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THE EFFECTS OF VAGAL STIMULATION ON THE PULMONARY.
CIRCULATION OF THE DOG, RABBIT, AND CAT AS SEEN
IN 35 mm FILM MOUNTED SERIAL SECTIONS

BY‘

HAROLD FRANKLIN ROTH

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Anatomy
1972

(1, ACKNOWLEDGEMENTS

A number of people have contributed to this study
by their encouragement, suggestions, and specialized
knowledge. To all who helped in any way I wish to express
my sincere gratitude and appreciation.

Dr. Robert Echt was especially patient and maintained
his interest and encouragement as advisor and chairman of
my masters committee. The other committee members,
Dr. Clifford W. Welsch, and Dr. Thomas W. Jenkins gave
valued advise, suggestions and guidance.

Valuable assistance was rendered by Mr. Robert
Paulson whose photographic professionalism aided the author
throughOut his program.

Special thanks is due to doctoral candidate
David O. DeFouw who was especially helpful and patient in
his assistance throughout the research program.

An expression of immeasurable gratitude is given to
the author's wife, Sherrie, for her continuous encouragement,

assistance, and sacrifices.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 4
Effects of Vagal Stimulation on Pulmonary
Vasomotion . . . . . . . . . . . . . . . . . . . 4
Isolated Perfused Technique . . . . . . . . . . 4
In §i33_Techniques . . . . . . . . . . . . . . . 9
Transillumination of Pulmonary Tissue . . . . 9
Thoracic Windows . . . . . . . . . . . . . . 12
In_§itu Pressure Recordings . . . . . . . . . 16
Isolated Pulmonary Vessels . . . . . . . . . . . l9
Subgross Pulmonary Anatomy . . . . . . . . . . . . 20
Anatomy of The Pulmonary Circulation. . . . . . . . 21
Pulmonary Arterial Supply . . . . . . . . . . ., 21
Pulmonary Capillary Network . . . . . . . . . . 25
Pulmonary Venous Return . . . . . . . . . . . . 27
Vagal Innervation of Pulmonary Airways and
Blood Vessels . . . . . . . . . . . . . . . . . 29
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 33
Experimental Dogs . . . . . . . . . . . . . . . . . 34
Histologic Preparation . . . . . . . . . . . . . 38
Microsc0pic Evaluation . . . . . . . . . . . . . 44
Stereologic Measurements . . . . . . . . . . . . 46

iii

Page

Volume Measurements . . . . . . . . . . . . . 46

Large Vessel Volume . . . . . . . . . . . 47

Capillary Ink Volume . . . . . . . . . . . 48

Airway Volume . . . . . . . . . . . . . . 49

Absolute Volume . . . . . . . . . . . . . . . 50

Vessel Length . . . . . . . . . . . . . . I 51

Statistical Analysis . . . . . . . . . . . . . . 53

Control Dog . . . . . . . . . .I. . . . . . . . . . 54

Experimental Rabbits . . . . . . . . . . . . . . . 54

Control Rabbits . . . . . . . . . . . . . . . . . . 56

Experimental Cats . . . . . . . . . . . . . . . . . 56

Control Cats . . . . . . . . . . . . . . . . . . . 57

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 64

Perfusion-Percentage Results . . . . . . . . . . . 64

Photographic Results . . . . . . . . . . . . . . . 64

Stereologic and Volumetric Results . . . . . . . . 64

Rabbit . . . . . . . . . . . . . . . . . . . . . . 84

Cat . . . . . . . . . . . . . . . . . . . . . . . . 86

Evaluation of The Experimental Technique . . . . . 88

SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 91

LITERATURE CITED . . . . . . . . . . . . . . . . . . . 94
APPENDIX A

Original data collected from the rabbit left
middle lobe . . . . . . . . . . . . . . . . . . 101

APPENDIX B

Original data from lobar volumetric analyses and
stereologic data measurements . . . . . . . . . 120

iv

Page
APPENDIX C

Analysis of covariance tables . . . . . . . . . . . 138

10.

11.

12.

LIST OF TABLES

Lobar perfusion-percentages for the
experimental dogs . . . . . . . . . . . . .

Lobar perfusion-percentages for the
experimental rabbits . . . . . . . . . . .

Lobar perfusion-percentages for the
experimental cats . . . . . . . . . . . . .

Lobar perfusion-percentages for the control
animals 0 O O O O O O O O O C O O O O O O 0

Absolute lobar volumes and total vessel
lengths for experimental and control
animals 0 O O O O O O O O O O O O O O O 0

Relative volume occupied by pulmonary vessels
(larger than capillaries) from the
stimulated rabbit left middle lobe. . . . .

Relative volume occupied by ink filled
pulmonary capillaries from the stimulated
rabbit left middle lobe . . . . . . . . . .

Length per unit volume of pulmonary vessels
larger than capillaries from the
stimulated rabbit left middle lobe. . . . .

Relative volume occupied by pulmonary airways
(larger than respiratory bronchioles) from
the rabbit left middle lobe . . . . . . . .

Original stereologic volume and length data
at varied section intervals of the
stimulated rabbit left middle lobe. . . . .

Original data from the volumetric water
displacement analyses of the experimental
dog middle lobes . . . . . . . . . . . . .

Original data from the volumetric water
displacement analyses of the experimental
rabbit middle lobes . . . . . . . . . . . .

vi

Page

66

67

68

71

75

102

106

110

114

119

122

123

Table

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Page

Original data from the volumetric water
displacement analyses of the experimental
cat middle lobes . . . . . . . . . . . . . . 124

Original data from the volumetric water
displacement analyses of the control
animals . . . . . . . . . . . . . . . . . . 125

Original data indicating the relative
volumes and vessel lengths in the middle
lobes of the experimental dog. . . . . . . . 126

Original data indicating the relative
volumes and vessel lengths in the middle
lobes of the control dog . . . . . . . . . . 127

Original data indicating the relative
volumes and vessel lengths in the middle
lobes of the experimental rabbit . . . . . . 128

Original data indicating the relative
volumes and vessel lengths in the middle
lobes of the control rabbit. . . . . . . . . 129

Original data indicating the relative
volumes and vessel lengths in the middle
lobes of the experimental cat. . . . . . . . 130

Original data indicating the relative
volumes and vessel lengths in the middle
lobes of the control cat . . . . . . . . . . 131

Original data indicating the absolute lobar
volumes and lobar vessel lengths of the
experimental dog . . . . . . . . . . . . . . 132

Original data indicating the absolute lobar
volumes and lobar vessel lengths of the
control dog. . . . . . . . . . . . . . . . . 133

Original data indicating the absolute lobar
volumes and lobar vessel lengths of the’
experimental rabbit . . . . . . . . . . . . 134

Original data indicating the absolute lobar
volumes and lobar vessel lengths of the
control rabbit . . . . . . . . . . . . . . . 135

Original data indicating the absolute lobar

volumes and lobar vessel lengths of the
experimental cat . . . . . . . . . . . . . . 136

vii

Table Page
26. Original data indicating the absolute lobar
volumes and lobar vessel lengths of the
control cat . . . . . . . . . . . . . . . . . 137
27. Analysis of covariance data for the dog . . . . 138

28. Analysis of covariance data for the rabbit . . . 140

29. Analysis of covariance data for the cat. . . . . 142

vii

10.

11.

12.

13.

14.

15.

16.

LIST OF FIGURES

Formalin pump apparatus . . . . . . . . . .
Microtome with tissue strip . . . . . . . .
Film transport device with Kinderman guide

and developing reel . . . . . . . . . .

'Tissue being placed on film strip . . . . .

