemoglobin at the time of the final three
samples in adult Single Comb White Leghorn hens sub-
jected to sustained hypovolemic hypotension and pre-
treated with propranolol or phenoxybenzamine ....... 61

6A. The change in plasma potassium concentration at the
time of the final three samples in adult Single Comb
White Leghorn hens subjected to sustained hypovolemic
hypotension ....................... 64

vii

* j

 

Figure

GB.

7A.

78.

LIST OF FIGURES

Page

The change in plasma potassium concentration at the

time of the final three samples in adult Single Comb

White Leghorn hens subjected to sustained hypovolemic
hypotension and pretreated with propranolol or pheno-
xybenzamine ....................... 64

The change in plasma glucose concentration at the W“
time of the final three samples in adult Single Comb
White Leghorn hens subjected to sustained hypovolemic
hypotension ........................ 68

The change in plasma glucose concentration at the time 2
of the final three samples in adult Single Comb White t
Leghorn hens subjected to sustained hypovolemic hypo-
tension and pretreated with propranolol or phenoxyben- _q
zamine .......................... 68

 

The changes in arterial pH, p02, and pCOz concentra-

tions from the time of the initial to the final sample

in adult Single Comb White Leghorn hens subjected to

sustained hypovolemic hypotension without pretreatment

and following pretreatment with propranolol or pheno-
xybenzamine ....................... 72

viii

Abbreviations Used:

ADH

ANG II

ANOVA
cAMP
C1
C0
CNS
CVS
DP
Hb
HCO-
HCT
HR
JGA
LVEDP
MABP
MDF
NADH
PBZ
Pc
pCOz
P02
ppm
PROP
RES

antidiuretic hormone.

angiotensin II.

analysis of variance.

adenosine 3'5' - cyclic phosphate.

cardiac index, ml/min/kg'73u

cardiac output, ml/min.'

central nervous system.

cardiovascular system.

diastolic pressure in mm Hg.

hemoglobin, gm% or mg/dl.

bicarbonate anion.

hematocrit, %.

heart rate in beats/min.

juxtaglomerular apparatus.

left ventricular end diastolic pressure in mm Hg.
mean arterial blood pressure in mm Hg.

myocardial depressant factor.

reduced nicotinamide-adenine dinucleotide.
phenoxybenzamine.

capillary hydrostatic pressure in mm Hg.

arterial partial pressure of carbon dioxide in mm Hg.
arterial partial pressure of oxygen in mm Hg.
parts per million.

propranolol.

reticuloendothelial system.

ix

SCWL single comb white leghorn.
T= time of a given sample in minutes.

TPR total peripheral resistance.

INTRODUCTION

The poultry industry is growing worldwide at a tremendous pace.
Any research providing insight into the chicken not only benefits com-
parative physiology, but the poultry industry as well. Many avian
diseases involve hemodilution and/or hemmorhage, including cocci-
diosis, fowl cholera, Marek's disease, aplastic anemia, leukosis, fatty
liver hemorrhagic syndrome, aortic aneurysm rupture, hemorrhagic ar-
teries, and cannabalism. Therefore, avian research concerning hemo-
dilution or hemorrhage has an application to the industry.

Perhaps more important is the role of such research in shedding
additional light, from a new perspective, on the shock phenomenon in
man.

Shock, in man, is a descriptive term characterized by hypotension,
pallor, collapse of superficial veins, altered mental status, and dimini-
shed urine formation. Hypovolemic (hemorrhagic) shock is the most
common of about seven types of shock. Cardiogenic shock, due to a
pumping insufficiency, rates second in prevelance in man. Septic shock,
due to a systemic gram negative endotoxin, and anaphylactisis, due to
allergic reaction, tie for third in prevelance.

Voluminous research involving rats, rabbits, sheep, cats, dogs,
and subhuman primates has provided much insight into the irreversible
shock phenomenon. Observations during experiments designed for other
purposes have suggested that the domestic fowl is quite resistant to
the deleterious effects of hemorrhage. This prompted Wyse and Nickerson
to provide the only research article on an avian species utilizing a
standardized shock protocol.

It should be mentioned here that the most advanced avian

l

cardiovascular (CVS) research has been conducted in the diving duck
by Scandanavian physiologists. Vascular responses in the chicken which
are thought to be quite different from the duck have not been docu-
mented. The chicken is hypertensive, is bipedal, has a CVS anatomy
similar to mammals, is prone to spontaneous atherosclerosis, and is of
a convenient size and availability, making it an interesting animal for
CVS research. \

This study was an attempt to elucidate some of the hemodynamic
and hematological events which occur while the hen was held in sus-
tained hypovolemic hypotension. Adrenergic receptors, electrolyte,

glucose, and blood gas alterations were of particular concern.

REVIEW OF LITERATURE

I. Circulatory Homeostasis

A. The Shock Phenomenon

Hypovolemic hypotension sustained beyond a given duration of time
results in myocardial and/or peripheral circulatory failure despite
reinfusion of all shed, and even additional, blood. Investigations
involving the duration of time until the onset of irreversibility are
of great practical importance. Certain sympathomimetic agents coupled
with volume repletion are the standard treatment for hemorrhage
(Carey gt $1,, 1971). It is generally understood that primates with-
stand shock better, vasoconstrict less, and survive longer than dogs
(Abel gt_g1,, 1967). Yet primate tolerance to hemorrhage apparently
does not compare to that of avian species where shock irreversible to
transfusion may not occur.

Shock is a condition where the circulatory system fails to provide
the various organs of the body with sufficient blood flow to meet their
metabolic demands. This circulatory deficiency is the result of either
an inappropriate cardiac output and/or peripheral resistance. Blood
loss causes a fall in the cardiac output and irrmediately initiates
remote neural reflex systems to increase the peripheral resistance in
an attempt to raise the arterial pressure. Some vascular beds, i.e.,
the skeletal muscles, possess local control mechanisms which, upon
sensing the fall in blood flow, initiate a local vasodilation which
keeps the blood flow to metabolic rate ratio constant. Hence, the final
degree of vascular tone is influenced by local and remote control systems.

The cardiovascular system makes several adjustments following acute

4

blood loss in an attempt to increase the cardiac output. Increases occur
in cardiac contractility, venoconstriction (to increase venous return),
arteriolar constriction, and transvascular fluid absorption (to in-
crease blood volume).

The latter adjustment is due mainly to a decrease in capillary
hydrostatic pressure (Pc), but is also due to hyperosmolarity of the
plasma due to the action of cathecholamines on the liver and a decrease
in pancreatic release of insulin. This reabsorptive process is self-
limiting and forces favoring net reabsorption progressively diminish.
The end result is a transvaScular fluid efflux which results in hemo-
concentration. This conversion from hemodilution to hemoconcentration
is an indicator of the onset of irreversible hemorrhagic shock.

The shock syndrome will lapse into a terminal state called "irre-
versible shock" if these compensatory mechanisms are not able to re-
store an adequate cardiac output. This "irreversible shock" is a
tremendously complex problem involving many interrelated systemic and
cellular mechanisms only a few of which are recognized and understood.

S Metabolic, central nervous, cardiac, and microvascular alterations may
all contribute to the irreversible shock phenomenon in mammals.

The acidosis resulting from tissue anaerobic metabolism can relax
vascular smooth muscle and decrease its response to cathecholamines.

A hyperkalemia results from both the cellular exchange of potassium

ions for hydrogen ions and from the inactivation of the eletrogenic
pump. A hyperkalemia not exceeding ten mEq/l. causes vasodilation
which may be partially responsible for the vascular decompensation which
leads to irreversibility to transfusion.

A heart is depressed during prolonged hemorrhagic hypotension by

cardiac hypoxia, acidosis, and the hyperkalemia. The ischemic pan-
creas releases a myocardial depressant factor which depresses cardiac
performance. Sympathetic tone is also depressed which, in turn, de-
presses the inotropic state of the myocardium. Other researchers,
however, have failed to demonstrate cardiac depression and have found
that, in fact, cardiac performance is increased in late shock. The
latter researchers attribute the circulatory collapse in late shock
to a failure of peripheral resistance.

The medullary vasomotor centers which are responsible for the main-
tenance of the peripheral resistance may begin to fail due to inade-
quate cerebral perfusion. The resultant decrease in vascular tone
causes venopooling and a reduced venous return. The inotropic state of
the myocardium is also reduced for the same reason.

The microcirculation may become damaged by the accumulation of vaso-
active metabolites such as histamine and bradykinin. Terminal ar-
terioles and precapillary sphincters lose reactivity to constrictor
stimuli. Blood is shunted from nutritional to non-nutritional vessels.
A hyperosmolarity can relax the vascular smooth muscle. Intravascular
thrombi may result from the now reduced flow velocity and hypercoagul-
ability. Cellular swelling, cellular deformities, and hemoconcentra-
tion all reduce the flow velocity.

The reticuloendothelial system (RES) also becomes depressed allow-
ing gastro-intestinal toxins to enter the blood stream. These toxins
reduce vascular tone which ultimately reduces the cardiac output.

The onset of irreversibility is marked by a shift from hemodilu-
tion to hemoconcentration in the mammal. This hemoconcentration is

thought to be due to myocardial depression (Crowell and Guyton, 1962)

and/or failure of some component of the vascular bed (Lansing and Steven-
son, 1957; Lillehei gt_gl,, 1964; Rothe and Selkurt, 1964; Bond gt_gl,,
1977). Mortality increases as decompensation progresses.

The body maintains the mean arterial blood pressure (MABP) during
blood loss by immediate (sympathetic) and delayed (renal) mechanisms. A
dog can adequately compensate for a ten percent blood loss. A ten percent
blood loss in the chicken will cause a large fall in the MABP which could
reduce the Pc shifting the Starling equilibrium favoring fluid influx
across the capillary wall. The resultant influx of low protein fluids,
primarily from the skeletal muscles, increases the plasma volume. This
is the cause of the hemodilution, i.e., the greatly reduced hematocrit
(HCT) and hemoglobin (Hb), seen during early hemorrhage in bird and mammal.

Hollandberg and Nickerson (1970) found Pc began to rise in the dog
at the onset of the decompensatory stage due to a fall in the pre/post
capillary resistance ratio which facilitated the efflux of fluids across
the capillary wall. This fluid loss into the surrounding tissues had to
be replaced by reinfusion of the shed blood to maintain the MABP at a
given level of hypotension. Once about 25 percent of the shed blood had
been reinfused, reinfusion of all of the shed blood was insufficient to
prevent the subsequent cardiovascular and circulatory collapse and death
in irreversible hemorrhagic shock. Avian species may not experience
this fall in pre/post capillary resistance ratio since reinfusion of the
shed blood will almost always reinstate normal cardiovascular function and
MABP.

The increase in Pc in a mammal for any given arterial or venous
pressure during decompensation is thought by some researchers to involve

an unchanged post capillary resistance and a decreased precapillary

resistance for a given flow rate. The fall in precapillary resistance
may be due to neurogenic failure (Rothe and Selkurt, 1964) or may be
linked to a hyperkalemia-acidosis induced dilatation of the vascular
smooth muscle (Bond gt_gl,, 1977). Diana and Laughlin (1974) found that
secondary and tertiary to the importance of increased Pc in influencing
the transvascular fluid movement were increased capillary surface area
and increased capillary porosity, respectively.

Djojosugito gt_gl, (1968) found that the duck had about three times
the capillary surface area of the cat. Folkow gt_gl, (1967) found that
submersion, in diving duck species, elicited an intense reduction in Pc.
These findings may partially explain why Kovach and Balint (1968) and
Wyse and Nickerson (1971) found a high rate of transcapillary fluid influx
in avian species. It should be mentioned here that the diving duck and
the chicken are quite dissimiliar in regard to certain hematological and
cardiovascular parameters.

Folkow gt 21, (1967) have found that the hind limb hyperemia which can
"break through" neurogenic vasoconstriction in mammals could not "break
through" neurogenic vasoconstriction in the diving duck species. This lack
of dilatation was due, in part, to the inherent differences in mamma-
lian and avian extramuscular arteries. Such avian vessels are narrower,
more numerous, and more densely adrenergically innervated. The adrenergic
nerves run along the vessel inasmuch as sectioning of the sciatic nerve
dkinot prevent the constrictor response. The turkey behaves more like
the mammal in regard to these responses.

Wyse and Nickerson (1971) found the post-hemorrhage transcapillary
fluid influx in the hen to be twice the rate and four times the volume

of the dog. They noted that small blood losses (4 to 5 ml/kg) lowered

the MABP by 20 mm Hg, and a 15 to 20 percent reduction in blood volume
reduced the MABP to 50 mm Hg. In the dog about 20 ml/kg of blood must
be removed to lower the MABP by 20 mm Hg (Grega gt_gl,, 1967). However,
dogs in alpha-adrenergic blockade showed an initial bleeding volume
(IBV) similar to a chicken. Half of a dog's blood volume must be re-
moved to lower the MABP to 50 mm Hg. The IBV is the amount of blood
removed to initially lower the MABP to 50 mm Hg and the secondary bleed-
ing volume (SBV) is the volume removed subsequently. The maximal bleed-
ing volume (MBV) is the total amount of blood removed from the animal
i.e. the sum of the IBV and SBV. The SBV/IBV ratio for the dog was
about 0.1 whereas the ratio was about 2.0 for the bird (Wyse and Nicker-
son, 1971). This suggested that avian regulatory mechanisms acted to
maintain blood volume, whereas mammalian regulatory mechanisms acted to
maintain the MABP.

Mammalian pressoreceptors communicate with medullary control centers
through afferent tracts within the vagus and glossophyaryngeal nerves.
These control centers then elicit the proper compensatory adjustments
in central nervous system outflow to maintain a constant MABP. The pri-
mary mammalian baroreceptorsare located at the bifurcation of the internal
and external carotid arteries and on the aortic arch. Low pressure re-
ceptors exist in the atria, ventricles, veno—atrial junctions, lungs, and
elsewhere within the thorax and abdomen. Compensation for decreased
blood pressure is by way of vagal inhibition and an enhanced sympathetic
outflow, i.e. a heightened cardiac inotropic state and peripheral vaso-
constriction. Rothe and Selkurt (1964) found there was a failure of
neurogenic control in late hemorrhagic shock characterized by a decrease

in total peripheral resistance, respiratory rate, and HR. Blood chemistry

is also sensed by chemoreceptors located on the carotid arteries, aorta,
lungs, and elsewhere.