Tissue loaded reel with staining dishes . .
Film holder with film strip . . . . . . . .
Modified Weibel stereologic viewing plate .
Assembled microscopic equipment . . . . . .
Viewing screen with Weibel plate and ocular
micrometer superimposed over the tissue
sample at 100x magnification . . . . . .

Tissue sample divided into unit areas . . .

Average lobar perfusion-percentages for the
experimental dog . . . . . . . . . . . .

Average lobar perfusion-percentages for the
experimental rabbit . . . . . . . . . .

Average lobar perfusion—percentages for the
experimental cat . . . . . . . . . . . .

Photographic analysis of experimental
dog lung . . . . . . . . . . . . . . . .

Photographic analysis of experimental
rabbit lung . . . . . . . . . . . . . .

Photographic analysis of experimental
cat lung . . . . . . . . . . . . . . . .

ix

Page
59

‘59

59
61
61
61
61

63
63
63
70
7O
7O
72
73

74

INTRODUCTION

A primary clinical manifestation of many respiratory
disorders is an abnormality in pulmonary vascular perfusion.
Knowledge of the factors controlling this hypo-or hyper-
perfused state would give the clinician a solid base on which
sound corrective therapy could be built. Unfortunately, this
controlling mechanism is an elusive one, and to date no
single group of regulatory factors has been universally
accepted.

The role of the autonomic nervous system in
regulating the pulmonary circulation has been intensively
studied for many years. The results of these investigations
have often been contradictatory and inconclusive. Two camps
of thought have been established. One, lead by Daly and
Hebb (1966a), sought to isolate the lung and perfuse it
with a regulatory pump. They measured neural effects by
monitoring changes in pressure and flow rate. Elimination
of the heart confined the observed response to the pulmonary
circulation but left a rather poor physiologic preparation.

The other camp measured pressure and flow changes
in intact animals. Their preparations were more physiologic

but left some speculation as to whether the neural effects

2
acted directly on the pulmonary vascular system or were
merely a passive reaction to changes in cardiac output.

Krahl (1968), an advocate of ifl.§i§3 techniques, has
developed a method whereby Pelikan ink can be injected into
the pulmonary vascular system. He concluded that mid-
cervical stimulation of the distal end of either severed
vagus nerve followed by ink injection would reveal the
effects of that stimulation on pulmonary vascular perfusion.

The present study is a combination of the ifl.§i§2
techniques deve10ped by Krahl and a much more expanded
histologic evaluation.. Its goal is to further define the
parasympathetic nervous system's role in regulating the
pulmonary circulation.

Electric mid-cervical stimulation of the distal end
of the severed left vagus nerve of dogs, rabbits, and cats
was followed by ink injection and in situ formalin fixation.
The middle lobes from both stimulated (left) and control
(right) lungs were evaluated grossly for the degree of
peripheral ink perfusion, and histologically by serial
sectioning from the hilus to the periphery. Histologic
sections were mounted on 35 mm leader film utilizing a
modification of a technique developed by Wilson and Pickett
(1970).

With the aid of an ocular micrometer and the Weibel,
Kistler, and Scherle (1966) method for stereologic measure-
ments, the following were calculated for both the stimulated

and control lobes: (l) the relative volume occupied by the

3
pulmonary arteries, arterioles, veins and venules (2) the
relative volume occupied by the ink filled pulmonary
capillaries (3) the relative volume occupied by the pulmonary
airways ranging in size from secondary bronchi to terminal
bronchioles and (4) the length per unit volume of the
pulmonary arteries, arterioles, veins, and venules.

The average volumes of right and left middle lobes
from three experimental animals of each species were
determined by the water displacement technique. The
calculated relative volumes were then multiplied by the
average lobar volume to determine the absolute volumes of
the pulmonary components being studied.

A representative animal from each species was
studied for control data. The surgical, histologic, and
microscopic evaluation procedures were identical to those
used in the experimental animals. The only difference was
that the control animals did not undergo vagal stimulation.
The control data was used to determine whether the
variations seen in the experimental animals were due to the
vagal stimulation or to the normal variations in lung

architecture.

REVIEW OF LITERATURE

Effects of Vagal Stimulation
on Pulmonary Vasomotion

 

 

Pulmonary vasomotor studies generally involve
chemical or electrical stimulation on either ifl.§i§2 or
isolated perfused preparations. The inherent disadvantages
of these techniques necessitates a cautious evaluation of
their results. Secondary or masking effects are often seen
in in sign studies where all bodily functions and inter-
relationships must be considered. The lack of this
physioligic setting in dealing with organs separate from the
whole is the cause of concern in isolated perfusion studies.
Fortunately these methods tend to complement each other and
valid conclusions can be drawn when both are viewed in light

of each other's weaknesses.

Isolated Perfused Technique

 

Daly and Hebb (1966b) summarized the technique most
commonly used in the study of isolated perfused pulmonary
tissue. This method employed two mechanical pumps which
fed the bronchial and pulmonary circulations. Only the
lungs and the extrapulmonary course of their nerves were
kept viable throughout the procedure. The two pumps were

fed by a single reservoir of perfusate (animal's own blood).

5

The first pump, replacing the right ventricle, was situated
between the right heart and the main pulmonary artery. Its
perfusate supplied the pulmonary circulation. The second
pump, replacing the left ventricle, was situated at the base
of the aortic arch and perfusion allowed down the thoracic
aorta to T7 or T8' Its perfusate supplied the bronchial
circulation. Blood returning from the pulmonary and
bronchial circulations was drained into the single reservoir
for later recycling. The free end of a cannula was then
inserted into the pulmonary artery and its opposite end
connected to a manometer.

This isolated perfused technique allowed perfusion
of the pulmonary and bronchial circulations at either a
constant volume or a constant pressure. When constant
volume inflow was employed, vasomotor responses to electric
or chemiCal stimuli were assesed according to changes in
the volume outflow by monitoring the volume of blood
draining into the reservoir. A decrease in the volume.
outflow would indicate a possible vasoconstriction, while an
increase in volume outflow would suggest a vasodilation.
When constant pressure was employed, vasomotor responses
to stimuli were assesed according to changes in either the
outflow volume or the pulmonary arterial pressure. An
increase in pressure suggested vasoconstriction and a
decrease in pressure indicated vasodilation.

According to Daly and Hebb (1966c) one of the first

isolated perfused studies of the pulmonary circulation was

6
performed by Cavazzani in 1891. He studied blood flow in
isolated perfused rabbit lungs before and after electric
mid-cervical stimulation of the vagus nerve. His results
suggested the presence of a vagally mediated pulmonary
vasoconstriction.

Hunt (1918) perfused cat and rabbit lungs with a
Ringer's solution containing acetylcholine and observed its
effect on outflow volume. He noted that small doses
(quantity not stated) of acetylcholine did not alter volume
outflow. Larger doses added to the Ringer's solution
produced a prolonged decrease in outflow volume which was
not affected by comparably large doses of atropine.

Von Euler (1932) electrically stimulated (primary
current of approximately 3 volts) the mid-cervical intact
vagus nerve on an isolated perfused preparation of rabbit
lung. Fifteen to thirty seconds of vagal stimulation
resulted in a rise in pulmonary arterial pressure, an
effect that was abolished by injection of atropine (5 mg).
Injection of 0.5 mg of acetylcholine into the pulmonary
artery also resulted in a significant rise in pulmonary
arterial pressure accompanied by a diminished outflow
volume. He concluded that the vagus nerve was at least
partially responsible for vaSOpressor activity in the
pulmonary circulation of the rabbit.

Daly and von Euler (1932), studying isolated perfused
canine lungs, electrically stimulated (current from a DuBois-

Reymond induction coil, coil distance = 7 cm) the vagus

7
nerve a distance of 6—10 cm above the inferior cervical
ganglion. Vasomotor responses to electric stimuli were
assessed according to changes in pulmonary arterial
pressure. Simultaneous stimulation of both the left and
right intact vagus nerves on three isolated perfused prepa-
rations resulted in an increase in pulmonary arterial
pressure (vasoconstriction) in two animals and a slight
decrease in pulmonary arterial pressure (vasodilation) in one
animal. In similar preparations, a vasodilation response
was observed after injection of 1.0 mg of acetylcholine into
the pulmonary artery of five experimental animals. One
preparation demonstrated a pulmonary vasoconstrictor
response which was abolished after injection of atrOpine.

Gaddam and Holtz (1933) studied the effects of
acetylcholine on isolated perfused dog and cat lungs. The
responses, seen as changes in perfusion pressure, were dual
in nature.‘ In both the dog and the cat small doses of
acetylcholine (1—10 ug) produced vasodilation while larger
doses (over 20 ug) resulted in vasoconstriction.

Alcock, Berry, and Daly (1935) observed isolated
canine lungs perfused with defibrinated blood and placed
under negative pressure ventilation. Injection of
acetylcholine (200 ug) resulted in a slight fall in pulmonary
arterial pressure accompanied by inconsistent changes in
pulmonary outflow. Larger doses (1.0 mg) produced a rise
in pulmonary arterial pressure accompanied by a slight

increase in outflow.

8

Borst, Berglund, and McGregor (1957) studied the
effect of acetylcholine injection on isolated perfused
canine lungs. Pressures in the left and right pulmonary
arteries were measured by electromanometers and continuously
recorded on an ocillograph. Responses to chemical stimuli
were assessed according to changes in pulmonary arterial
pressure. The lungs were ventilated with 100% oxygen and
'allowed to collapse immediately prior to the drug injection.
Acetylcholine (3-9 ug per kg of body weight) was injected
into the pulmonary artery of the non—ventilated lungs of
six animals. Two animals exhibited vasoconstriction,
another two vasodilation, one demonstrated vasoconstriction
followed by prolonged vasodilation, and one animal gave no
response. All responses were of a small magnitude. Borst
33 31. concluded that the lack of consistent pulmonary
vasodilation after injection of acetylcholine may be due
to the fact that the tone of the pulmonary vascular bed was
low before the drug was injected.

Daly and Hebb (1966d) summarized their observations
on the responses of isolated perfused canine lungs to
injections of acetylcholine. They found that injection of
10—100 ug of acetylcholine resulted in pulmonary vascular
dilation seen as a fall in pulmonary arterial pressure.
Injection of acetylcholine in units ranging from 5-1000 ug
resulted in pulmonary vascular constriction seen as an

increase in pulmonary arterial pressure.

9
Hague, Lunde, and Waaler (1966) infused acetylcholine
into the pulmonary artery of isolated perfused rabbit lungs.
A marked increase in pulmonary arterial pressure was
observed after injection of acetylcholine in doses down to
5.19 ug. They considered that this increase in pressure was
due to a marked pulmonary vasoconstrictor response to

acetylcholine injection.

In Situ Techniques

 

The majority of in_§itu studies employ one or a
combination of three possible techniques; transillumination
of pulmonary tissue, thoracic windows, and pulmonary
arterial or venous pressure recordings. Common to all is
the requirement that the animal's lungs, cardiovascular
system, peripheral, and central nervous systems be kept
intact. Preparations may involve either open or closed
chested surgery.

Transillumination of Pulmonary
Tissue

 

According to Wagner and Filley (1965) attempts to
observe alveolar function date back to 1661 when Malphigi,
using the Open thorax technique, transilluminated frOg lungs
with candle light. More than two centuries passed before
Hall (1925) expanded the Malphigian technique in his studies
on the rabbit and cat. After light anesthesia the animals
were pithed and their lungs aerated by a modification of
the Meltzer's (1909) method of intratracheal inflation.

The necessary portions of the rib cage were removed and the

10

inflated lobe elevated and its margins held in place by
clips. The marginal areas of the lobe were transilluminated
and the pattern of blood flow observed. Hall noted that
flow in the larger arterioles and veins was pulsatory while
flow in the smaller arterioles, venules, and capillary
networks was continuous. He then painted the heart with
nicotine to eliminate any changes in cardiac rate during
subsequent vagal stimulation. Electric vagal stimulation,
which on non-nicotine treated animals caused a slowing in
heart rate, had no effect on cardiac rate, the caliber of
any pulmonary vessel being observed or the velocity of blood
flow within these vessels.

Melvin Knisely (1934, 1938, 1967) has developed and
improved the technique of studying living tissue through
the use of fused quartz rod transillumination. William
Knisely (1969), in conjunction with the work of Irwin (1958),
applied the technique of quartz rod transillumination in his
studies on pulmonary tissue. His experimental procedures
were as follows: After surgical anesthesia and tracheal
cannulation a thoracotomy was performed allowing the edge
of a lobe to be visualized. An oxygen catheter (diameter
less than that of the cannula) was inserted into the
traCheal cannula and the flow of oxygen regulated so that
the lungs remained constantly inflated. Gases were allowed
to escape around the inner edges of the tracheal cannula.
The tip of a fused quartz rod was then placed under the edge

of the lobe in question. A beam of light falling upon the

11
base of a clean, smooth, polished quartz rod is reflected
inside the rod until the majority of it reaches the opposite
end. Knisely has likened this property to the way a hose
conducts water. The rod is broken near its light source to
prevent heat from reaching the tissue by conduction or
convection. To protect the tissue from heat delivered by
the transformation of radiant energy a flowing wash
solution of Ringer's lactate was applied to the exposed
tissue by means of two glass tubes. The first tube brought
the wash solution to the tip of the quartz rod so that the
tissue being placed above it would never come into direct
contact with the quartz. The second wash was delivered
to the upper surface of the tissue. All microscopic
observations were made from this upper surface. William
Knisely has utilized this technique to further advance the
anatomical knowledge of the pulmonary circulation. The
results of his observations will be included in the review
of the pulmonary anatomy.

Irwin and Burrage (1954) utilizing fused quartz rod
transillumination of the rabbit lung observed that inter-
mittent linear blood flow occurred in the pulmonary
arterioles, capillaries, and venules. This flow was
observed in vessels up to 160 u in diameter.

Wilson (1970) suggested replacing the fused quartz
rod with a rod made of borosilicate glass. Borosilicate,
while having similar light transmission prOperties, is
cheaper and conducts less heat than quartz and may be

pigmented to increase photographic contrast.

12

Thoracic Windows

 

In 1934 Wearn, Ernstene, Bromer, Barr, German, and
Zachiesche developed a technique of directly observing the
smaller superficial blood vessels and air sacs in the closed
thorax of the cat. A circular layer of cOstal parietal
pleura (0.6-1.2 cm in diameter) was exposed by stripping
away the skin and muscle in the mid-axillary line between
the eighth and ninth ribs. A similar window was made
directly Opposite the first by dissecting the muscle away
from the abdominal surface of the diaphragm, exposing the
disphragmatic parietal pleura. Pulmonary tissue was
transilluminated through this diaphragmatic window with
light from a fused quartz rod. All microscopic observations
were made through the chest window.

Wearn gt a1. noted intermittent blood flow through
pulmonary arterioles less than 100 u in diameter. They
also found that the velocity of blood flow increased after
atropine injection into the femoral vein.