Though avian species do have carotid bodies and pulmonary chemo-
receptors homologous to mammalian species, the existance of carotid or
aortic baroreceptors has not yet been documented. The avian homologue
for the carotid sinus is within the thoracic cavity on the carotid
artery proximal to the vertebral artery and distal to the subclavian
artery. Avian baroreceptors have been identified histologically (Chowd-
hany, 1953) but not physiologically. McGinnis and Ringer (1967a) per-
formed bilateral occlusion of the carotid and/or vertebral arteries in
the hen and found no reflex tachycardia or pressor response. Prolonged
bilateral ligation of carotid and/or vertebral arteries for two weeks
produced no apparent brain damage and no cardiac hypertrophy (McGinnis
and Ringer, 1965). Occlusion studies in mammals generally produce
cardiac hypertrophy (Best and Taylor, 1961). Bilateral carotid and/or
vertebral occlusion in the hen produced no effects typical of a bara-
receptor when the cerebral perfusion pressure, measured by a carotid
cannula inserted craniad, decreased by 64 percent, i.e. to about 30 mm
Hg (McGinnis and Ringer, 1967b). This all indicated that a functional
baroreceptor does not exist in the head of the fowl. Results following
bilateral vagotomy were similar. Avian baroreceptors will be elaborated

upon subsequently in this review.

8. Alpha-Adrenergic Blockade

The alpha-adrenergic antagonist phenoxybenzamine, trade name
Dibenzylene, behaves the same in mammals and birds. Phenoxybenzamine
(PBZ) produces a noncompetitive blockade of alpha receptors, i.e.

increasing the dosage of an alpha-agonist will not overcome the blockade.

10

This is due to the drug binding in a very stable manner to the receptors
of other nearby structures in a persistant manner. Phentolamine, another
alpha-adrenergic antagonist, causes a competitive blockade of alpha
receptors that can usually be overcome by increasing the availability of
an agonist.

The drug has a multitude of actions. It causes postural hypotension
in humans. Phenoxybenzamine inhibits reflexogenic pressor responses by
preventing transmission of nerve impulses to the blood vessel cells.

The positive inotropic and chronotropic effects of catecholamines on
heart muscle is not affected by PBZ, but it may reduce the arrhythmias
caused by catecholamines. '

Phenoxybenzamine, administered by slow i.v. infusion has been suggest-
ed as a treatment procedure for preventing ischemia of the organs and
microcirculation during shock in animals.

Phenoxybenzamine has been shown to reduce the mortality of shock due
to trauma, bacterial endotoxin, adrenaline infusion, and superior
mesenteric artery occlusion (Gregerson and Root, 1947; Lillehei gt_gl,,
1961; Nickerson, 1961; Lillehei gt_§l,, 1964; and Hollandberg gt_gl,,
1970). The usefulness of treating hemorrhagic shock with PBZ is uncer—
tain. PBZ has been shown to increase the blood volume (Nickerson, 1961)
and prevent the severe vasoconstriction of shock (Clauss and Ray, 1968;
Hollenberg and Nickerson, 1970; and Carlson et_g1,, 1976). Hollenberg
and Nickerson (1970) found PBZ pretreatment delayed the onset of decom-
pensation and reduced the rate of reuptake of blood from the reservoir,
and so prolonged survival.

However, Grega gt_g1, (1967) found PBZ decreased the rate of trans-

capillary fluid efflux, but not at a rate sufficient to increase the

11

survival time when administered as an intermediate treatment. These
researchers did find intermediate treatment with strong beta-adrenergic
agonists to be beneficial. Chien (1967), in his comprehensive review,
also held doubts about the merit of PBZ in treating shock. In view of
this PBZ controversy, a brief review of shock related articles concern-
ing P82 is in order.

Nickerson (1961) found PBZ would increase the blood volume in normo-
tensive dogs. Stekiel gt 21, (1967) found PBZ had no effect on plasma
volume in normotensive dogs, but did increase the plasma volume in
hypertensive dogs. Williams and Rodbard (1960) found PBZ caused a 26
percent increase in plasma volume in the normotensive chicken, producing
an overall increase of 13 percent in blood volume.

Carlson gt_gl, (1976) found a maldistribution of coronary blood flow
during hemorrhagic shock in the dog. This involved a fall in the endo-
cardial/epicardial flow ratio. Phenoxybenzamine, 5 mg/kg, delayaibut
did not prevent this decrease in subendocardial blood flow. The resultant
subendocardial ischemia may lead to subendocardial hemorrhages, ne-
crosis, and zonal contraction lesions sometimes seen after shock. The
lesions may contribute to the disruption of the contractile machinery of
the myocardium, and to the eventual cardiac failure that follows, al-
though this has not been proven experimentally. Hackel gt_gl, (1974)
demonstrated that large exogenous or endogenous catecholamine conCen-
trations can cause the lesions. Hyperbaric oxygen during the hypotensive
phase (Ratcliff gt_gl,, 1963) or beta-adrenergic blockade (Entman gt_gl,,
1969) prevented the lesions. Phenoxybenzamine has no effect upon the
lesions (Martin gt_al,, 1969). Birinyi gt_gl, (1977) have shown PBZ

produced a 32 percent increase in myocardial blood flow in hemorrhaged

12

dogs indicating that the sympathetic nervous system limits the maximal
coronary dilatation during shock.

Fitch gt_gl, (1975) have shown PBZ decreased the vasoconstriction and
increased the blood flow to the head of the hemorrhaged baboon by dilating
the extraparenchymal cerebral arteries at the base of the skull. This
indicated that the sympathetic nervous sytem can regulate the lower limit
of cerebral autoregulation of blood flow. Fitch used the radiolabeled
xenon gas technique which is not as accurate as other methods for measur-
ing cerebral blood flow, i.e. the Repella-Green method.

Birds, like reptiles, amphibia, and fish, have a renal portal system.
Located at the junction of the renal vein and the iliac artery is a
valve that governs the flow of blood into the renal vein. Histamine
and acetylcholine have been shown to close the value jg_!jtrg_and epin-
ephrine opens the valve. The avian kidney possesses nephrons with long
and intermediate length loops of Henle, like the mammal, and also loop-
less reptilian-type nephrons confined to the cortex. Birds do possess
a juxtaglomerular apparatus (JGA) and utilize the renin-ANG II-aldo-
sterone-ADH mechanism as do mammals.

Plasma renin activity (PRA) increased tenfold in mammals in hemor-
rhagic shock (Jakschik gt_gl,, 1974). Du Charme and Beck (1971) found that
the renal pressor system reduced the vascular capacity by about 60 per-
cent that of the nervous system. Alpha-adrenergic blockade alone, there-
fore, does not overcome the severe vasoconstriction of shock, and ANG II
antagonists may be beneficial. Errlington gt 31. (1973) found that the
administration of ANG II converting-enzyme inhibitors increased the survival
of dogs in hemorrhagic shock.

Feigen gt 11. (1977) reported that PBZ, 2.0 to 2.5 mg/kg, greatly

l3

reduced the severe increase in renal vascular resistance seen in shock
and so increased renal blood flow, glomerular filtration rate, and urine
flow rate. Presumably PBZ dilated the renal afferent arterioles yield-
ing a 50 percent increase in urine flow rate. Phenoxybenzamine in-
creased the renal blood flow by 83 percent above control shock dogs.
Therefore, PBZ and an ANG II inhibitor, e.g. cysteine - ANG II, given
together may prevent the prolonged ischemia of organs, such as acute
tubular necrosis, seen during shock.

Phenoxybenzamine caused a tenfold increase in urinary sodium concentra-
tion and did not affect urinary potassium levels in control dogs.
Neither urinary electrolyte concentration was changed significantly in PBZ
pretreated shocked dogs, however the urine flow rate increased by 50
percent, giving a twofold increase in potassium excretion rate. This
potassium loss is beneficial in shock because it alleviates the hyper-
kalemia (Feigen gt_gl,, 1977).

Kashyap gt_gl, (1975) found the arterial free fatty acid (FFA)
concentration fell significantly in dogs and rabbits, while there were
continuous increases in adipose FFA's until death, in hemorrhagic shock.
Phenoxybenzamine pretreatment had no effect upon prehemorrhagic mean
arterial or adipose FFA concentrations, but did cause significant post-
hemorrhage increases in both of these parameters. Therefore, hypoper-
fusion of adipose may play an important role in the decreased supply of
this major body fuel in shock. Avian plasma FFA levels are comparable
to other vertebrates except for the laying hen whose plasma FFA concen-
tration is very high (Christie and Moore, 1972).

Haggendal gt_gl, (1976) found the dilating effect of PBZ was poten-

tiated by supplemental hydrocortisone. Conversly, the dilating effect

14

of hydrocortisone was greater following PBZ treatment. This latter
effect indicated that the vasodepressor effect of the steroid was not
due to an alpha-blockade, but rather, it increased the concentration of
neurotransmitter at the smooth muscle receptor site. This was accom-
plished by either inhibiting extraneuronal degradation mechanisms or by
preventing reuptake into the neurone. Phenoxybenzamine is known to
inhibit the extraneuronal uptake of neurotransmitters (Avakian and
Gillespie, 1968). The lingering catecholamines may continue to stimulate
beta-adrenergic receptors and initiate a further dilatation, which is
supported by the fact that beta-adrenergic blockade has prevented the
hydrocortisone-induced vasodilation after PBZ treatment.

Vargish gt 91, (1977), in their attempt to identify the most bene-
ficial form of steroid therapy in hemorrhagic shock, found dexamethazone,
15 mg/kg, provided the best protection in purebred beagle dogs. Second
best was methylprednisolone at 15 and 30 mg/kg.

Harvey gt_gl, (1954), in their early studies with P82 in the chicken,
found the drug to produce vasomotor reversal when given at 20 to 60
mg/kg. Nickerson (1961) found the reversal phenomenon in mammals during
alpha-blockade to be due to the unmasking of beta-effects initiated by
a non-specific agonist, epinephrine in the case of Harvey. Bunag and
Walaszek (1962a) found the dose administered by Harvey to be lethal to
hens. Peterson and Ringer (1968) administered P82, 39 mg/kg, to adult
hens while measuring feather intrafollicular pressure. The drug produced
hypotension but no mention was made of the degree of inhibition produced.
Kovach gt g1, (1969) found the pigeon capable of surviving a 100 percent
blood volume depletion if the hemorrage was extended over a six hour

period. Peripheral resistance increased in response to hemorrhage

15

(Kovach and $2552, 1968) although treatment with PBZ had no effect on
survival (Kovach gt_gl, 1969). The significance of this latter result
is unclear since, in the pigeon, PBZ fails to block the vasoconstriction
caused by epinephrine. Kovach and Balint (1969) noted that hemodilution
occurred continuously until death in the pigeon, whereas in the rat, the
hemodilution stopped after about fifteen minutes of hemorrhage. Block-
ade of alpha-receptors eliminated vasoconstriction in the skeletal
muscles of the duck and led to a greatly retarded restoration of blood
volume (Djojosugito §t_gl,, 1968). Alpha-adrenergic blockade also
abolished the intense vasoconstriction in the sciatic vascular bed in
the submerged duck (Butler and Jones, 1971). The duck showed a large
postdive hyperemia in carotid blood flow which was likely due to the intense
postdive tachycardia.

Szeto gt_al, (1977) produced accurate dose-response curves for the
chicken for PBZ, PROP, and atropine. Doses of 5 mg/kg PBZ were found to
produce 75 percent blockade of the phenylephrine-induced rise in arterial
diastolic pressure. They also found PBZ to be effective within 15 minutes
in the hen, unlike the very slow induction in mammals.

Harvey gt Q1, (1954) and Peterson and Ringer (1968) both mentioned
the extreme variation in various CVS parameters in birds as compared to

the dog or cat.

C. Beta-Adrenergic Blockade

Propranolol, tradename Inderal, causes a competitive blockade of
beta-adrenergic receptors. Therefore, large doses of beta-agonists can
overcome the beta-blocking effects. The drug produces similar effects in

flammals and birds. It prevents the positive chronotropic and inotropic

16

effects of catecholamines on the myocardium. Propranolol (PROP) has
the "quinidinelike" effect of stabilizing cell membranes. It also
causes a prolongation of the atrioventricular conduction time and
decreased upstroke velocity and overshoot of the cardiac action poten-
tial. Its advent in the late sixties as an antiarrhythmatic and anti-
hypertensive medication had tremendous impact upon the treatment of
various CVS disorders in man. It has reduced the mortality in humans
due to arrhythmias by 50 percent (Rowe, 1974).

Investigators advocating alpha-adrenergic blockade in shock seek
to obviate the deleterious effects of prolonged vasoconstriction leading
to the ischemic damage of vital organs. Conversely, other investigators
recommend beta—adrenergic blockade based on the supposition that failure
of peripheral vasoconstriction after prolonged hemorrhagic hypotension
leads to death. Evidence supporting the latter hypothesis is scant.

Berk gt_a1, (1967) found equal mortality (78 percent) in untreated
dogs and dogs in beta-blockade subjected to hemorrhagic shock. Increased
survival was observed only if the PROP was coupled with administration
of atropine, oubain, hypertonic glucose, sodium bicarbonate and calcium
chloride. Zierott gt 11. (1969) found PROP reduced the oligemic period
and was not beneficial. Halmagyid gt Q1, (1967) found that PROP and
P82 given together provided additional protection against shock in dogs.
Later, Wood gt_g1, (1974) found likewise and suggested that this protec-
tive action was the result of the combined drugs' suppression of the
large endogenous catecholamine concentration during compensation.

It is generally conceded that PROP does not have any beneficial
effects for the mammal in hemorrhagic shock. Beta-blockade has relieved

the systolic reduction in coronary blood flow seen in hemorrhagic shock

17

and decreased the incidence of subendocardial lesions by relieving the
severe beta-adrenergic stimulation (Rowe, 1974). These effects are far
outweighed by the following deleterious effects: reduced absolute coronary
blood flow, decreased cardiac output (C0), increased coronary vascular
resistance, depressed general inotropic state of the myocardium, de-
creased heart rate and decreased stroke work.

Most birds have two main coronary arteries, while some avian species
have three or four coronary arteries. The right coronary artery is al-
ways larger. There are four primary coronary veins. The avian heart
is similar anatomically to the mammalian heart except the right AV
valve consists only of a heavy muscular flap. The avian heart also
represents a much higher percent of the body weight than in mammals.

Basal resting heart rate in the chicken is about 300 b/minute. Myo-
cardial beta-receptors in the chicken are associated with positive ino-
tropic and chronotropic effects. The vagi exert a powerful tonic
inhibiting influence on heart rate and the ventricular inotropic state.
The avian myocardial tissue does not have t-tubules.