I Terry (1939) develOped the technique of implanting
a permanent artificial thoracic window. His device,
consisting of a short hollow bronze cylinder one end of
which was covered by a glass plate, was surgically installed
in the right fifth intercostal space of the cat. Using a
binocular microsc0pe and an arc lamp, direct observations
were made on the pulmonary tissue.

Krahl (1962) eliminated the irritating reactions of

direct contact of metal and glass on the pulmonary visceral

l3
pleura by jacketing a lucite window frame in transparent
teflon. In this case, the transparent teflon served as the
viewing port. By observing rabbit lungs through various
areas of the costal parietal pleura he noted an area on the
right side at the level of the third rib where the movement
of the lungs was at a minimum. Installation of the thoracic
window at this static area required prior surgical amputation
of the right thoracic limb.

When Krahl observed the surface of the right lungs
through the teflon window he noted polygonal areas light
pink in color surrounded by wider areas having a deeper
pink hue. He concluded that these lighter areas corre-
sponded to the bases of the pyrimidal units of the lung
referred to as secondary pulmonary lobules. By the‘
transient nature of the lighter pink areas he suggested
that a mechanism possibly exists which regulates pulmonary
blood flow on a lobular basis.

According to Miller (1950), a primary lobule
consists of one alveolar duct and the atria, alveolar sacs,
and alveoli which arise from it. Approximately fifty
primary lobules are grouped together to form a secondary
lobule. This subgross anatomical division is surrounded by
a layer of connective tissue which separates it from
adjacent secondary lobules.

Krahl (1963) injected a mixture of India ink,
saline, and KCl directly into the beating right heart.

Post mortum examination of the lung surfaces revealed a

14
mosaic pattern of ink distribution. He concluded that the
lighter mosaic areas were underperfused and the darker
areas well perfused when functional cardiac pumping ceased.
Subsequent histologic evaluation demonstrated ink perfusion
in both the arterioles and capillaries of the darker areas.
In the lighter areas ink perfusion ceased at the right
angled branching precapillary arterioles leaving the
capillary networks void of ink.

His next step (1965) was to electrically stimulate
the distal end of the mid—cervically severed right vagus
nerve while injecting a mixture of India ink and saline
(1:1) through the external jugular vein. The animals were
terminated by either increasing the current to produce
cardiac arrest, or adding a few crystals of KCl to the
India ink saline mixture. Examination of the surfaces of
the lungs revealed that the vagally stimulated right lung
was considerably lighter than the control left lung.
Histologic sections of the lighter areas of the stimulated
lung demonstrated a constriction of a circular layer of
smooth muscle at the precapillary arteriolar site. This
constriction stopped the flow of ink to the capillary
network. Subsequent histologic evaluation of the black
areas of the control (left) lung revealed that the sphincter
mechanism was relaxed allowing ink to freely enter the
pulmonary capillary networks.

Further experiments (1966) involved intracardiac

injection of acetylcholine (0.01 mg/kg of body weight of

15
a 1:25,000 solution of acetylcholine) just prior to the
injection of ink. The surfaces of these lungs appeared
nearly as pink as the normal non-injected lungs.

From the results of both the electric vagal
stimulations and injections of acetylcholine, Krahl con—
cluded that blood flow to the pulmonary capillary networks
was at least partially regulated by vagally induced
constriction of the precapillary muscular Sphincter.

Krahl (1969) discussed the clinical significance
of this vagally mediated pulmonary vasoconstriction. He
suggested that atropine (parasympathetic blocking agent) be
used in treating infants with respiratory distress syndrome
to counteract the clinically established pulmonary
vasoconstriction. (Chu gt 31., 1965).

Wagner and Filley (1965) implanted a thoracic window
(plexiglass and vinyl) on the right side of the canine lung
at the level of the second and third intercostal spaces.
The thickness of the lung at this static site did not
permit transillumination and a Leitz Ultropack incident
illuminator microscope was used for all observations. They
observed a pulsatory flow of blood through the arterioles
(up to 100 u), capillaries, and venules (up to 200 u).
Vagal stimulation (20 volts/15 cycles per second) resulted
in a significant decrease in the velocity of red blood cells
in all vessels with no change in vessel lumen size. The
stimulation effects were qualitatively evaluated by

observing cinemicrographs taken during the stimulation.

16

In Situ Pressure Recordings

 

Rudolph, Kurland, Auld, and Paul (1959) injected
acetylcholine and observed its effects on the pulmonary
circulation of the dog. Responses were seen as changes in
pulmonary arterial pressure. Injection of acetylcholine
(40—50 mg/kg/min) resulted in a slight rise in pulmonary
arterial pressure. Similar injections of acetylcholine in
pulmonary vessels which were preconstricted by a prior
injection of serotonin (500 ug) resulted in a significant
decrease in pulmonary arterial pressure.

Rudolph gt gl,‘studied the effects of similar
injections of acetylcholine on two open chested preparations
in which left pulmonary venous and pulmonary arterial wedge
pressures were recorded. Drug injection resulted in a mild
increase in pulmonary arterial wedge and pulmonary venous
pressure. Injection of acetylcholine while the pulmonary
arterial pressure was in an elevated state due to continuous
injection of S-hydroxytryptamine creatinine sulfate
(75-100 ug/kg/min) resulted in a decrease in pulmonary wedge
and venous pressures.

Braun and Stern (1967) in their review of Rudolph
gt 213's work (1959) concluded that the response of the
pulmonary venous system to drugs may depend on pre—existing
venous tone. . '

Shimomura, Pierson, Krstulovic, and Bell (1962)
studied the effects of acetylcholine injection (0.5-10 ug)

on the perfusion pressure of a catheter wedged in a small

17
pulmonary artery of the dog. The perfusate (heparinized
autogenous venous blood) was delivered to the wedge segment -
at a constant flow. The catheter was designed to both
perfuse and measure the wedge arterial pressure. A micro—
catheter was inserted to the tip of the wedge catheter to
permit direct injection of acetylcholine. Injection of a
single bolus Of acetylcholine (0.5-1.0 ug) resulted in a
rise in perfusion pressure with no change in cardiac rate,
left atrial or systemic blood pressures. When larger doses
(2-10 ug) were injected, a rise in perfusion pressure was
also noted before the drug was able to decrease systemic
pressure. Shimomura gt a1. concluded that the rise in
pressure seen after injection into the perfused wedged
segment of the canine lung was ample proof for a direct
vasoconstrictor effect of acetylcholine on the pulmonary-
vascular bed of the dog.

Rudolph and Scarpelli (1964) studied the effects of
acetylcholine injection (1-25 ug/min) on the pulmonary
arterial pressure of closed chested dogs. Electromagnetic
flow transducers were placed around the main and left
pulmonary arteries. Catheters were situated in the right
ventricle, the main and left pulmonary arteries. Infusion
of acetylcholine into the right ventricle or main pulmonary
artery resulted in no significant changes in the total
(main pulmonary artery) or left pulmonary artery blood flow.
Similar infusions into the left pulmonary artery resulted

in a significant decrease in left pulmonary flow and no

18
changes in total flow. Rudolph and Scarpelli interpreted
these responses to mean that acetylcholine caused a direct
pulmonary vasoconstriction in the dog.

Harris (1957) studied the effects of acetylcholine
injection (0.25-8.0 mg) on normal and diseased humans. Of
the three normal subjects studied, no significant change
in pulmonary arterial pressure was observed after drug
injection. Of the forty-two patients suffering from
various abnormalities, eighteen demonstrated a fall in
pulmonary arterial pressure, two responded with a rise in
pressure, and twenty-four gave no response. Harris noted
that the decrease in pressure was found most frequently in
patients who's normal pulmonary arterial pressure was
elevated. He concluded that pre-existing vessel tone must
be considered in evaluating the effects of acetylcholine
on the pulmonary circulation.