St. Petery gt_g1, (1977) confirmed the existance of alpha- and
beta-receptors in the three day chick embryo. Beta—adrenergic blockade
was achieved in the chicken by Peterson and Ringer (1968) using dichloro-
isoproterenol. They found hypotension. Bulton and Bowmen (1969) found
alpha-blockade inhibited the hypertension caused by catecholamines and
that beta-blockade slightly increased the pressor response in hens.
Therefore, the hen has alpha- and beta-adrenergic receptors, but the beta-
receptors are dominated by the alpha-receptors. The beta-receptors do,
however, have an inhibitory effect upon pulmonary arterial alpha-receptors.

Here beta-blockade produced maximum isotonic contraction lg vitro because

18

it removed the beta-adrenergic inhibition of alpha-receptors. Szeto gt
31, (1977) found PROP to have an effect of short duration in birds, as
in mammals. They obtained an 82 percent blockade of isoproternol-
induced tachycardia by administering a .25 mg/kg bolus of PROP followed
by a 5 ug/kg/minute infusion.

Edens (1974) found the normally-occurring increase in plasma
corticosterone in chicks in heat stress was nonexistant after alpha-
blockade. He also found PROP, 4 mg/kg, or reserpine would prevent the
fall in corticosterone which usually occurs after 80 minutes of heat
stress.

When Kregenow gt_gl, (1976) added norepinephrine to an isotonic
medium containing duck erythrocytes it initiated a very rapid bidirec-
tional movement of sodim and potassium ions into the erythrocyte. A
hypertonic solution caused the erythrocytes to shrink and the cells re-
adjusted their volumes by an adenosine 3'5'-cyc1ic physphate (cAMP)
facilitated change in cation permeability. Isoprotenenol bound to beta-
receptors and increased cAMP, but the binding was not necessary. PROP
has been shown to be a potent inhibitor of adenyl cyclase activity, but
a weak inhibitor of binding in the turkey erythrocyte (Bilezikian and
Aurbach, 1973).

Beta-adrenergic blockade did not have any effect on the CVS response
to submersion in the duck, but did reduce the immediate hyperemia and
abolished the rise in MABP normally occurring upon emersion (Butler and

Jones, 1971).

D. Normovolemic Anemia
Sustained hemorrhagic hypotension produced a 50 percent reduction in

HCT and plasma protein concentrations in the fowl (Wyse and Nickerson,

19

1971), thus a brief discussion of normovolemic anemia is in order. Acute
normovolemic anemia is accomplished by exchanging whole blood with six
percent dextran-70.

In the dog a dextran exchange sufficient to reduce the HCT from 36
to 13 percent produced a 91 percent increase in C0. The HR, stroke
volume, and LVEDP all increased, whereas the TPR decreased. Beta-
adrenergic blockade at this time decreased the C0 and HR and increased
the TPR. Therefore, the sympathetic nervous system plays a large role
in the physiological response to normovolemic anemia.

Nightengale (1976) produced acute normovolemic anemia in the chick
by replacing one percent of the body weight, i.e. about one-sixth of
the blood volume, with six percent dextran-70. This reduced the HCT by
50 percent. Tissue oxygen delivery was maintained by increased extrac-
tion of the oxygen coupled with increased stroke volume and CO. The
TPR decreased. The HR, right atrial pressure and oxygen consumption
(V02) were not changed. Further decreases in HCT and Hb resulted in
CVS collapse as indicated by a rapidly fallen CO, stroke volume and V02.
Wyse and Nickerson (1971) found that a similar CVS collapse, occurring
at any point in time during the hypovolemic procedure, could be corrected
by a rapid reinfusion of the shed blood.

A two percent dextran-7O exchange provided the maximum CVS adjust-
ment in the chick. Exchanges of four to six percent of the body weight
were required in the dog to produce the same hemodilution effect.
Dextran-7O exchanges of one through three percent of the body weight
produced progressive increases in the plasma sodium and potassium con-

centrations in the chick (Nightengale, 1976).

20

E. Other Factors in Shock

It has been well established that extracellular osmolarity and the
concentrations of potassium, hydrogen and magnesium ions become elevated
in far advanced hemorrhagic shock (Schwinghamer gt_gl,, 1970). These
may be involved in the gradual diminution of the compensatory constric-
tion since all of these electrolytes caused vasodilation in the systemic
circulation in test systems. Bond gt_gl, (1977) found that hyperkalemia
in shocked dogs may be due to hypoxic inactivation of the electrogenic
pump activity, but is more likely due to a passive movement of potassium
ions out of the muscle cell with the bicarbonate anion in response to
the intracellular accumulation of hydrogen ions. The direct inhibition
of vascular smooth muscle by hyperkalemia and acidosis may be one of the
causes of vascular decompensation during late shock in mammals.

Corticoids augmented intracellular sodium, which increased smooth
muscle tone and contractile responsiveness which,in turn, increased the
MABP (Clauss and Ray, 1968). The corticoids also caused a rapid ex-
trusion of intracellular potassium, which diminished the transmembrane
gradient of potassium, resulting in increased CVS contractility and
MABP in mammals. Electrolyte changes during hemorrhage in avian species
have not been documented.

Histamine, released from the mast cells during trauma, infection,
exercise, etc., has been shown to be a potent vasodilator of arterioles
and caused increased capillary permeability. Elevated histamine levels
resulted in degeneration of the microvasculature. Trauma resistance
has been obtained from the injection of spleen extracts prepared from
trauma-conditioned rats, RES stimulation, and trauma conditioning. This

may have been due to the RES which recognized high histamine levels and

21

elaborated mediators to prevent the histamine-induced destruction of
the microvasculature beds (Galvin _t_gl,, 1977). Harvey et_gl, (1954)
demonstrated that histamine was released in response to serotonin (5-HT)
in avian species. Bunag and Walaszek (1962b) reported that histamine
was found in high concentrations in avian plasma, was readily released,
and was depressor in the chicken.

The humoral agent "myocardial depressant factor" (MDF) is another
consideration in shock. Discovered in mammals, MDF is thought to be a
small peptide produced by lysosomal hydrolases in the ischemic pancreas.
MDF depresses both the inotropic state of the myocardium and the RES.
Lefer and Martin (1970) found that the MDF titer correlated inversely
with survival time in hemorrhaged dogs. Phenoxybenzamine may cause
hyperperfusion of the pancreas and dininish MDF release. This factor has
not been isolated, and may not exist, in avian species.

Rothe and Selkurt (1961) have demonstrated the existance of dilating
agents of intestinal origin in the portal blood of the hypovolemic dog.

Splanchnic pooling is another consideration in irreversible hemor-
rhagic shock. Abel §t_gl, (1965) reported the primate did not demon-
strate the congestion and bowel necrosis encountered in the dog. Pul-
monary pooling may be another site of blood loss from the circulating
blood volume although Abel gt_g1, (1967) have cast serious doubt on this
theory.

Plasma glucose concentration increased initially in hemorrhagic and
endotoxin shock in the mammal then quickly fell to acute hypoglycemia
(Strawitz et_gl,, 1961, Hinshaw gt a1,, 1976). The hypoglycemia was
coupled with a decreased sensitivity to insulin which decreased glucose

transport. Moffat gt gl_(1968) found that a glucose infusion late in

22

shock can prolong survival beyond that of control animals. Drucker
gt_gl, (1975) showed that a low protein/high carbohydrate diet decreased
the tolerance to hemorrhagic shock. High protein diets did not increase
the tolerance.

Plasma glucose concentrations in man and hen are 80 mg% and 180 mg%,
respectively. The total plasma protein concentration of the chicken is
about 5.3 gm%, whereas this value is 7.3 gm% in man. The colloid osmotic
pressure of the hen is only 11.1 mm Hg (Albritton, 1952) compared to 28
mm Hg in man. The difference is due to the low albumen/globulin ratio
in the chicken (0.8) as compared to that of man (2.0). Wyse and Nickerson
(1971) and Kovach and Balint (1968) have reported that hemorrhage can
reduce the plasma total protein concentration by as much as 35 percent in

the fowl.

II. Respiratory Homeostasis

A. Avian Blood Gases and pH

Mammals in hypovolemic shock have generally maintained arterial
p02 indicating an adequacy of oxygen exchange (Bond gt_g1,, 1977). A
mild respiratory alkalosis in early shock in mammals resulted in a low
pCOZ in the arterial blood. This alkalosis soon converted into a severe
acidosis and the arterial pH dropped as low as 7.00 (Bond gt 21,, 1977).
The changes in blood gases during hemorrhage in avian species have not
been documented.

The measurement of avian pH with direct-reading radiometer electrodes
must be corrected for the high avian body temperature, i.e. 40.500. If
the radiometer bath is at mammalian body temperature, i.e. 37°C, the
Rosenthal (1948) formula makes the correction as follows:

Blood pH = th - .0147 (37 - t).

23

Shepard gt El, (1959) reported arterial pC02 values of 40 mm Hg in
the deeply anesthetized hen. This is similar to man at rest. However,
numerous subsequent investigators found lower pCOZ levels. Edens and
Siegel (1974a) reported pCOz values of 26 mm Hg in lightly anesthetized
hens. Choidi and Terrman (1965) may have been the most accurate in
their assessment of blood gases from the locally anesthetized, lightly
restrained hen. They found a pCOz of 32.8 mm Hg and an arterial pH of
7.49 by comparing samples to reference pH-pC02 curves obtained from two
aliquots equilibrated at different known C02 levels.

This variation in reported values may be due to stress hyperventi-
lation. Hyperthermia-induced panting in adult hens increased the arterial
pH above 7.70 reducing the pCO2 below 15 mm Hg and the C02 content to be-
low 10 mEq/L (Calder and Schmidt-Nielsen, 1968; Frankel and Franscella,
1968). This suggests a need for telemetric studies with in-dwelling
cannulas for unstressed blood sampling.

Account should also be taken for wide swings in acid-base balance
associated with age, nutrition and laying cycle. Helbacka gt_gl, (1964)
demonstrated significant effects of feeding and starvation on plasma
pCOz. Egg calcification over a 16 hour duration has been shown to
decrease plasma bicarbonate content and to decrease plasma pH from 7.52
to 7.42 (Mongin and Lacassagne, 1966). Sodium phenobarbital anesthesia

did not affect pH, pCO , or bicarbonate levels in avian species, but

2
may have decreased the p02 slightly (Edens and Siegel, 1974b). Cohen
and Horwitz (1974) have found a high sodium diet will increase plasma pH

in hens.

B. Intrapulmonary Receptors in Aves

Early researchers thought that high C02 inhalation decreased

24

respirations in the hen, bit this was only due to the stimulation of chemo-
receptors in the nares, tongue, and pharynx.

Birds breathing a zero percent CO2 gas mixture in air experienced
apnea after hyperventilation (Ray and Fedde, 1969). A five percent C02
mixture produced normal respirations in deeply anesthetized hens (Fedde
gt $1,, 1963). A 20 percent C02 mixture produced an increase in MABP
and a 200 to 300 percent increase in muscle vascular resistance reaching
a maximum in three to five minutes in the diving duck species (Peterson
and Fedde, 1968a). Attempts to create this vasoconstriction in the cat
or turkey with a 20 percent CO2 mixture were unsuccessful (Hiestand
and Randall, 1941). Inspiratory minute volume, tidal volume and hydro-
gen ion concentration all increased as the inspired C02 increased
(Osborne and Mitchell, 1978).

The existance of intrapulmonary chemoreceptors was demonstrated by
Hiestand and Randall (1941). They found a rapid decrease in inspired
C02 would produce a rapid fall in ventilation within 0.5 seconds even
when the pulmonary veins and arteries were occluded. Conversely, the
administration of high C02 concentrations via low trachae (Ray and Fedde,
1969) or humori (Jones and Purves, 1970) stimulated respirations. The
maximal respiratory response occurred at an arterial pCOZ of 40 to 55
mm Hg.

Avian intrapulmonary chemoreceptors sensitive to C02 play a major
role in the control of breathing during normocapnic and hypocapnic
conditions (Osborne gt_gl,, 1977). The impulse frequency in vagal
afferent fibers from these intrapulmonary chemoreceptors increased when
the intrapulmonary C02 was decreased. The receptors were not sensitive

to hypoxia, hyperoxia (Tschorn and Fedde, 1974), lactic acid, sodium

25

cyanide, acetylcholine (Fedde and Peterson, 1970),or changes in intra-
pulmonary pressure.

Afferent fibers from intrapulmonary mechanoreceptors run centrally
in the vagus nerve. Unlike the mammalian slow adapting stretch receptors
which respond to changes in both C02 and mechanical stimuli, the avian
receptors are not sensitive to changes in C02 concentrations (Fedde

g_t__a_i_.1974).

C. Intracardiac Receptors in Aves

Cardiac ventricular C02 receptors have been identified in the bird
(Estravillo and Burger, 1973). Such chemoreceptors may extend into the
aorta. The magnitude of the tidal volume is inversely related to the
ventricular-receptor discharge frequency along the afferent fibers of
the middle cardiac nerve. Electrical stimulation of this nerve depressed
the respiratory rate in the same manner as low C02 inhalation.

A transient rise in MABP, which increased the rate of receptor
activity, also produced a decreased respiratory rate for the duration of
the pressure rise (Estravillo and Burger, 1978). Information supporting
any functional description for ventricular mechanoreceptors is lacking.
However, recent work has shown that inflation of a balloon in the left
ventricle produced inhibition of spontaneous breathing in closed-chest

dogs on cardiac bypass (Kostreva gt 21,, 1977).

D. Other Receptors in Aves

Rodbard and Saiki (1952) hypothesized that an intracranial barore-
ceptor mechanism may control cerebral blood flow in the chicken.

Although nerve fibers from the nodose ganglion of the vagus nerve

terminate in the region of the carotid sinus and aortic wall, no functional

26

baroreceptor has been identified in these areas.

The avian carotid body is innervated by a branch of the vagus nerve
which arises from the nodose ganglion. Hollandberg and Uvnas (1963)
presented evidence that indicated stimulation of the carotid bodies was
responsible for the circulatory changes, i.e. bradycardia, increased
blood pressure, decreased Splanchnic and cutaneous blood flow and little
change in skeletal muscle blood flow, which occurred in diving ducks
during submersion asphyxia. Carotid body denervation abolished these
responses. The carotid bodies of the chicken do not appear to play a role
in respiratory control inasmuch as ventilatory sensitivity to inhaled
C02 was not greatly affected by hypoxia (Jones and Purves, 1970).