Soderholm and Werko (1958) injected acetylcholine
(3.0-14.5 mg/min) into the pulmonary artery of thirteen"
patients with mitral valve disease. Drug injection
produced a statistically significant decrease in pulmonary
arterial pressure and systemic oxygen saturation,
accompanied by an increase in cardiac output and no
significant differences in cardiac rate. They concluded
that the decrease in systemic arterial saturation was due to
the dilation of pulmonary arterioles in poorly ventilated

areas of the lung.

l9

Schlants, Tsagaris, Robertson, Winter, and Edwards
(1962) observed the effects of acetylcholine on the human
pulmonary circulation of twenty~one patients suffering from
various abnormalities. Arterial blood gases and pressures
from the brachial and main pulmonary arteries were recorded
throughout the experiment. Cardiac output was calculated by
the Pick principle and minute ventilation was recorded with
Douglas bags. Infusion of acetylcholine into the pulmonary
artery or right ventricular outflow (20-75 ug/kg/min)
resulted in a decrease in mean arterial saturation, an
increase in mean cardiac index, and a decrease in calculated
mean total pulmonary resistance and total systemic
resistance with no significant changes in pulmonary and
brachial arterial pressures.

Schlants gt_al. (1962) reviewed the work of Chidsey
(1960) in which acetylcholine (3 mg/min) was injected into
the right atrium of patients with pulmonary emphysema.
Chidsey observed that systemic arterial saturation decreased
in nine out of thirteen patients. He concluded that
acetylcholine dilated pulmonary vessels constricted by
hypoxia thus increasing the perfusion of poorly ventilated

areas of the lung.

.Isolated Pulmonary Vessels
Franklin (1932) placed rings cut from isolated
pulmonary blood vessels of the dog in Ringer's solution

(370C) containing acetylcholine (up to l:l,000,000). An

20
optical device was used to record changes in ring diameter.
All vessels studied had a circumference larger than 4 mm.
He observed relaxation of the extrapulmonary arteries, no
response from intrapulmonary arteries, mostly contraction
of intrapulmonary veins, and consistant contraction of the
extrapulmonary veins.' He concluded that parasympathetic
stimulation may result in engorgement of the lungs by
dilating the arteries and constricting the veins.

Bohr, Goulet, and Taquini (1961) studied the effects
of acetylcholine on helical strips of rabbit and dog
pulmonary arterial vessels (200-300 u in diameter). The
helical strips were placed in Krebs solution and tension
measured on a Grass displacement transducer. Significant
constriction was observed in the pulmonary vessels of both
the rabbit and the dog when placed in Krebs solution

containing acetylcholine (30—100 ug/L).

Subgross PulmonaryyAnatomy

 

McLaughlin, Tyler, and Canada (1961) injected latex
into the pulmonary arteries, bronchial arteries, pulmonary
veins, and pulmonary airways in their studies on the
subgross anatomy of rabbit, dog, and horse lungs. Basic
anatomical similarities were observed between the dog and
the cat. The subgross anatomy of the horse lung appeared
to be very similar to that Of the human. Their

observations were as follows:

21

Dog and Cat--- Secondary lobules and interlobular septa
were absent. The membranous pleura was
extremely thin, and its blood supplied
via the pulmonary artery. No bronchial
artery-pulmonary artery anastomoses were
observed.

Horse --------- Secondary lobules were incompletely
developed and interlobular septa were
thick. The pleura was thick and its
blood supplied via the bronchial artery.
One bronchial arteriolar-pulmonary
arteriolar anastomosis was observed.

Anatomy Of The Pulmonary Circulation

 

According to Wagner and Filley (1965) anatomical
studies of the pulmonary circulation date back to the
sixteenth century when Malphigi, observing transilluminated
frog lungs, noted that the systems for blood and air were
anatomically independent of each other. Malphigi was the
first researcher to observe and describe pulmonary
capillaries. Subsequent anatomical studies involving more
elaborate equipment have allowed the researcher to examine
all compartments of the pulmonary circulation. This
expanded knowledge has been accompanied by an overlap in
nomenclature. Thus, the researcher has learned to place
more stock in a concise anatomical description of the
vessel rather than relying on a term as misleading as

pulmonary arteriole.

Pulmonary Arterial Supply

 

Brenner (1935) studied the post-mortum anatomy of
the pulmonary circulation of fifteen human subjects.

Through the use of histologic sections, he was able to

22

distinguish the microscopic components of the pulmonary

arterial vessels. Brenner noted that the primary component

of pulmonary arteries larger than 1000 u in diameter was
elastic tissue. He also found that smooth muscle was the
consistant feature in arterial vessels 100-1000 u in
diameter while arterial vessels 33—66 u in diameter were
mainly endothelial.

Ferencz (1969) expanded the work of Brenner (1935)
by using thick serial sections (120-500 u) to study the
pulmonary arterial supply of the human, rabbit, dog, and
cat. She noted that the basic histolOgic structures of
the pulmonary arterial vessels of all four were quite
similar. Her evaluation was summarized as follows:

Elastic arteries (>500 u) Capacitance vessels
consisting mainly of
elastic fibers

Transitional arteries (100-500 u) Resistance vessels having
an incomplete muscular
wall with scattered
elastic fibers

Muscular arteries (30-115 u) Resistance vessels having

a complete smooth muscle
wall covered by an elastic

lamina
Endothelial arteries (15-40 u) Channeling vessels
(arterioles) consisting of an

endothelial lining
supported by a single
elastic lamina
Ferencz concluded that the pulmonary arterial supply
of the dog and cat were quite similar to that of the human

in that the vessels tapered very smoothly showing no abrupt

gross or histologic alterations. The transition from one

23
vessel type to another in the rabbit lacked this uniform
tapering quality. The rabbit also exhibited numerous right
‘angled branching of the endothelial vessels and large
irregular amounts of smooth muscle in the transitional and
muscular arteries.

William Knisely (1960, 1969), utilizing the
principle of fused quartz rod transillumination, observed
the normal morphology of the surface alveoli and the small
pulmonary blood vessels on open chested inflated lungs of
dogs, rabbits, and cats via the still lung technique (Irwin,
1958). Direct observations of the pulmonary vasculature
revealed a unique architectural pattern in which the
arterioles gave off numerous branches and rapidly tapered
ending in rounded, blunt tips. Exposing the pulmonary
arterial vessels to a shower of blood emboli resulted in
the majority of these emboli being trapped in the blunt
ends of the pulmonary arterioles. Knisely postulated that.
these pulmonary arteriolar tips could act as a catch-trap
mechanism which would contain pulmonary blood emboli until
they were disolved. Knisely concluded that surface
alveoli vary in size and shape from one species to another.
A right angled pattern of branching was seen in pulmonary
arterioles up to 100 u in diameter. He also observed that
a single alveolar capillary network may be supplied by more
than one arteriole, and one arteriole may supply many

alveoli.

24

Von Hayek (1960a), in his review of human pulmonary
circulation, stated that precapillary pulmonary vessels
often arise at right angles to their parent artery. The
length of these precapillary vessels appeared to be void
of smooth muscle, but sphincter-like muscular bands were
observed at their right angled origins. Von Hayek considered
that these muscular sphincters played, at least a partial
role, in the regulation of pulmonary circulation.

Reeves, Leathers, and Quigley (1965) injected a
suspension of barium sulfate in gelatin (50 mm Hg pressure)
into the pulmonaryarteries of excised rabbit lungs. The
lungs were fixed by intratracheal inflation of 10 percent
formalin and 50 u thick sections radiographed and the
plates viewed under a microscope. The pulmonary arterioles
demonstrated regular right angle branching into vessels of
prOgressively smaller size. The capillary networks were
filled by short right angled branching arterioles (10-20 u
long and 10-15 u in diameter).