Atland (1961) found that the hypoxic tolerance of the fowl was lower
than other small animals. His birds died as a result of exposure to an
inspired p02 of 46 to 73 mm Hg. In support of Atland, Richards and
Sykes (1967) found that alterations in the avian electroencephlogram
began at about 70 percent oxygen saturation. This was much higher than
in mammals (Brechner gt_gl,, 1965). The hyperventilation alkalosis in
hens produced vasoconstriction which may have offset the local dilation
of the cerebral arterioles occurring in hypoxia (Paff and Boucek, 1958).
This may have resulted in failure of the medullary cardiovascular and
respiratory centers. ,

Much research by the Scandanavian group has been directed at deter-
mining the relative roles of chemoreceptors and baroreceptors in the CVS
responses to diving in the duck (Blix et_§l,, 1975). Jones (1973) found
that the bradycardia, obtained during a l to 2 minute submergence in the
chronically denervated duck, was identical to that in intact ducks, though

the MABP fell greatly in the denervates due to a relatively lower vascular

27

resistance. Even so, the sciatic vascular resistance in the denervates
was still half that of the intact ducks indicating that half of the in-
crease in the TPR was achieved independently of baroreceptor stimulation.
Jones and West (1978) performed a constant-flow hind limb perfusion on
the duck during submergence and at the same time electrically stimulated
the one intact depressor nerve innervating the baroreceptor. Submer-
gence caused bradycardia and increased vascular resistance, and nerve
stimulation caused a fall in HR, MABP, and perfusion pressure.

Hind limb perfusion studies have not been reported for the chicken.

Mechanoreceptors may exist in many of the visceral organs of the bird
(Duke et_gl,, 1977). The function of these receptors is unknown.

Aortic bodies have been found in the connective tissue between the
ascending aorta and the pulmonary artery of the chicken (Tcheng et_gl,.
1963). These researchers suggested a possible chemoreceptor or barore-
ceptor function for these bodies in the bird. They are innervated by a

branch of the vagus nerve.

E. Hypoxia in Aves

Whereas hypercarbia produced linear increases in MABP and HR in the
duck, Ray and Fedde (1969) reported that hypoxia decreased the arterial
diastolic blood pressure. Besche and Kadono (1978) have shown that the
mean femoral blood pressure decreased linearly in progressive hypoxia.
The hypoxia-induced hypotension involved an unchanged HR (Butler and
Taylor, 1974), bradycardia (Sturkie, 1970), or tachycardia (Besche and
Kadono, 1978). The latter have reported that an eight percent reduction
in inspired oxygen, i.e. from 21 to 13 percent, caused a 35 percent reduc-
tion in TPR in the adult Leghorn-type hen. Recently, Grubb and Schmidt-Neil-

son (1978) employed the xenon clearance technique for measuring cerebral

28

blood flow in the duck during hypoxia. The hyperventilation-induced
respiratory alkalosis and hypocapnia diinot alter cerebral blood flow in
the duck, whereas mammals vasoconstrict under similar conditions re-
ducing the cerebral blood flow by 50 to 75 percent.

Although the fowl can adapt well to chronic hypoxia, it has little
tolerance to acute hypoxia compared to mammals and other species of birds.
The effects of acute hypoxia in the chicken include: decreased body
temperature and oxygen consumption, increased respiratory rate, plasma
volume, and HR. However, Richards and Sykes (1967) have reported a
decreased respiratory frequency in the hen in acute hypoxia. The cardiac
output did not change during acute hypoxia in the chicken (Besche and
Kadono, 1978). The decreased blood pressure coupled with normal CO
values suggests that marked changes may have occurred in the vascular
beds, i.e. possibly a redistribution of blood flow.

Sturkie and Abati(l978) showed that a large drop in cardiac con-
tractility occurred in various avian species' isolated hearts when made
hypoxic by substituting 95 percent nitrogen for 95 percent oxygen in
the perfusion fluid. The drop ranged from 15 percent of normal for the
chiCken to 52 percent of normal for deep-diving ducks. The pigeon was
intermediate at 31 percent. This demonstrated how demand for, and utiliza-
tion of, oxygen, and the tolerance to oxygen deficiency differ greatly

between diving, flying, and terrestrial birds.

OBJECTIVES

To determine the effects of propranolol or phenoxybenzamine on heart
rate, arterial blood pressure, cardiac index, stroke volume, stroke
work, and total peripheral resistance in adult SCWL hens thirty

minutes after treatment.

To determine the effect of sustained hypovolemic (hemorrhagic)
hypotension without pretreatment and following pretreatment with
phenoxybenzamine or propranolol in adult SCWL hens on heart rate,
arterial blood pressure, cardiac index, stroke volume, stroke

work, total peripheral resistance, bleeding volumes, survival times,

and mortalities.

To determine the effect of sustained hypovolemic hypotension without
pretreatment and following pretreatment with phenoxybenzamine

or propranolol in adult SCWL hens on hematocrit, hemoglobin, plasma
potassium, plasma magnesium, plasma sodium, and plasma glucose

concentrations.

To determine the effect of sustained hypovolemic hypotension without
pretreatment and following pretreatment with phenoxybenzamine or
propranolol in adult SCWL hens on arterial pH, arterial pCOz, ar-

terial p02, and the incidence of respiratory arrest.

29

II.

III.

MATERIALS AND METHODS

Experimental Stock -- Mature SCWL hens (1.5 to 2.2 kg.), after a
year of egg production at the Poultry Science Teaching and Research
Facility, were housed in laying batteries at a constant environ-
mental temperature for at least five days prior to use. Not all
birds were producing eggs. Water and cage layer ration were
supplied éfl.li§1£!fl: Feed was withdrawn twelve hours prior to each
experiment.. The birds were on a 14 hour light cycle. The research
was conducted in the spring and summer. Thirty hens were divided

into five equal groups.

Anesthesia -- Each animal was lightly restrained in a supine position
on a small animal board. The legs were tied down and a wire was
inserted through the nares to restrict movement of the head. The
hens were anesthetized with sodium phenobarbital, 100 mg/kg, via

the brachial vein. ~This plane of anesthesia is considered "light
anesthesia." Toe pinch would produce slight withdrawal and comb
pinch would produce vigorous head shaking. This anesthetic was
chosen for its long action and margin of safety. Each bird then
received an intracutaneous injection of about 0.5 ml of a two per-
cent procaine solution at the surgical site on the ventral surface
of the upper neck. After all surgical procedures, a piece of towel-
ing was placed over the birds' head because this can produce a

calming effect.

Surgical Procedure -- The carotid arteries and the jugular veins were
exposed by making a three centimeter midline incision on the ventral

surface of the neck in the upper cervical area. The right carotid

30

31

artery was then dissected free from the connective tissue and M. longus
ggllj_at about the level of the second cervical vertebra. A permanent
ligature was placed around the cranial end of the exposed artery. A
nick was made in the artery and a fluid filled polyethylene cannula
(Intramedic Clay-Adams, New York, N.Y., PE-90: 1.0. = .034", 0.0. =
.050") containing sodium heparin (0.25 mg/ml) was inserted about five
centimeters toward the heart.

The right jugular vein was teased from all connective tissue. The
ramifications of the vagus and glossopharyngeal nerves were carefully
teased from a two centimeter segment of the vessel. The vessel was
then looped with suture material for ease of identification for the sub-
sequent dye injections.

Low-neck tracheotomy was performed to prevent the congestion in
the larynx region often seen in avian species and for ease of artificial
ventilation should the need arise. The tracheal tube was suctioned
periodically with a suction catheter.

The animals were heparinized with 2.5 mg/kg sodium heparin via
the carotid cannula upon completion of the surgical procedures. He-

parin was readministered at two hour intervals.

IV. Drug Administration -- The treatment groups were set up in the follow-

ing manner:

 

 

GRP # TREATMENT DOSE ._g_

1 sham operated, sample ---- 6
replacement
hemorrhaged ---- 6
hemorrhage and P82 5 mg/kg 6

4 hemorrhage and PROP .25 mg/kg + 6

5 ug/kg/min
5 sham operated, no sam- ---- 6

ple replacement

32

Group #1 sampling blood losses were immediately compensated for by the
infusion of equal volumes of donor blood.

All drugs were administered via the brachial vein. Phenoxybenzamine
was injected 30 minutes prior to hemorrhaging. Birds receiving PROP
underwent left brachial vein cannulation (Intramedic, PE-90) in the
direction of the heart. A continuous infusion was necessary due to the
shortness of action of this drug. The brachial cannula was attached by
way of a three-way stopcock to a five ml glass syringe fitted into an
infusion/withdrawal pump (Model 940, Harvard Apparatus Co., Millis,

Mass.). The infusion was started 30 minutes prior to hemorrhaging.

V. Shock Protocol -- The experiment lasted 225 minutes. Every 45
minutes, beginning at time zero (T=0), blood samples were obtained and
arterial blood pressure and EKG were recorded.

Starting at T=O, blood was withdrawn from the bird through the
carotid cannula. This blood was shunted through the pressure trans-
ducer, through a dye tracer cuvette, and then into a lubricated glass
10 m1 syringe fitted into a second infusion withdrawn pump (Model 950,
Harvard Apparatus Co., Millis, Mass.). Bleeding rate was about one
ml/kg/minute. Blood was quickly pumped from the infusion/withdrawal
pump in ten ml intervals into a graduated cylinder sitting in a con-
stant temperature water bath (Buchler Instruments, Fort Lee, N. J.).
Reserve blood was agitated by a stream of room air.

Blood was removed until the mean arterial blood pressure (MABP), as
measured periodically from the carotid cannula, reached 50 mm Hg. The
amount of blood removed to reach this level of hypotension was designated

the Initial Bleeding Volume (IBV). The IBV was generally obtained within

33

the first 15 minutes of the experiment. Subsequent small amounts of
blood had to be removed to maintain the MABP at 50 mm Hg. This volume
was designated the Secondary Bleeding Volume (SBV). The total volume
of blood removed from the bird prior to death, or upon surviving 225
minutes of hypotension, was designated the Maximal Bleeding Volume (MBV).

Birds entering respiratory arrest were artificially ventilated
(Harvard Small Animal Respirator, Harvard Apparatus Co., Millis, Mass.)
for no longer than ten minutes, after which, the respirator was dis-
continued and the bird observed.

When a bird entered cardiovascular (CVS) collapse, as indicated by
a rapid fall in the MABP, the right jugular vein was cannulated (Intra-
medic, PE-l60, 1.0. = .045", 0.0. = .062") and the reserve blood rein—
fused at a rate not exceeding 2 m1/kg/minute. This would generally
re-establish CVS status provided the fall in MABP was not of sufficient

magnitude to prevent reversal.

VI. Blood Pressure Measurement -- The blood pressure was monitored via
the carotid cannula by a Statham physiological pressure transducer (PA-
23AC) connected to a Grass 7A polygraph. The MABP was obtained by
electronically dampening the pressure oscillations. This was later
determined more accurately by the following formula:

MABP = diastolic pressure + 3/8 pulse pressure (Sturkie, 1967).

Calibration of the recorder was accomplished by using either a
pocket anaeroid barometer connected directly to the transducer or by

the internal calibration mechanisms contained within the polygraph.

VII. Heart Rate Determination -- The HR was obtained from the 8P trac-

ings. Measurements were made over a six or ten second interval and

34

multiplied by ten or six,respectively. A three lead EKG was taken
every 45 minutes to check for arrhythmias.

A chart speed of 10 or 25 mm/minute was used throughout the ex-
periment, but every 45 minutes the chart speed was accelerated to 25 mm/

second to obtain accurate BP and HR measurements.

VIII. Blood Chemistry Analysis -- Every 45 minutes, beginning at T=0,
a 1.5 ml arterial sample was drawn through the carotid cannula after
clearing the cannula dead space. TWenty microliters of this whole blood
was used to determine the hemoglobin (Hb) concentration by the cyano-
methemoglobin method (Lynch et_gl,, 1969). The remaining blood was
separated by centrifugation at 10009 for 15 minutes at 10°C (Sorvall
RC-5 Superspeed Automatic Regrigerated Centrifuge). The plasma thus
obtained was placed into small, capped glass vials that had been previously
rinsed with triple distilled deionized water. These plasma samples were
then frozen for subsequent analysis of magnesium, potassium, sodium, and
glucose concentrations.

Every 45 minutes, beginning at T=0, three to five tenths of a ml
of arterial blood was carefully withdrawn from the carotid cannula into
a one m1 syringe. This syringe was quickly capped and placed into an
ice bath in a freezer for subsequent blood gas and pH analysis.

Synchronous with the arterial samplings, blood was withdrawn by
venipuncture of a small shank or wing vein into a heparinized Micro-
Capillary tube. This was centrifuged for ten minutes using an International
Micro-Capillary Centrifuge (International Equipment Co., Boston, Mass.).
Hematocrit values (HCT) were then determined on an International Micro-
Capillary Reader.

Plasma sodium and potassium concentrations (mEq/L)were determined

35

in duplicate on a Beckman Flame Photometer (Model 105) using a 900 part
per million lithium internal standard and appropriate plasma dilutions
(Appendix A). Plasma magnesium was determined on a Perkin Elmer
Atomic Absorption Spectrophotometer (Model 2908 BMS3 Mark-2) using a
lanthanum oxide diluent (Appendix A). Plasma glucose was determined

by a glucose oxidase/peroxidase enzyme method (Sigma Chemical Co. as
per Technical Bulletin #510). Blood gas and pH determinations were done
on a direct-reading radiometer (BMS-3, Mark-2 Blood Micro System with

a PHM Mark-2 Digital Acid Base Analyzer, Copenhagen, 0k).

IX. Cardiac Output Determinations -- Cardiac output was measured by a
dye dilution technique (Hamilton gt_gl,, 1932) at T=0, T=90, and T=180.
The two groups receiving drug treatment had cardiac output (C0) deter-
minations at T=-30 and T=0 only, because preliminary experiments showed
the procedure was often fatal at a later time. During the C0 procedure
about four to five m1 of blood were withdrawn from the carotid cannula
through a dye tracer cuvette (General Medical Electronics, Middleton,
Wisc) at a rate of 10.3 m1/minute into a lubricated glass syringe fitted
into an infusion/withdrawal pump (Model 950, previously mentioned).