Sobin, Intaglietta, Frasher, and Tremmer (1966)
injected silicone rubber (25 mm Hg pressure) into the main
pulmonary artery of dogs, rabbits, and cats. The lungs
‘were fixed by intratracheal inflation of 10 percent formalin
until their distension matched that at end inspiration.
Frozen serial sections (47 u thick) were taken from various
lobes. Arterial vessels large enough to be seen by the
unaided eye down_to precapillary alveolar branches showed a

consistent pattern of right angled branching with minimal

25
changes in the direction of the parent vessel. An
examination of the precapillary arteriole revealed that its
average diameter was between 18—25 u. The lack of an
increase in the amount of smooth muscle nor a specific
orientation of the muscle cells at the right angled branch-
ing origin of the precapillary vessels lead Sobin gt al.
to negate the presence of a functional precapillary muscular
sphincter. These observations were essentially consistent

for all three species.

Pulmonary Capillary Network

Pulmonary capillaries are primarily thin-walled
endothelial tubes surrounded by a basement membrane
(von Hayek, 1960b). In man and most laboratory animals,
they exhibit a diameter of 7-10 u which is sufficient to
allow passage of red blood cells (Daly and Hebb, l966e).
They lack smooth muscle and it is generally agreed that
their vasomOtion is passively influenced by either upstream
or downstream events.

The vast network of pulmonary capillaries serves
in the transportation of nutrients and wastes across the
blood air barrier. According to Wagenvort (1964a) this
barrier consists of five anatomical layers:

1. Alveolar epithelium

2. Alveolar basement membrane

3. Tissue space containing occasional reticular fibers
4. Capillary basement membrane

5. Capillary endothelial layer

26

Clements (1957) and Pattle (1967) would amend the
work of Wagenvort by adding a sixth anatomical layer to the
blood air barrier. This physiologically vital component is
the continuous film of surfactant that lines the alveolar
epithelium. In a review of the pulmonary surfactant system,
they discussed the functional significance of this alveolar
lining. A simplified version is as follows: There is a
direct relationship between the thickness of the surfactant
film and the surface tension exerted on pulmonary alveoli.
When the alveoli are partially deflated at end expiration,
the film of surfactant is relatively thick and the
resultant surface tension low. As the alveoli expands
during inspiration the film of surfactant streaches with
the alveoli. As the film thins out, alveolar surface
tension increases to the point where partial alveolar
collapse occurs and expiration ensues. The probable source
of the surfactant is considered to be the type II alveolar
epithelial cells.

Goldenberg, and Buckingham (1967) examined the
pulmonary ultrastructure of rats after bilateral cervical
vagotomy. The most consistent ultrastructure response was
a decrease in the amount of osmiophilia in the inclusion
bodies of the type II alveolar epithelial cells. This was
accompanied by atelectasis, focal edema, and capillary
congestion. Their results suggested that a lack of vagal
tone would result in atelectasis by decreasing the
production of surfactant thus increasing alveolar surface

tension.

27

Krahl (1969), in studies on the rabbit, suggested
that vagally mediated pulmonary vasoconstriction would give
rise to atelectasis by decreasing the nutrients to the
type II alveolar cells. He concluded that the decrease in
nutrients would slow surfactant production resulting in an
increase in alveolar surface tension.

Staub (1966) has presented data indicating that
pulmonary blood flow is directly related to the cyclic
changes in respiration. He concluded that the increase
in alveolar Surface tension seen during inspiration would
result in an increase in the diameter (volume) of the
larger (elastic) pulmonary arteries, and veins, due to the
increase in transpulmonary pressure. Inspiration will also
result in the partial collapse of the capillaries on the
flat surface of the alveolar walls. Capillaries and larger
vessels located at the curved junction of the alveolar
walls are protected from the increase in alveolar pressure
by the increased surface tension across the curvature.
Staub concluded that surface tension and thus the factors
which control the production and release of surfactant can
have a definite influence on the patency of pulmonary

vasculature.

Pulmonary Venous Return

 

Most authors will agree that the anatomical
differences between pulmonary venous and arterial vessels
less than 100 u in diameter are slight and functionally

insignificant (Harris, 1962). Franklin (1937) and

28
Wagenvort (1964b) in their extensive studies of the pulmonary
venous system observed the following Species variations:
Human pulmonary veins larger than 80 u in diameter possess
an irregular circular muscular layer, while the corre-
sponding veins in both the dog and the cat demonstrate even
distribution of muscular fibers. Pulmonary veins often lack
the external elastic layer seen in the arteries. In general,
the media of the veins contains more elastic and fibrous
tissue and less muscle than arteries of comparable size.
The elastic tissue in the larger intrapulmonary arteries of
man, dog, and cat was found to exceed that in the larger
veins. Pulmonary veins lie in the interlobar septa and,
unlike the arteries, are associated with the bronchial tree
only at the hilus. Ferencz (1969) reported that the veins
of the rabbit were uniformly thin, a rather striking
difference from the thick walled muscular arteries.

If one uses the premise that smooth muscle is a
prerequisite for vasoconstriction, then it becomes rather
easy to speculate that pulmonary veins (larger than 100 u)
have considerably less vasomotor control over pulmonary
circulation than do their corresponding arteries. The work
of Franklin (1932), previously reviewed, questions the
validity of such speculation by his observation that inter-
as well as extrapulmonary venous rings reSponded with
significant constriction when placed in a Ringer's solution

containing acetylcholine.

29

Vagal Innervation of Pulmonary
Airways and Blood Vessels

 

The efferent pathway of the vagus to the lungs is
structured with an upper and lower motor neuron complex.
The cell bodies of the upper neurons are located in the
rostral third of the dorsal motor nucleus of the vagus
(Getz, 1949). Efferent preganglionic vagal fibers exit the
skull through the jugular foramen, transverse either side
of the trachea, and synapse with their lower motor neurons
in the peribronchial tissue, within the walls of the
bronchi, and alongside the larger veins in the pulmonary
septal tissue (Spencer, 1964). The resultant postganglionic
fibers are extremely short. A smaller portion of the
parasympathetic preganglionic fibers synapse with post-
ganglionic cell bodies in the perineural tissue or within
the vagus nerve as it courses toward the lungs (Hirsch and
Kaiser, 1969). These vagal efferents demonstrate long
postganglionic fibers.

Larsell (1921) and Larsell and Dow (1933) using
methylene blue and silver staining techniques evaluated
human and rabbit pulmonary innervation. Unmyelinated
fibers were assumed to be postganglionic efferents. The
smaller of the unmyelinated fibers were considered to be
sympathetic. Vagal efferent fibers were observed leaving
their postganglionic cell bodies to innervate the smooth
muscle and epithelial cells of the bronchioles.
Innervation of the pulmonary blood vessels, including

capillaries, was primarily by very thin unmyelinated fibers

30
which were considered to be sympathetic. Numerous myelinated
afferent fibers were distributed throughout the pulmonary
tissue down to the level of the atria.
spencer and Leof (1964) using vital methylene blue

and silver impregnation were able to demonstrate the general
distribution of autonomic nerves to pulmonary airways and
blood vessels in the human.. Pulmonary nerves entering the
hilus branched_several times and accompanied either the
bronchi, the pulmonary arteries, or the pulmonary veins in
their course toward the periphery. Parasympathetic motor
fibers leaving the peribronchial ganglia were observed
penetrating smooth muscle in airways down to respiratory
bronchioles. The innervation of the pulmonary vasculature
was evaluated by the presence of either thick or thin
fibers. The results were as follows:
1. Elastic arteries were poorly supplied with both thick

and thin nerve fibers lying between the media and

adventitia.