The output signal of the GME dye tracer cuvette control unit was
amplified by a Grass Model 7PL low level DC preamplifier and ultimately
was recorded on an Esterline Angus Single input analog recorder (Model
E1101E with a twelve inch scale) at a chart speed of twelve inches per
minute. This apparatus was calibrated by passing jg_!jtgg_blood samples
from each bird containing zero or ten mg of dye per liter of blood through
the dye cuvette at the end of each experiment.

The baseline was set as blood was drawn through the cuvette. Two

to three tenths of a mg of the concentrated cardio-green dye (Hyson,

36

Westcot, and Dunning, Inc., Baltimore, Md.) was injected rapidly into the
exposed right jugular vein. When the recirculation effect was evident
in the dye concentration curve, the pump was reversed and the blood re-
infused.

The area under the curve was estimated with a compensating polar
planimeter (Model 4236, Keuffel and Esser Co., Hoboken, N.J.) following
extrapolation of the declining limb of the curve onto semilogarithmic
paper and replotting the limb to within one percent of the baseline to
eliminate recirculation effects (Appendix 31- The C0 and cardiac
index (CI) were then calculated by the following formulae:

mg dye injected

Cardiac Output (C0) = area under curve
mg min/liter

L/mihute

CO in ml/minute .73“
Cardiac Index (CI) = (Body weight in kg)
(Speckman and Ringer, 1963)

L/min./kg°731+

Stroke volume was estimated by dividing the CI by the HR. Stroke
work was determined by multiplying the stroke volume by the MABP. Total
peripheral resistance (TPR) was determined by dividing the MABP by the CI.

All birds, those that died during the hypotensive period and those
that survived, were necropsied to determine the placement of cannulae,
egg laying status, and the occurrenCe, if any, of subendocardial, hepatic

or gastrointestinal hemorrhagic lesions.

X. Statistical Analysis -- One-way analysis of variance (ANOVA), using
an f test, was utiliZed to determine if a significant difference existed
between two means.

Comparison of the percent change from the initial value for the

final three samples prior to death within and between treatments was done

37

by split-plot repeated measure ANOVA. Dunnett's test determined if
significant differences existed between the hemorrhage group and each of
the other three groups. This was followed by linear and quadratic ortho-
gonal polynomial contrasting within each treatment for the final three
sampling times to determine if average and/or curvilinear trends existed
(Appendix Cl).

A similar two-way block-design repeat measure ANOVA was used to
analyze variation from the initial CI values, and the variance of para-
meters derived from the CI, e.g. TPR, stroke volume, and stroke work

(Appendix C2) .

RESULTS

EXPERIMENT I: A total of twelve hens were utilized in this experiment.
All were anesthetized and cannulated as described previously. They were
allowed to stabilize for ten minutes before initial measurements of HR,
MABP, and C0. Immediately after these initial measurements, at T=-30
minutes, the appropriate drug treatment was administered. One group re-
ceived PROP (0.25 mg/kg followed by a 5 ug/kg/minute infusion i.v.),

the other PBZ (5 mg/kg i.v.). Thirty minutes later, at T=0, these
parameters were remeasured.

A. Effect of PROP: One-way ANOVA within threatments showed PROP
caused significant reductions in HR (P<0.01), C0 (P<0.5), CI (P<0.05),
MABP (P<0.01, and stroke work (P<0.05). The only parameters not affected
were stroke volume and TPR (P<0.05), although these did not have a non-
significant tendency to be reduced. ANOVA between treatments showed that
PROP caused a significantly greater fall in HR (P<0.01) and CI (P<0.05)
than did PBZ. There was also a tendency, although not significant, for
the MABP of the PROP-treated animals to be lower than the PBZ-treated
animals. The TPR did not differ between the two drug treatments, although
the PBZ-treated animals have a nonsignificant tendency to have a lower TPR
(Tables 1A and 18 and Figure l).

B. Effect of P82: One-way ANOVA showed that PBZ did not signifi-
cantly change any of the measured parameters except for a reduction in
the MABP (P<0.05). There was a tendency for stroke work and TPR to be
reduced. The HCT was significantly reduced (P<0.05) by 8.7 percent of

the initial value (Table 5). The Hb value was not reduced.

38

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40

FIGURE 1: The effect of a thirty minute pretreatment with propranolol
or phenoxybenzamine on the cardiac index. mean arterial
blood pressure, heart rate, and total peripheral resistance

of adult SCWL hens.

41

 

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EXPERIMENT II:

A. Hemodynamic Results: This experiment utilized 24 hens divided
into four equal groups designated; control, hemorrhage, PROP + hemorrhage,
and P82 + hemorrhage. Inasmuch as some of the hens did not survive the
entire 225 minutes of the experiment, the final three determinations
of several parameters prior to death were recorded. These were labeled
"sample 1" (90 minutes prior to the final sample), "sample 2“ (45 min-
utes prior until the final sample), and “final sample" (prior to death).
The actual times to the final sample are listed in Table 7. This tem-
poral form of sampling synchronized the terminal changes in the measured
parameters for each animal regardless of survival time. All electrolyte,
HCT, Hb, glucose, and HR results were tabulated in this manner. The
three samples were then compared to the initial pre-hemorrhage values
to obtain accurate terminal percent changes. These percent values
were then analyzed statistically as shown in Appendix C.

1. Heart Rate -- The hemorrhage group had a significantly higher

 

(P<0.05) HR than the control group. One-way ANOVA of the absolute final
HR value yielded the same level of significance. Dunnett's test in-
dicated that the PROP + hemorrhage group had a significantly lower
(P<0.01) HR than the hemorrhage group. The PBZ + hemorrhage group HR
values did not differ from the hemorrhage group. These data are tabu-
lated in Table 3 and are shown graphically in Figures 2A and 28.

Neither average trend nor curvilinearity existed between sampling
times. This means that, once the initial change in HR occurred, there
would be no further change over time.

2. Cardiac Index -- The cardiac index was obtained at 90 minute

 

intervals in the control and hemorrhage groups. The drug treatment groups

43

.>.e .mx\me mm
.cocmaccw .=*e\m¥\m= m »n eczo_Poc .>.a m=_on oX\ae mu.
_

 

 

 

 

mm: m:—
m.~n ¢.mn m.wn o.mh -m~=mnxxo=mga
P.~p+ m.op+ m.~+ ~.¢mm + mmagceosm: m
~.¢n 0.0“ a.mn N.mn Ppopocmcaoca
o.-- o.m_- o.mp- o.om~ + muesccosm: o
~.mn m.mn m.¢n o.mph
a._~+ e.o~+ “.mp+ N.eom consccOEmI e
m.en m.en m.mn P.m_n
¢.p+ m.o+ m.~- m.mom pocucou o
orgasm Faced N m—msmm _ mpmsmm one o in» acmEDmmc» mew;
~.cws\awl mo
mum; beam; meuwcw seem manage ucmucma muma “cam: .oz
PmmuwcH

 

 

.mcmsm~:maxxocmgn co Fopocmcaoea new: ucmEummcumea mcwzoppoe new ucwEummepmea
“segue; commcouoazz uvmmcecoeo; umcwepmam op cmaumnnam new; 430m “Faun cw .mpm>cmucw masses m>wm
-xueoe um emcwmuno .mwpnsmm omen» peeve mew mo we?» we» we mums acme; cw mmmcmso eo.m.mn cam: .N mgm<e

45

FIGURE 2A: The change in heart rate at the time of the final three
samples in adult SCWL hens subjected to sustained hypo-

volemic hypotension. I

FIGURE 28: The change in heart rate at the time of the final three
samples in adult SCWL hens subjected to sustained hypo-
volemic hypotension and pretreated with propranolol or

phenoxybenzamine.

% CHANGE IN H.R.

7. CHANGE IN we.

 

46

 

 

 

 

 

 

 

130~ l-
...... + ........ i ............... l
110- - ..'....
T l *r
90- )-
w-i r-
CONTROL
TOJ F" """"" “RM.
w p
,9
433 mind i i «he!
"can 2A ‘
1w“ -
12m - ......... I. .................
“°"‘ "' ’0". I
100- - K.
\\
9°" \
\
\ ’ ’ a- , \ \
ao« — \ d ._ , \\
T, \+
10~ -
......... HEM.
60" r- - -— _ PROP ll HIM.
, , _..nz a HEM.
A l t l 1 l J
l I l I r
'30 Inlilll 1 3 "u.

FIGURE 2|

47

had CI determinations only twice, i.e. at T=-30 and T=0 (immediately
prior to hemorrhage), as previously described in Experiment 1. Cardiac
output values not corrected for differences in body weight are meaning-
less, therefore only weight-standardized CI values will be considered
in this thesis.

A two-way block-design repeated measures ANOVA (Appendix 02) was
used to compare the second and third CI determinations to the first
within a treatment. The control group showed no significant difference
(P>0.05) between C15. The hemorrhage group did show a significant
(P<0.05) decrease in the last two CI determinations below the initial
CI value. These data are tabulated in Table 3 and are expressed graphi-
cally in Figure 3A.

3. Stroke Volume -- This parameter was analyzed in the same manner
as the CI determinations. The T=90 and T=180 determinations did not
differ significantly from the initial value in the control group, but did
become significantly lower (P<0.01)for the latter two sampling times in
the hemorrhage group (Table 3 and Figure 38).

4. Stroke Work -- This parameter was analyzed in the same manner

 

as the CI determinations. No change occurred in the control group, but
a highly significant reduction (P<0.01) did occur in the hemorrhage group
in the latter two sampling times (Table 3).

5. Total Peripheral Resistance -- The TPR of the hemorrhage group
decreased significantly (P<0.05) over the first ninety minutes, whereas
the control group did not change significantly. However, the magnitude
of the change in the first 90 minutes in the hemorrhaged group, when
compared to the change occurring in the control group, was not signifi-

cant (Table 4).

48

 

6. Bleedjgggyolumes -- No significant difference existed in the
IBV, SBV, or the MBV between the three hemorrhaged groups. The two drug
treated groups did show a nonsignificant tendency to have lower IBVs
and higher SBVs than the hemorrhage group. This is especially evident
in the PROP + hemorrhage group (Table 5). Though no significant dif-
ferences existed in the SBV/IBV ratios between treatments, there was
a tendency for the ratio to be higher in the PROP + hemorrhage group
(Table 4).

7. Survival Times and Mortalities -- No significant difference
(P >0.05) existed between survival times among the three hemorrhaged groups.
There was, however, a nonsignificant tendency for the PROP + hemorrhage
group to have a lower survival time than either of the other hemorrhaged
groups (Table 4). Four out of six birds survived the hemorrhage pro-
cedure in both the hemorrhage and the PBZ + hemorrhage groups. Only
two birds in the PROP + hemorrhage group survived. Mortalities for the
hemorrhage, PBZ + hemorrhage, and PROP + hemorrhage groups were 33, 33,
and 67 percent, respectively (Table 4).

8. Lesions and Arrhythmias -- Petechial hemorrhages were observed

 

in the endocardium of two of the PBZ + hemorrhage birds, however, both
of these birds lived the entire duration of the experiment. No hepatic
or gastrointestinal lesions were observed in any of the other birds.

The EKG had arrhythmias in only one bird. This was in the PROP +
hemorrhage group and this bird died early.

Traube-Herring waves were often markedly exaggerated after the pri-
mary fall in blood pressure. These were most prevalent in the hemorrhage
group (occurring in three birds) but also occurred in each of the other

hemorrhaged groups. They had a frequency of about one per minute. Small

49

m

c

.1

 

 

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Amo.ovav o u e soc» mucmcmccwe ucmuwcwcmwmp
m. F.mn Fmo.n mmo.w
Nam.m~ m.Pm Ne—me. Nun. omp u e
m.¢ w.~w mmo.w Nmo.w
Ne.m m.mm «wee. mms. om u e

m N.en ppo.w mac.“

m «.mm ops. mum. N o n e
mmmneeoew: Focpcou mmmcecosm: pocucou mmeceeosm: A.:wsv week
A .ax\ummn\FvaAam<zv Aemh.mx\ummn\PEv .mx\:ws\Fsv

emu

ecoz exocpm

manpo> mxocum

xmccm umwuemu

 

 

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eowca mam; 430m “Faun cm xeoz mxocpm can .m53po> mxoeum .xmucw omwuemo cw mmmcmsu mo.m.mh meow:

.m m4m<b

50

FIGURE 3A: The change in cardiac index in adult SCWL hens subjected

to sustained hypovolemic hypotension.

FIGURE 38: The change in stroke volume in adult SCWL hens subjected

to sustained hypovolemic hypotension.

INDEX [avian/3,334]

CARDIAC

VOLUME [av hot/lo-n‘]

STROKE

F) .

51

um -

220i-

 

2oo— — I

140d !— 'Oooao-o-I....+
CONTROL

'20" '- '..-.I..-. "EM.

 

 

90
TIME (min.)
FIGURI 3A

 

 

 

CONTROL

 

.......... “EM.

 

I 1

90
TIME (min.)
FIGURE 3.

go

52

 

wmo.h
0mm.

mac.“
mum.

omp

 

 

mes.“ wee.“
one. ¢mo.
use.“ . mmo.w
omm. «mm.
cm 0

 

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mmmzegosm:

Poeucou

 

hzm2H<mmh

 

 

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mmu==_e om .ou eomca new; 430m p_=ue cw mm=Fe> oucaumvmwc Pmcmsawcma Pepe» use coe.m.mn came one

53

blood losses would causethese waveforms to become greatly exaggerated.

B. Hematological Results: Blood chemistry data were analyzed and
recorded as described previously in Experiment IIA (Appendix Cl).

1. Hematocrit -- The control group had a significantly greater
(P<0.01) average HCT than any of the hemorrhaged groups. No significant
difference (P<0.05) existed between any of the hemorrhaged groups.
Analysis at individual times indicated that "sample 1" (the sample 90
minutes prior to the final sample before death) of the control group
did not differ significantly from the hemorrhaged groups at that time.
This means that the truly significant alterations in HCT occurred pri-
marily in the hour preceding death (Table 6 and Figure 4A and 48).

Only the hemorrhaged groups showed a significant (P<0.01) linear
trend. This was in a curvilinear manner (P<0.01) for the PROP + hemor-
rhaged group. i

2. Hemoglobin -- The average Hb for each of the hemorrhaged groups

 

was significantly (P<0.01) lower than the control group. However, com-
parison within each of the hemorrhaged groups indicated that only the
final two sampling times were significantly different from the control
group. "Sample 1" for each of the hemorrhaged groups showed a non-
significant (P>0.05) tendency to be lower than the control group, "sample
2" was lower (P<0.05), as was the "final sample" (P<0.01), (Table 7 and
Figures 5A and 58). No difference existed between the hemorrhaged groups.
No linear trend existed in the control group. The hemorrhage group
showed a signigicant (P<0.05) linear trend, but in a curvilinear manner
(P<0.05), with most of the change occurring one to two hours prior to
death. Both drug treatment groups showed a significant (P<0.01) linear

trend without curvilinearity.