2. Transitional and muscular arteries, down to the
arterioles, were invested only with thin fibers.

3. Large pulmonary veins were richly innervated with thin
fibers, some of which ended just below the endothelium.
From their position with its close proximity to blood
flow they were thought to be chemoreceptive.

4. Smaller veins were innervated by thin fibers.

Hirsch and Kaiser (1969) utilizing the principle of

Wallerian degeneration and histologic silver staining

techniques compiled an extensive review of the innervation

of the mammalian lung. Vagal nerves, seen entering the

hilar portion of the lungs, continued toward the periphery

31

in the adventitia surrounding the airways and associated
blood vessels to the level of the terminal bronchioles.
The majority of nerves penetrating the pulmonary tissue were
associated with ganglionic cells, thus linking them to the
parasympathetic nervous system. Using the affinity of
myelin for silver stains they were able to determine that
most of the nerve fibers were enclosed within a thick
myelin sheath. This suggested the presence of a large
number of vagal afferents. Fasicles of axons (motor and
sensory) left the adventitia to supply the airways and blood
vessels. These fibers were evaluated by noting the
anatomical site of their termination and the structural
complex at their endpoints. Fibers terminating in the
smooth muscle of the airways and blood vessels were assumed
to be both motor and sensory. Fibers terminating in the
endothelium of the blood vessels were thought to be
afferent chemoreceptors because of their close association
with blood flow. Fibers terminating in the subepithelial
tissue of the bronchioles, the alveolar ducts, and the
alveolar sacs were thought to be afferent stretch receptorsa
reacting to distension of the pulmonary airways. From
their studies on the innervation of the canine lung, Hirsch
and Kaiser concluded that vagal innervation was substantially
ipsilateral.

' The most definitive study on parasympathetic
innervation of the pulmonary airways and vasculature was

done by Hebb (1969). These histochemical studies utilized

32
the presence of acetylcholinesterase to determine the
distribution of vagal fibers. They concluded that:

l. The dog, rabbit, and cat demonstrated more numerous
cholinergic fibers in the bronchial tree than in the
arteries or veins.

2. Cholinergic fibers extend down to arterial vessels
30—40 u in diameter in the dog and the cat, while
the rabbit exhibits cholinergic fibers only in
arterial vessels larger than 100 u.

3. The cholinergic fibers of the rabbit and cat appear
more extensive in the larger vessels.

4. The pulmonary veins of all animals appears to be
moderately innervated.

MATERIALS AND METHODS

Experimental studies were performed on dogs, rabbits,
and cats, in groups of five. In each case, the distal end
of the severed left vagus nerve was mid-cervically stimu—
lated followed by ink injection and i£.§iEE formalin
fixation. Left vagal fibers were chosen for stimulation
because they inhibit cardiac activity to a lesser degree
than do right vagal fibers (Truex, 1955). The vagal effect
on pulmonary circulation was evaluated grossly by the degree
of peripheral ink perfusion and histologically with serial
sections. In all cases, the right or contralateral lung,
served as a control. All experimental animals utilized in
this study had undergone cardiac catheterization and three
one minute vagal stimulations just prior to the initation
of ink injection procedures. The parameters for the three
vagal stimulations were within the ranges of 2—10 volts/
2.5-6.0 msec/lO-30 cycles per second and had a duration of
one minute.

A control animal for each species was studied in a
similar manner. These animals did not undergo vagal
stimulation and the data obtained was compared to that from

the stimulated animals.

33

34

Experimental Dogs

 

Five mongrel dogs, each weighing between seven and
eleven kg, composed the first study group. The anatomical
orientation in the dog is such that a mid-cervical stimu-
lation of only vagal fibers would be extremely difficult
due to the fact that both sympathetic and parasympathetic
nerves are intertwined in one comron truck. Stimulation of
this vagosympathetic nerve trunk would thus result in the
activation of both sympathetic and parasympathetic nerve
endings. To remove the undesired sympathetic effect,
phenoxybenzamine hydrochloride (alpha—adrenergic blocker)
and prOpranolol hydrochloride (beta-adrenergic blocker) were
administered prior to surgery. Used in the proper dosage,
these two drugs have been reported to be sufficient to
block all sympathetic activity (Goodman and Gilman, 1970a).

Each dog was anesthetized with sodium pentobarbital
(32 mg/kg of body weight) via the left cephalic vein. The
ventral cervical and thoracic regions were then shaved with
electric clippers. A mixture of propranolol hydrochloride
(1 mg/kg) and forty ml of normal saline was infused through
intravenous (I.V.) drip via the left cephalic vein over a
period of forty-five minutes. Upon completion, the I.V.
drip was replaced with a solution of phenoxybenzamine
hydrochloride (2 mg/kg) and forty ml of normal saline which
was also infused over a forty-five minute duration.

The animal was then restrained in a supine position

on the surgical table and a ventral cervical incision made

35
through the skin and superficial fascia. The incision began
at the larnyx and was extended caudad a distance of approxi-
mately 7.5 cm. Using a blunt hemostat, the sternohyoid and
sternothyroid muscles were separated at their midventral
lines thus exposing the trachea. The left vagosympathetic
trunk and right common carotid artery were separated from
their respective carotid sheaths and a loop of umbilical
tape placed around each structure. The trachea was then
cleaned of any excess fascia and lOOped in a similar manner.
Blunt dissection was continued to expose and loop the right
external jugular vein.

Cannulation of the right external jugular vein was
initiated by placing a loop of 2-0 silk around the vessel
and ligating it as far cranial as the surgical exposure
would permit. A probe was then placed under the caudal
extent of the vessel, just caudad to the loop of umbilical
tape. Using the probe and one end of the 2—0 silk, tension
was applied to the vessel. A cut extending through approxi-
mately three fourths of the vessel's diameter was made and
the tip of a 25 cm piece of polyethylene (P.E.) 190 tubing
maneuvered a distance of approximately 5 cm down the vessel.
The tubing was then secured by tying the umbilical tape
around both the vessel and catheter approximately 1 cm
caudal to the catheter's insertion. The free end of the

P.E. 190 tubing was connected to a plastic syringe which

36
contained physiologically buffered Pelikan ink1 (volume
approximated animal's total pulmonary blood volume,
Schermer, 1967).

A similar procedure was used in the cannulation of
the right common carotid artery. The only differences were
that a Lehman 5F ventriculography cardiac catheter2 was used
and that this catheter was maneuvered into the left ventricle
before being secured. The free end of this catheter was
connected to a Stathem P23AC transducer which recorded on a
model 5D direct writing Grass polygraph. Left ventricular
pressure recordings taken throughout the experiment were
used to determine the degree of effectiveness of the vagal
stimulation. The left ventricular catheter was periodically
flushed with heparinized saline (8 units/cc) to maintain
the integrity of the pressure recordings. A syringe con-
taining one cc of a 50% carbamylcholine chloride (carbachol)
solution was then connected to the left ventricular catheter
via a two—way stopcock on the Statham transducer.

A tracheotomy was performed by elevating the trachea
with the previously placed umbilical loop, placing a blunt
hemostat under it for stability, and incising its ventral
aspect carrying the incision up its lateral walls. A glass
cannula was then placed in the tracheal lumen and secured by

tying the umbilical tape.

 

lJohn Henschel Co., 141 Albertson Ave., Albertson,
N.Y. 11507 -

2United States Catheter Corp., Glenn Falls, N.Y.