54

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.mmpacws mmm mm umecou
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.>.P mx\me m
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55

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.>.P mx\me mm
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remumcH

 

 

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mam; 430m “Faun cw spawn o» eowca .mpm>cwpcw mpzcms me am cmxmp .mmpqemm omega Pee?» men Co
we?“ we“ no mapm> Papaya? we» seem manage pcmoema on» ecu .umeuoumem; Pavuwcw we“ mo.m.mw new: .0 mem<e

56

FIGURE 4A: The change in hematocrit at the time of the final three
samples in adult SCWL hens subjected to sustained hypo-

volemic hypotension.

FIGURE 48: The change in hematocrit at the time of the final three
samples in adult SCWL hens subjected to sustained hypo-
volemic hypotension and pretreated with propranolol or

phenoxybenzamine.

% CHANGE IN HCT

IN HCT

°/. CHANGE

?

801-)-

70' '-

40-i-

 

100'"-

«H-

 

 

CONTROL
..I-OIIII HEM-

 

I
l
Inlilcl

57

 

i
1
nouns M

i

 

III-II... "EM.
. _. ... . PROP 8: HEM.

— PBZ & HEM.
l

l
iniilcl

I
I

1

FIGURE 43

b
1—

58

Page Omitted

59

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page we azocm Foepcou msp soc; acmememmu xpucmuwwwcmwmm

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e

.>.F \m¥\me mN

 

 

 

 

 

 

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mom;
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em.¢n “a...“r mm.¢n mNdH mmm:geo&mz
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mo
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meuwca
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umcwmumam o» cmuumnnam mew; 43mm upsum cw .mpm>emucm muscve me am :mxmp .mmpaEmm omega chwm we
we?“ on» as mapm> Popuwcw asp soc» mucosa ucmucma ecu use cwnopmoeo; meuwcw on» Co.m.mn came on» .N m4m<e

60

FIGURE 5A: The change in hemoglobin at the time of the final three
samples in adult SCWL hens subjected to sustained hypo-

volemic hypotension.

FIGURE 58: The change in hemoglobin at the time of the final three
samples in adult SCWL hens subjected to sustained hypo-
volemic hypotension and pretreated with propranolol or

phenoxybenzamine.

73 CHANGE IN Hb

“I. CHANGE IN Hb

 

 

 

 

 

 

 

 

100-)-
90‘ r- ‘+
80‘ " To.
'10—. e I
oo- -
50" - a... ...- ........... +
40- - CONTROL
......... HEM
ao~ L
V
’f . 1 I l
iniIIoI 1' l2 "I'IOI
FIGURE 5A
100- —-
80" r
ro- -
60- *-
so— —
4o- - HEM.
"" "" " PROP In HEM.
30- :- -—- P82 8: HEM.
)1!
f l L 1 1
I F l I
iniilol 1 2 IIIIIII

. 62

3. Plasma Potassium -- The control group plasma potassium level
was lower than the hemorrhage group (P<0.05) when averaged over the
three sampling times. Likewise, the P82 + hemorrhage group had a lower
potassium level than the hemorrhage group, though this trend was not
significant (P<0.05) (Figure 6A).

One-way ANOVA of the differences in the initial and final samples
between treatments indicated that the hemorrhage and PROP + hemorrhage
groups showed significantly greater differences than the control
(P<0.05) or the P82 + hemorrhage (P<0.05) groups. The P82 + hemorrhage
group had a significantly lower (P<0.05) average potassium level than
the PROP + hemorrhage group (Figure 6B and Table 8) and a significantly
(P<0.05) higher potassium level than the control group.

One-way ANOVA of the initial versus final potassium levels within
each treatment indicated that the concentration increased over time in
the control group (P<0.01), the PBZ + hemorrhage group (P<0.01),
and in the PROP + hemorrhage group (P<0.05) (Table 8 and Figure 68).

Hence, potassium was elevated in all of the groups. The magnitude
of the increase in the hemorrhage and PROP + hemorrhage groups was
significantly greater than the remaining two groups.

Only the hemorrhage and PROP + hemorrhage groups showed a linear
trend (P<0.01) over time. None of the groups varied in a curvilinear
manner.

4. Plasma Magnesium -- The plasma magnesium concentration did not
vary significantly over time or treatments. One-way ANOVA comparing the
initial and final samples within each treatment also showed no signi-
ficant change (Table 8).

Trend analysis at individual sampling times indicated that the

.63

FIGURE 6A: The change in plasma potassium concentration at the time
of the final three samples in adult SCWL hens subjected to

sustained hypovolemic hypotension.

FIGURE 68: The change in plasma potassium concentration at the time
of the final three samples in adult SCWL hens subjected
to sustained hypovolemic hypotension and pretreated with

propranolol or phenoxybenzamine.

64

l
l

 

 

 

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.. . a -
... E
u n
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III-II’HIIII L I [1 F P I.
L ...: O 2
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o ... M M
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m ..m m . ..
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WWW

1

FIGURE 63

I

IIIIIIIII

65

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s... 9:9. mN

 

 

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me Nm— m.m~+ N.NNW m.mpN N.N w o.¢mp .Em; + aoma on:
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Foe?» o» “amoeba Paced meuwcH
we?» cam:

 

 

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page; on» o» m.mn weep come we» use .cwwcwsu mmmcmeu “smegma on» .m.mw cams peeve use meuw:_ one .m m4m<e

66

hemorrhage group had a nonsignificant tendency (P>0.05) to have a
linear trend. The PROP + hemorrhage group did show a significant (P<0.01)
linear trend, but in a curvilinear manner (P<0.01) with most of the
change occurring two to three hours prior to death. The other groups
showed no trends.

The initial versus final plasma magnesium concentrations are
tabulated in Table 8.

5. Plasma Sodium -- Dunnett's test and one-way ANOVA failed to

 

show any significant change in plasma sodium concentrations between or
within treatments, with the exception of the P82 + hemorrhage group,
where one-way analysis did show that a significant increase (P<0.05)
occurred between the initial and final samples (Table 8).

No linear or curvilinear trends occurred in plasma sodium concentra-
tion over time.

6. Plasma Glucose: Dunnett's test showed all of the hemorrhaged

 

groups showed a nonsignificant tendency for plasma glucose to be higher
than the control group (Figures 7A and 7B). The hemorrhage group had the
largest tendency to increase (P>0.05). One—way ANOVA indicated that the
change between initial and final samples was significant in the hemor-
rhage (P<0.01) and the PBZ + hemorrhage (P<0.05) groups (Table 8). The
PROP + hemorrhage group tended to increase (P<0.05).

No linear or curvilinear trends existed over the final three sampl-
ing times. This indicated that the change which occurred in plasma glu-

cose concentration must have occurred early in the experiment.

C. Respiration Data:

1. Arterialng -- One-way ANOVA between initial and final samples

 

67

FIGURE 7A: The change in plasma glucose concentration at the time of
the final three samples in adult SCWL hens subjected to

sustained hypovolemic hypotension.

FIGURE 78: The change in plasma glucose concentration at the time of
the final three samples in adult SCWL hens subjected to
sustained hypovolemic hypotension and pretreated with pro-

pranolol or phenoxybenzamine.

68

 

 

I—CONTROL
IIIOIIIII "EM.

L _

Wm

 

3003va

O.
O
O
O.
0
O
0
O
O
O
O
D
O
O
G
O
G
O
O.
G

—
m
<22

—
w
...—

». _.
n m
z. 3256

 

"
m
x

I2

E‘

 

FIGURE 7A

 

 

 

 

— — -PROP G HEM.
PIZ II HEM

l
Ila-I

12

 

 

m.

M
b.hLKW+T.
wmmmnmmw

umOUNSO <23 2. NOV-(:0 x

FIGURE 1|

69

within a treatment indicated that only the PROP + hemorrhage group showed
a significant (P<0.05) increase in arterial pH over time. All of the
other groups had tended to increase over the course of the experiment,
with the hemorrhage group having the greatest trend of the three (Table 9
and Figure 8). Similar testing indicated that no difference existed
between any of the initial or final pH values across the four treatments.

2. ArterialpCO2 -— The pC02 was reduced in all of the groups, but
only in the hemorrhage group was it significant (P<0.01). The PBZ +
hemorrhage group showed a strong nonsignificant tendency to decrease
(P>0.05) (Figure 8 and Table 9).

3. Arterial p02 -- No significant difference existed between or
within treatments over time (Figure 8 and Table 9).

4. Respiratory Arrest -- The PROP + hemorrhage group had less of a
tendency to become apneic. None of the PROP + hemorrhage birds died as
a result of respiratory failure. The four deaths in this group were =
a result of cardiac failure as evidenced by a rapidly falling MABP and
HR. Reinfusion of the shed blood reinstated normal, albeit hypotensive,
CVS status in only one of these birds. As previously discussed in
Experiment 116, two birds died in both the hemorrhage and the PBZ +
hemorrhage groups. In each case, one death resulted from respiratory
problems and the other from cardiac problems. The birds in the PBZ +
hemorrhage group appeared to have more respiratory problems than the
other hemorrhaged groups, as evidenced by the fact that three of these
birds required some respiratory assistance during the experiment. This
artificial ventilation never exceeded ten minutes in duration, but in
two of the three instances this was sufficient to restore normal respira-

tion. Though respiratory rates were not recorded, there was a general

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FIGURE 8:

71

The changes in arterial pH, p02, and p002 concentrations
from the time of the initial to the final sample in adult
SCWL hens subjected to sustained hypovolemic hypotension
without pretreatment and following pretreatment with pro-

pranolol or phenoxybenzamine.

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73

tendency for the animals to become progressively tachypneic over time,
especially in the hemorrhaged groups. The animals were hyperventilating

by the end of the experiment.

0. Group #5 and Overall Grouped Data

1. Group #5 -- Group #5 was a control group, i.e. a nonhemor-
rhaged group, in which sampling losses were not replaced with donor blood.
This group experienced a large fall in MABP and HCT, and an increase in
HR when compared to the transfused control group (Table 10). The CI
did not differ between the two control groups.

2. Overall Grouped Data -- The mean initial values for CI, HR,

 

MABP, HCT, Hb, and plasma potassium, magnesium, sodium, and glucose
concentrations for all the birds utilized in all of the experiments

(n=30) are listed in Table 11.

74

 

 

 

 

 

 

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DISCUSSION

1. EFFECTS OF PROP + PBZ 0N HEMODYNAMIC PARAMETERS

It is well documented that PROP greatly reduced the inotropic and
chronotropic state of the myocardium in mammals. In view of the large
reductions in cardiac performance during beta-adrenergic blockade,
myocardial beta-receptors also play an important role in avian species.

The hypotensive action of PROP in the hen was primarily mediated
by its negative inotropic and chronotropic effects on the myocardium.
The greatly reduced HR and stroke work resulted in the greatly reduced
CI and MABP The peripheral vasculature was not altered by PROP inas-
much as the TPR did not change (Figure 1).

Conversely, the hypotensive effect of P82 in the hen, as in the
mammal, was mediated by peripheral vasodilation without significant
positive inotropic effect on the myocardium. Though both drug treat-
ments caused singificant reductions in the MABP, the PROP caused signi-
ficantly greater reductions than the P82. This indicated that cardiac
performance in the hen, not altered vascular tone, was the primary
regulator of arterial blood pressure.

This latter supposition is logical since avian species have been
shown to have a low arterial capacitance and low compliance in the ab—
dominal aorta (Speckmann and Ringer, 1964). This low aortic compliance
was reflected in pulse pressures often exceeding 100 mm Hg during the
hypotensive phase in research for this thesis. The relatively stiff
arterial system may represent a compromise situation related to the ex-
tremely high HR in the chicken, so the net result of smoothing flow in
the periphery is still achieved. The low aortic compliance is likely
due to the amount of collagen in the vessel walls and to the accumulation

76

77

of atherosclerotic plaque common in birds.

PBZ reduced the HCT by 8.7 percent of the initial value, indicating
an increase in plasma volume. Avian extramuscular arteries are more
numerous and more densely adrenergically innervated than in mammals
(Folkow gt g1,, 1967). Williams and Rodbard (1960) hypothesized, upon
finding that PBZ caused a large increase in plasma volume in the hen,
that the drug could conceivably release trapped plasma in extramuscular
vessels by blocking the effectors of postganglionic neurons. Therefore,
changes in nerve density or impulse frequency could effectively change
the pressure-volume relationship in these vessels which could, in turn,
regulate the effective circulating plasma volume. Though Williams and
Rodbard (1960) did not find hypotension after PBZ treatment, reasearch
for this thesis found hypotension, and numerous other researchers have
found likewise (Harvey gt g1,, 1954, Peterson and Ringer, 1968).

The fact that PBZ caused plasma volume expansion in the chicken
and not in the dog (Stekiel et_gl,, 1967) could be due to a larger capi-
llary surface area in the bird. Folkow gt_g1, (1967) estimated that the
duck has three times the capillary surface area of the cat. Hence, if
the fowl is similar to the duck in this regard, the fowl could be ex-
pected to experience large changes in transcapillary fluid movement
following small changes in TPR.

II. EFFECT OF HEMORRHAGE IN THE FOWL WITH AND WITHOUT PRETREATMENT WITH

PROP OR PBZ.

A. Hemodynamic Parameters -- The birds used in this research were
normal and apparently in good health as evidenced by the overall mean
initial hematological values listed in Table 11.

Whereas flying and diving avian species showed intense vasoconstriction

78

and maintained the MABP during large hemorrhages (Djojosugito et_gl,,
1968), the chicken showed large and rapid falls in MABP with only mini-
mal blood losses. The hen showed better survival after blood loss than
the mammal, yet fell considerably short of the phenomenal tolerance of
the duck or pigeon (Kovach et 91,, 1969). The sensitivity of the chicken
is exemplified in Table 10, where a ten ml blood loss over a 225 minute
duration caused large changes in HCT, Hb, and MABP. This confirmed the
findings of Kutola et 21, (1967) who f0und an eleven ml blood loss over
two hours reduced the venous HCT by 18 percent. Though the hen cannot
maintain the MABP after blood loss, she does have the ability to endure
long periods of hemorrhagic hypotension without entering vascular de-
compensation.

The onset of decompensation in the mammal may result from a failure
of the intense neurogenic vasoconstriction. This dilatation may be due
to failure of the CNS-mediated neural activity, or is possibly the re-
sult of the accumulation of metabolic vasodilator substances, which may
include MDF, C02, lactate, potassium ions, hydrogen ions, adenosine,
histamine, bradykinin, or prostaglandins. Neurogenic vasoconstriction
after initial blood loss is apparently not very intense in the hen since
PBZ did not reduce the IBV with near the magnitude it has in the dog. The
possibility then exists that vascular decompensation, if indeed it even
does occur in the hen, would have a much less dramatic effect on the
MABP than it does in the dog.

The hen does show a degree of vasomotor tone, as evidenced by the
small fall in TPR upon alpha-adrenergic blockade (Figure l). Neuro-
genic constriction in the duck is intense, inhibited by alpha-adrenergic

blockade, and cannot be overcome by the accumulation of metabolic

79

vasodilator metabolites (Folkow et_gl,, 1967). Sciatic vascular resis-
tance increased eightfold during submersion in the duck (Butler and Jones,
1971). Isolated hind-limb perfusion studies have not been reported in the
hen. Such studies in the turkey have shown that a skeletal muscle hy-
peremia does occur during the vasoconstriction caused by breathing a 20
percent C02 gas mixture. Similar studies in the duck have failed to
demonstrate such a hypermia. Hence, large variations exist among bird
species, with the most efficient MABP homeostasis being achieved by

flying and diving birds.

The IBV is an indicator of an animal's ability to maintain the MABP.
The chicken IBV is one half that of a dog when compared on a percent of
initial blood volume basis (Table 12). This again indicates that vaso-
constriction in the chicken is not as important as it is in the mammal with
regard to blood pressure homeostasis.

Kovéch and $2552 (1968) reported that a loss of 54 percent of the
initial blood volume in the pigeon resulted in only a 30 mm Hg drop in
the MABP. Roughly 22 percent of the initial blood volume of the dog had
to be removed to lower the MABP by 20 mm Hg (Hollenberg et 21,, 1970),
however, after PBZ treatment only five percent had to be removed to ob-
tain the same effect. Research for this thesis indicated that a blood
loss of less than 10 percent was all that was necessary to reduce the
MABP by 20 mm Hg in the hen, and P82 did not change this value.

The SBV represents the movement of fluid into the vascular space in
avian and mammalian classes. The magnitude of the SBV in a bird varies
with the duration of the experiment. The experimental duration, i.e. the
survival time, depends upon the level of hypotension and the bleeding rate.

The bleeding rate in this research was twice that of Wyse and Nickerson

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81

(1971) and the resultant bleeding volumes were somewhat smaller. The
canine SBV is limited by the onset of decompensation. The SBV in the
chicken is limited by a rapid CVS collapse culminating in the death un-
less the shed blood is quickly reinfused. The mean canine SBV from

the five research articles cited in Table 12 is approximately 14 percent
of the initial blood volume, as compared to 38 percent in the chicken.

The hemorrhaged chicken has been reported to mobilize 40 to 52
percent of the initial blood volume before dying (Wyse and Nickerson,
1971), whereas the dog mobilized only 10 percent. Wyse and Nickerson
(1971) reported the plasma mobilization rates for the dog and chicken
for the first 90 minutes of hypotension to be 6.7 and 15 percent of the
initial blood volume per hour, respectively. The volume of fluid
mobilized in the research for this thesis, as indicated by the fall in
the HCT, was roughly 35 percent of the initial blood volume over a three
hour duration, or about 11.7 percent per hour. Hence, the rate of trans-
capillary fluid influx is higher in avian species than in mammals and
continues until death.

The duck has been shown to almost entirely replace a 40 ml blood
loss (14 percent of the initial blood volume) in 20 to 25 minutes
(Djojosugito gt gl., 1968). This high rate of transcapillary fluid in-
flux in the hemorrhaged duck did subside over time despite a continued
increase in flow resistance. This reduction could be a consequence of
the reduced plasma oncotic pressure and increased tissue oncotic pressure.
The duck, like the chickens in this research, may experience hyper-
glycemia which would increase the plasma osmolarity. This, coupled with
a reduced Pc, could shift the Starling equilibrium, favoring fluid absorp-

tion despite the large fall in plasma oncotic pressure. Eventually the

82

forces could become balanced and a fluid efflux could occur in the termi-
nal stages of hypovolemic hypotension (Wyse and Nickerson, 1971), though
this was not observed in research for this thesis.

The TPR apparently falls, or at least does not increase, in the
hemorrhaged chicken. How, then, does the chicken utilize the Starling
forces to absorb fluid faster than the hemorrhaged dog? The low arterial
pressure may cause some precapillary vessels to collapse, facilitating
fluid absorption. The oncotic forces also favor fluid absorption. The
respiratory alkalosis and the associated hypocapnia, may cause vasodila-
tion(unlike the mammal), shifting the Starling equilibrium favoring fluid
absorption.

Irreversible shock in mammals is thought to be partially the re-
sult of low tissue flow due to a low MABP and high TPR. The latter did
not occur in the chicken and this may offer this species some protection.

Phenoxybenzamine treatment reduced the rate of transcapillary fluid
influx in the hemorrhaged duck. PBZ also reduced the rate of fluid in-
flux and then efflux across the capillary wall in the hemorrhaged dog
(Stekiel et_g1,, 1967). Hence, PBZ reduced the IBV and increased the
SBV in dogs. The drug did not significantly change the IBV or SBV in
the chickens in this research, though small trends did exist in the same
direction as those observed in the dog (Table 5). Therefore, alpha-
adrenergic receptors play a role in all three species, but to a much
lesser extent in the chicken.

Beta-adrenergic blockade with PROP prior to hemorrhage greatly re-
duced the MABP, which could directly reduce the IBV as seen in Table 5.
The mean SBV of this group of birds was the highest of all three hemorrhaged

groups, even though their survival time was 50 minutes less than either

83

of the other hemorrhaged groups. Inasmuch as the rate of hemodilution
in this group, as evidenced by the uniformity of the slopes for HCT and
Hb (Figures 48 and 58) after temporal adjustment for differences in
survival times, is identical with the other hemorrhaged groups, one

can assume that death resulted from similar vascular responses. The
decreased survival time in this group appears to have been due to the
negative chronotropic effects of the drug on the myocardium.

The question immediately arises as to whether or not the PROP +
hemorrhage birds were stressed more than the other groups. However,
the more relevant question might be why was it necessary to remove blood
much more rapidly in these birds in order to maintain the MABP constant.

The ratio of SBV/survival time, which is an indicator of the volume
of blood removed per kilogram per minute to maintain the MABP at 50
mm Hg for the PROP + hemorrhage group and the hemorrhage group was .136
and .084, respectively. If the SBV reflects the transcapillary fluid
influx, PROP enhanced the rate of fluid mobilation almost twofold. This
could have been due to alterations in either cardiac performance or
peripheral vascular tone. The cardio-inhibitory effects of PROP alone
could reduce the Pc facilitating plasma volume expansion. Betaeadrener-
gic blockade has been shown to produce a degree of vasoconstriction in
mammals.

The SBV/IBV ratio was reduced in the dog pretreated with PBZ, in-
dicating that alpha-adrenergic receptors are involved with MABP regulation.
PBZ pretreatment did not change the SBV/IBV ratio in hens. Hence, the
mechanisms involved with blood pressure regulation in the chicken depend
less upon alpha-adrenergic innervation than on local control mechanisms

and/or differences in anatomical design of the peripheral vasculature.

84

It should be noted here that the SBV/IBV ratios for the dog and
chicken are .32 and 1.80 (Table 12), respectively. This again indicates
the intrinsic differences in the control mechanisms between the two
species; the dog acting primarily to maintain MABP and the chicken act-
ing to maintain the intravascular volume.

The MBV's for avian and mammalian classes are remarkably similar.

The MBV is obtained at a time when the critical events leading to death
occur in both classes. Immediately following the MBV in the dog hemo-
concentration begins to occur, and mortality increases as hemoconcentration
progresses. The fowl, on the other hand, enters sudden death at the time
the MBV is obtained.

Irreversible hemorrhagic shock in mammals is due either to cardiac
failure or failure of the peripheral resistance. Inasmuch as the chicken
apparently neither vasoconstricts nor enters vascular decompensation it
appears that mammalian irreversible shock may result from failure of
the peripheral resistance.

8. Hematological Parameters -- The hematological events which
occur in the hemorrhaged dog up to the onset of decompensation are very
similar to the events occurring in fowl. The primary differences in the
two species are the rapid rate of fluid mobiltation and long duration to
the MBV in the fowl. The HCT, Hb, and plasma protein concentrations fall,
while plasma potassium and glucose increase and plasma sodium and magnesium
remain quite constant in both species during the interval of time leading
to the MBV.

These trends all continue in fowl up to the time of death. The
dog, however, undergoes a reversal of events: the HCT increases by 30

percent of the initial value. the Hb and plasma protein levels rise, the

85

glucose suddenly falls to severe hypoglycemia, but the plasma potassium
continues to rise.

The failure of avian species to decompensate or enter shock
irreversible to transfusion raises questions concerning the present under-
standing of the shock phenomenon, particularly in regard to the develop-
ment of hyperkalemia and hypoglycemia in late shock. Before these
questions can be addressed, a review of some of the biological differences
between dogs, ducks, and chickens is necessary.

The skeletal muscle response for vasoconstriction during a reduc-
tion of the MABP in the dog is overcome, and a constant blood flow is
maintained, by intrinsic neurologic responses, i.e. Baylis response, and
by the accumulation of vasodilator metabolites. Mellander (1963) and others
have hypothesized that after continued hypotension in the mammal, pre-
capillary sphincter (if indeed such a sphincter exists outside of the
bat wing) reactivity began to fall, while at the same time the reactivity
of the postcapillary resistance vessels and capacitance vessels remained
high. Eventually capacitance vessel reactivity fell, causing venopooling,
decreased venous return, decreased C0, and a further fall in reactivity
completing a vicious circle. It should be mentioned here that many sub-
sequent researchers have failed to demonstrate changes in the pre/post
capillary resistance ratio during shock in the mammal.

As mentioned previously, the duck is thought not to show an active
hyperemia, or "neurogenic breakthrough," since no ascending vasodilation
was seen in constant-flow hind-limb perfusion studies during neurogenic
vasoconstriction. The chicken may behave more like mammalian species
than the duck, though this his not been confirmed by experimental evidence.

The slapes of the HCT and Hb curves and the magnitude of the SBV

86

(both indicators of the rate of fluid transfer into the vascular system)
were not affected by PBZ pretreatment in hemorrhaged chickens. Pheno-
xybenzamine reduced the rate of post-hemorrhage fluid influx in the
duck. It is tempting to speculauethat the difference between the chicken
and duck in the rate of fluid mobilization under the influence of P82 is
somehow related to the ability of the duck to tolerate a more severe
hemorrhage. Before such speculations may be made, it must be realized
that many intrinsic physiological differences exist between the duck
and chicken beside the vascular dissimilarities.

The duck has a much higher HCT and Hb, and an oxygen dissociation
curve shifted considerably to the left of that of the chicken. The
duck also has a higher degree of oxygen saturation in the arterial blood
than the chicken, which has a high degree of unsaturation. The adult
duck also has a higher level of adenosine triphosphate (ATP) in its
erythrocytes than the chicken,about 12 versus 3 umoles ATP/m1 of ery-
throcytes, respectively. The duck has all red muscle and a high myo-
globin concentration. Vagotomy produced a 65 to 200 percent increase in
HR in the duck, and only a 20 to 40 percent increase in the chicken, in-
dicating that parasympathetic tone is more intense in the duck. Intra-
cardiac glucagon (mammalian) caused an intermediate to severe hyper-
glycemia in the mammal or bird. However, the concomitant increase in
plasma insulin seen in man, dog, and duck did not occur in the chicken.

The submerged duck maintained some degree of skeletal muscle blood
flow, though primarily through non-nutritional vessels. This enhanced
the utilization of venous oxygen stores, and the cortical concentration
of reduced nicotinamide-adenine dinucleotide (NADH) increased only

slightly after a minute dive (Jones and West, 1978). The chicken

87

tolerated a minute of submergence poorly by showing CVS difficulties
(Bond, 1961) and greatly increased cOrtical NADH concentrations. If

such large differences exist in the diving responses of these two species,
it is not unreasonable to suspect large differences may also exist in
their response to hemorrhage. ’

The increased potassium in hemorrhaged mammals is likely the result
of a passive movement of potassium ions out of the skeletal muscle cells
with the bicarbonate anions (HC05) in response to the intracellular
accumulation of hydrogen ions. This hyperkalemia may be one of the pri-
mary initiating factors in reactive hyperemia in the mammal. A hyper-
kalemia-induced vasodilation in mammals may be involved in vascular de-
compensation in shock.

The hyperkalemia seen in the hens in this research was of a magni-
tude similar to that of mammals late in decompensation. Bond et 91, (1977)
reported plasma potassium in the hemorrhaged dog rose from 4 to 6 mEq/L
(a 50 percent increase). The potassium in hens increased 20 to 55
percent (Table 8 and Fgures 6A and 68) during the duration of the experi-
ment. Potassium levels of a 6 mEq/L in the mammal (but not exceeding 12
mEq/L) are vasodilator (Haddy and Scott, 1975), but will not cause nearly
the vasodilation seen during exercise hyperemia. Therefore, the hyper-
kalemia probably plays only a small role in initiating vascular decom-
pensation in late shock in the mammal. Nothing is known about the effect
of eletrolyte alterations in the chicken during exercise, or active, hy-
peremia, or even if such forms of hyperemia occur.

The hydrogen ion concentration decreased while the extracellular
potassium ion concentration increased in these hens. This is contrary

to the popular opinion that the hyperkalemia is the result of the passive

88

movement of potassium ions out of the cell in exchange for accumulating
hydrogen ions. It may be that the hen possesses a more dynamic buffering
system than the mammal, even though Morgan and Chichester (1935) con-
cluded that the buffering capacity of avian blood was about the same as
that of man. Inhibition of the Na-K electrogenic pump could be another
explanation for the hyperkalemia. The high heart rate also may have
contributed to the hyperkalemia.

The production of carbonate for the eggshell results in an excess
of hydrogen ions and reduces the blood pH. Since shell formation takes
20 out of the total 26 hours required for egg formation as a whole, and
an adult hen will lay 250 eggs in a year, it can be appreciated that for
half of its life the hen is faced with an incipient acidemia. Renal
and pulmonary mechanisms play a major role in the preservation of the
neutrality of the blood. Urinary excretion of photons in birds is by
way of dihydrogen phosphate (as in mammals) and by the production of
ammonia. Both of these mechanisms respond to experimentally-induced
acidemia by hydrochloric acid infusion. Metabolic acidosis can also be
compensated for by hyperventilation.

Alpha-adrenergic receptor stimulation inhibits insulin secretion
(Akerblom et_gl,, 1969). Insulin inhibits the efflux of potassium ions
from both the liver (Mortimore, 1961) and the skeletal muscles (Zierler,
1959). Hence, PBZ may reduce plasma potassium levels. Alpha-adrenergic
blockade can also increase pancreatic perfusion, which could lead to
increased insulin release and plasma insulin concentrations, thereby de-
creasing the magnitude of a hyperkalemia. Alpha-adrenergic blockade can
also decrease plasma corticosteroid concentrations (Edens, 1974) and,

therefore, could decrease the extrusion of intracellular potassium

89

(Clauss and Ray, 1968). These may be the reasons why the increase in
plasma potassium in the P82 + hemorrhage group was of a lesser magnitude
than seen in the other hemorrhaged groups (Figure 68).

The hypoglycemia in mammals in late hemorrhagic and endotoxin shock
is due to decreased glucose production by the liver and kidney, increased
peripheral utilization of glucose, decreased pancreatic blood flow, and
increased insensitivity to insulin. These all result in decreased intra-
cellular glucose and cell death.

The injection of enough glucose to cause a significant hyper-
glycemia in the chicken doubled or tripled insulin levels within thirty
minutes. The hyperglycemia from one catecholamine injection did not
affect insulin release (Langslow and Hales, 1971). Catecholamine in-
jections caused hyperglycemia in both the fowl and mammal, yet while
the plasma FFA concentration fell in the mammal, it did not change in
the chicken. There is generally an inverse relationship between glu-
cose and FFA concentrations in the mammal, whereas the FFA concentration
stays constant in the chicken. Massive doses of mammalian insulin did
not affect plasma FFA concentrations, and resulted in only a slight hypo-
glycemia in the hen. This difference is not due to pancreatic glucogon
since results following pancreatectomy are the same. The avian in-
sensitivity to exogenous insulin is well documented.

The avian liver has a well developed insulin-destroying mechanism.
Differences in mammalian and avian nervous systems may also contribute
to the avian insensitivity to insulin. Some researchers have observed
that avian hepatic and skeletal muscle glycogen stores are decreased
by repeated administration of insulin. These results are the opposite

of what occurs in mammals, and can also be produced in the fowl by the

90

continuous administration of catecholamines. Thus, insulin-induced
hypoglycemia in the hen may encourage the adrenal-medullary release of
glycogenolytic catecholamines, which may favor the release of glucose
into the blood stream. Likewise, the massive adrenal-medullary dis-
charge during hemorrhagic shock causes large and continous increases
in plasma glucose (Figures 7A and 7B).

Beta-adrenergic blockade with PROP has been shown to depress the
insulin increase in response to elevated glucose and to prevent the
catecholamine-stimulated lipolysis and FFA release 1__vivo and ig_!jt!g_

(Akerblom et 21,, 1969). In glucose-loaded rats, the insulin/blood

 

glucose ratio, an indicator of the response of the pancreatic islet

cells to glucose stimulation, increased threefold in controls and did

not change in PROP-treated rats. Therefore, beta-adrenergic receptors
must be intact for complete insulin response to hyperglycemia to be
elicited in the mammal. Though Figure 78 appears to show that the PROP +
hemorrhage group had high glucose levels, one-way ANOVA showed that this
was the only hemorrhaged group failing to show a significant hypergly-
cemia (Table 8). The two PROP + hemorrhage birds which survived the
experiment had the largest increases in plasma glucose for that group.
This is because the birds, dying early in the procedure due to the cardio-
depressive effects of beta-adrenergic blockade, did not have time to
become hyperglycemic.

It cannot be ascertained if the elevated glucose levels were the
result of hypoinsulinemia or decreased reactivity to insulin. It is clear
that no trend toward hypoglycemia existed in the hemorrhaged chickens as
it did in the mammal (Strawitz et_gl,, 1961).

It is tempting to speculate that hyperglycemia in the fowl raised the

91

plasma osmolarity enough to provide a considerable osmotic gradient for
fluid influx from the interstitial and intracellular spaces. As mentioned
previously, this gradient, coupled with a reduced Pc, could eventually
reach an equilibrium with the decreased plasma oncotic pressure. The

rate of fluid influx would then decrease, or even reverse. Wyse and
Nickerson (1971) reported a nonsignificant tendency for the plasma pro-
tein and HCT to increase in the terminal stages of hemorrhagic hypoten-
sion in their hens, though this was not seen during research for this
thesis.

The lower degree of hyperglycemia in the PBZ + hemorrhage group
(Figure 78) than the hemorrhage group could be the result of the drugs
insulin-elevating effects, as discussed previously. This is supported
by the fact that PBZ has been shown to increase the plasma FFA concen-
tration during shock in mammals (Kashyap et_gl,, 1975). Alpha-adrenergic
blockade has also decreased the cortisone level in the chicken, which
could also reduce the plasma glucose (Edens, 1974).

The normal value for the oxygen (02) combined with Hb for the chicken
is about 12 ml 02 per 100 m1 of blood (or 12 vol%) at a Nb of 10 grams
per 100 ml of blood (10 gm%), pCOz of 40 mm Hg, p02 of 95 mm Hg, if it
is assumed that one gram of Hb will combine with 1.34 ml of 02 (as in
mammals). The 02 combined with Hb value below which electrocortical
alterations begin to occur in the hen is about 8.0 vol%. This value was
obtained by using the lower critical p02 of 70 mm Hg (Richards and
Sykes, 1967) and the O2 dissociation curve of Choidi and Terrman (1965).
If, as in this research, the Hb was reduced by 50 percent, the pCOz was
20 mm Hg due to hyperventilation (shifting the 02 dissociation curve far
left), and the p02 was 90 mm Hg, then the 02 combined with Hb value would

92

be about 6 vol%. This is considerably below the lower limit for normal
electrocortical behavior. In fact, a reduction of Hb by more than 30
percent, while the pCO2 was below 20 mm Hg, could cause cortical dys-
function. If this is truly the case, the birds in this research began
showing electrocortical alterations for the last one and one-half hours
of the experiment (Table 7).

Although this cerebral ischemia may be a contributing factor in
the CVS collapse (or possible peripheral vascular failure) in the fowl,
it is not the primary cause of death. This was proven by Wyse and Nicker-
son (1971), who reinfused all the resuspended shed erythrocytes in
their hemorrhaged hens after two hours of hypotension and found the sur-
vival times were not changed. Therefore, although hypoxia caused a large
fall in TPR (Besche and Kadono, 1978) and the birds in this research
were quite hypoxic, this does not appear to be the ultimate cause of death.

Recent research has indicated that, following hemorrhage in the hen
the intraerythrocytic concentration of l,3,4,5,6 -- myoinsitol pento-
phosphate increased, which shifted the 02 dissociation curve far to the
right and enhanced 02 delivery to the tissues (Jones et_gl,, 1978).

This is different from mammals, where the main controller of O2 trans-
port is increased intracellular concentrations of 2,3 -- diphospho-
glycerate. These researchers also found the rate of erythrogenesis
following hemorrhage in the hen was comparable to that of the dog.

Many other factors could be involved, such as direct inhibition of
the vascular smooth muscle by hypoxia, metabolic acidosis, and/or
catecholamine depletion. There may be a decreasing adrenergic effect on
resistance vessels. Plasma prostaglandin E], which increases between 90

and 180 minutes after shock in mammals, and can cause inhibition of

93

adrenergic constriction, may be involved. Cellular swelling and a
catecholamine refractoriness due to low pH are other possibilities. MDF,
histamine, and/or bradykinin may also be involved.

Further studies involving local blood flow regulation in the gut
and skeletal muscles of the chicken should be conducted. Capillary
filtration coefficients (CFC) and other perfusion studies should be per-
formed on the hen to determine the effects of hypoxia, histamine,
prostaglandin, adenosine, electrolytes, and bradykinin on local blood
flow. The duration and magnitude of active and reactive hyperemias in
the chicken also needs to be determined. Studies elucidating the pro-
cess of local blood flow regulation in the chicken would not only benefit
shock research, but expand current understanding of poultry physiology

as well.

 

SUMMARY AND CONCLUSIONS

Propranolol significantly reduced heart rate, arterial blood pressure,
cardiac index, and stroke work, thirty minutes after administration.
There was a tendency for stroke volume and total peripheral resis-
tance to be reduced. Phenoxybenzamine pretreatment did not change

the heart rate, mean arterial blood pressure, cardiac index, stroke
volume, stroke work, or hemoglobin. There was a tendency for total
peripheral resistance to be reduced. Hematocrit was reduced by

nine percent of the initial value by PBZ treatment.

Hemorrhage reduced cardiac index, stroke volume, stroke work, and
total peripheral resistance. Heart rate increased in all the
hemorrhaged groups except for the PROP + hemorrhage group, where
heart rate remained subnormal throughout the experiment. Bleeding
volumes were not affected by alpha- or beta-adrenergic blockade.
Survival time was lower in the PROP + hemorrhage group and mortality
was higher (67%). Mortality for both of the other hemorrhaged

groups was 33%.

Hemorrhage caused the hematocrit and hemoglobin to fall progressively
throughout the duration of the experiment. Mammals do the opposite

in late shock. Plasma potassium and plasma glucose concentrations in-
creased progressively throughout the duration of the experiment in

the hemorrhaged groups. Small increases also occurred in the control
group. Mammals demonstrate severe hypoglycemia after hemorrhage.
Plasma magnesium and plasma sodium Concentrations did not change in

the hemorrhaged groups.

94

95

Hemorrhage caused progressive reductions in the arterial p002, and

a concomitant increase in the arterial pH. The arterial p02 was not
changed by hemorrhage. One death occurred in each of the hemorrhaged
groups due to respiratory arrest, except for the PROP + hemorrhage
group where all deaths appeared to be of cardiovascular causes.

The birds in this latter group experienced no respiratory difficulties,
whereas each of the other hemorrhaged groups required artificial venti-

lation in at least one instance to keep a bird alive.

96
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106
APPENDIX A
PROCEDURE FOR PLASMA ELECTROLYTE DETERMINATIONS

Sodium Concentration: Fifty ul of plasma was added to lo ml of a 90
ppm lithium internal standard diluent. Analysis by automatic
feed flame photometer.

Potassium Concentration: Fifty ul of plasma was added to lo ml of
a 90 ppm lithium internal standard diluent. Analysis by auto-

matic flame photometer.

Magnesium Concentration: 0.2 ml of plasma was added to 3.8 ml of

 

lathanum oxide diluent. Analysis by automatic feed atomic

absorption spectrophotometer.

107
APPENDIX B

SAMPLE CALCULATION OF CARDIAC OUTPUT, CARDIAC INDEX,
AND TOTAL PERIPHERAL RESISTANCE

Body weight = l.7 kg
Dye Injected = 0.2 mg
Area under curve = 6.5 in2

Calibration - 10 mg/l. = 6 in.defl.

Chart speed l2 in./minute
MABP = 100 mm Hg

Cardiac Output =

 

.2 mg
6.5 1.n2(‘|0mgfl.élgninJmin.)

0.2‘mg
.903 mg min/l

0.222 l./min. = 222 ml/min

Cardiac Index =
DYE 222 mllmin.
CONC. 11773 kg
= l50.5 ml/min./kg°73“
Total Peripheral Resistance =
100 mm Hg

150.4 ml/min./kg73#

= 0.66 arbitrary units

 

 

TIME

 

 

108

APPENDIX C
STATISTICAL ANALYSIS':
1. Split-plot repeated-measure ANOVA

 

 

 

 

Source of Variation d.f. SS MS
i = Treatments 3 SSt MSt
El = i/j = Birds/treatment' 20 5551 M55]
k = Sampling times 2 , SSS MSs
Treatment X Sample 6 SSts "Sts E"
E2 = Birds X Sample 40 SSEZ MSEZ :
Total SSy
F-testing: MSt/MSE] vs. fa,3,20 ---------- treatments .
l.
MSSIMSEZ vs. fa,2,40 ---------- samples
”Sts/MSEZ vs. fa,6,40 ---------- treatment-sample
interaction
ssy = zzzyZijk - jgzzyijk)2/72 C.F. = Correction Factor
h—‘C-F- yin = trt. total of l8
SSt = zyzi../l8 - C.F. observations
y... = bird total of 3
ssE1 = zzyZij./3 - zin../18 ‘3 observations
y..k = sampling time to-
SSS = zyz..k/24 - C.F. tal of 24 observa-
tions
SStS = 22y21.k/6 -(C.F. + SSt + $55) Yi-k = trt. total at one

time (6 obs.)
SSEZ = SSy ‘ (SSt + SSE] + SSS + SStS)

Treatment comparisons: (Averaged over sampling times) using Dunnett's
test with group #2 as the 'control.‘

t] = y] - {92/ /2MSE1/18}
t3 = 93 ' {92/ /2MSE1/18} 311 V5. tdga,3,20

109
APPENDIX C (continued)

Sampling time comparisons: (averaged over treatment) using ortho-

gonal polynomials (L, 0).

sample times

 

; I45 minutes qL Y3 - y]

3 I45 minutes

4 death qQ = 93 + 9] - 292

FL = qE/ZMSE2/24 vs. fa,l,40 ---------- average trend
FQ = q5/6MSEZ/6 vs. fa,l,40 ---------- curvilinear

2. Block design repeated measure 2-way ANOVA

SS.y

(ssi
sst

ssE

Dunnetts

 

t

Sources of variation d.f. SS MS
Individuals 4 $51

Sampling times 2 SSt

Error 8 SSE MSE

zzyzij - (ZZyij)2/l8

\‘---C F Yi- = total on 1 indiv.
2 ' ' (3 observations)
zyi ./3 ' C.F.

zygj/6 - C.F. y.j = total at l time
(6 observations)

ssy - ssi - sst

est: 0 vs. 90: tD '§.1 - 9.2/ JensE/s

0 vs. 180: to y.1 - y.2/ J2MSE/6

both vs. tD,a/2,2,10

 

"IIIT'IIIIINIINIT)“