37

The left vagosympathetic trunk was then elevated with
the umbilical loop, clamped with a blunt hemostat, and
severed just cranial to the hemostat. The distal end of the
nerve was then stimulated with a monophasic square wave
current produced by a Grass S—8 stimulator. All stimulations
throughout the experiments were within the parameters of
2-10 volts/2.5-6.0 msec/10-30 cycles per second, and had a
duration of one minute. The criteria for parameter
selection was that range which decreased left ventricular
rate by one third of thecontrol readings.~ After fifteen
seconds of stimulation, the ink was injected, a process
taking another fifteen seconds. As the last few cc of ink
were being infused, the carbachol was injected into the left
ventricle followed by a five cc saline flush. Carbachol's
prolonged parasympathomemetic action is more resistant to
the enzymatic breakdown by cholinesterase than is
acetylcholine chloride. Its high concentration and dura-
bility caused immediate cardiac arrest by hyperpolarizing
the cardiac muscle fibers (Goodman and Gilman, 1970b). This
procedure insured that the greater majority of ink remained
in the lungs. Vagal stimulation continued for a duration of
one minute. The glass tracheal cannula was then immediately
connected, via polyethylene tubing, to a reservoir of 20%
phosphate buffered formalin (Carlton, 1967) held at 25 cm
above the animal. The formalin used throughout all
subsequent experimental procedures had the same concentration

and buffer system. The gravitational pull on formalin held

38
at this height was sufficient to allow infusion into the
trachea at a pressure of 25 cm of water which is considered
to be the optimum pressure for fixation of the lungs in a
physiologically inflated state (Heard, 1962). The formalin
was allowed to infuse into the lungs for five minutes, after
which the trachea was clamped and the lungs removed.

Lung removal was accomplished by stripping the Skin
and muscle away from the thorax and severing the costo-
chrondral junctions just lateral to the sternum. The ribs
and diaphragm were then manually retracted and the remaining
fascia and vessels severed thus freeing the lungs and heart.
The external surface of the lungs was then gently washed
with tap water and evaluated visually for areas of ink
penetration.

Eash lobe was carefully studied for the percent of
ink perfusion. Pink areas were considered non-perfused,
and black areas perfused. These rather subjective
measurements were taken by two people at different times and
the inter—observer percentages averaged.

The lungs were then photographed from the dorsal,
ventral, left lateral, and right lateral surfaces and the

film stored for later reference.

Histologic Preparation
Preservation of normal lung architecture was
accomplished by utilization of the Weiss and Tweeddale (1966)

method of infusing formalin down the trachea at a constant

39
pressure. This insured the integrity of the tissue by
keeping the alveoli in a physiologically inflated state.

The lungs were placed in a plastic pan (Figure 1)
containing six liters of formalin. The glass tracheal
cannula was connected to an outflow valve at the base of a
two liter pyrex aSpirator bottle by means of a 30 cm piece
of 5/16 inch P.E. tubing. The aspirator bottle was then
supported in a manner such that when filled its upper level
of fluid was 25 cm above the level of fluid in the plastic
pan. A 40 cm piece of 5/16 inch P.E. tubing was connected
to the outflow valve at the neck of the aSpirator bottle and
allowed to rest in the bottom of the plastic pan. A small
aquarium pump3 Was then placed with the lungs in the plastic
pan. One end of a 45 cm 5/16 inch piece of P.E. tubing was
connected to the pump outflow valve and the other end was
allowed to enter the top of the aspirator bottle coming to
rest about 5 cm from the bottom of the bottle. This tubing
was enclosed in a double layer of #1 penrose tubing; one
end of which rested freely in the formalin pan at the level
of the pump valve, the other end being secured around the
neck of the aspirator bottle. The penrose tubing acted as
a second overflow valve. The aquarium pump was then
activated thus inflating the lungs. The lungs remained under
constant pressure (25 cm of water) for twenty-four hours
after which they were removed and stored in formalin for a

period of at least three days.

 

3Little Giant Pump Co., Oklahoma City, Oklahoma.

40

The above procedures were repeated until five canine
lungs rested in formalin storage. The left ventricular
pressure recordings were then evaluated for the degree of
effectiveness of the vagal stimulation and the speed with
which carbachol arrested the heart. One animal was chosen
which demonstrated the most rapid cardiac arrest, and a
uniform decrease of left ventricular rate by one third of
the control readings. This set of lungs was then removed
from the formalin and the right and left middle lobes
severed at the hilus. The peripheral edges of each lobe
were then trimed to a rectangle having sides of approximately
two and two and one half cm respectively. The main
pulmonary artery and bronchus supplying each lobe was
oriented in the center of their respective rectangle.

The tissue was then cleared and infiltrated as

 

follows:
70% alcohol overnight
80% alcohol one hour under 15 ins. Hg. vacuum

 

9 S % alcoho l ' n n u u n n n

 

'95% alcohol u n u u u u n

 

100% alcohol I: n n n u u u

 

100% aléohol " n u u n u u

 

100% alcohol " " n u n u u

 

TO luene . " II n

 

To luene " H II II u u u

 

50% paraffin & 50% toluene " " " " " " "

Paraffin " n H II u u n

 

41

Paraffin one hour under 15 ins. Hg. vacuum

 

Paraffin N u n u n n n

 

Embed

The paraffin embedded tissue was then serially
sectioned and mounted in a manner similar to that described
by Wilson and Pickett (1970). The paraffin blocks were
first mounted on a Spencer rotary microtome. Extending down
from the microtome blade was a rigid strip of paper which
aided in guiding the serial sections (Figure 2). The micro-
tome, set to cut at 10 microns, was engaged and a strip of
paraffin encased tissue allowed to slide down the paper.
Cutting continued until approximately thirty sections had
passed onto the paper guide at which time the paraffin strip
was out leaving only five sections attached to the knife.
This process was repeated until all of the tissue had been
sectioned. The ribbons of tissue were stored sequentially
on a nearby table. Using a scalpel, the strips of tissue
were then subdivided into units each containing approximately
six sections.

In order to mount the tissue, a film transport
device was constructed in our laboratory using a pattern
similar to that developed by Wilson and Pickett (1970). The
unit was made of inexpensive plexiglass, and a Kinderman

guide and developing reel4 (Figure 3). The transport device

 

4Ehrenreich Photo-Optical Industries Inc., Executive
Office, 623 Sterart Avenue, Garden City, N.Y. 11530.

42
was lowered into a 7 1/2 inch lo-boy water bath kept at a
constant 48°C. A reel of 100 feet of P40b Cronar motion
picture leader film5 was placed on the free end of the
transport device. The exposed end of the film was then
slightly rounded and guided under the rollers of the
transporter, through the Kinderman film guide and secured
in the center of the develOping reel. The film was
continually coated with egg albumen before entering the
water bath to insure pr0per adherence of the tissue.

The first strip of tissue was then placed in the
water bath (Figure 4). It was aligned directly above the
roller guides with the tip of the first section just touching
the center of the film as it emerged from the water. The
reel was then slowly turned and the paraffin strip gently
guided onto the film until all sections were resting on a
film base. This process was repeated until the reel held
its maximum of five feet of film. The film was then cut and
the reel removed and stored in a dry place for twenty-four
hours. The mounting procedure was continued until all
sections had been placed on the film.

After twenty-four hours had elapsed, each reel was
stained in hematoxylin and eosin and coated with RC905
methacrylate plastic.6 A blunt hemostat was attached to

the center of each reel and used in elevating them in and

 

5Available from E.I. duPont De Nemours & Co.,
Wilmington, Delaware.

61bid.

 

43

out of the Staining dishes (Figure 5). The method of

staining and plastic coating was as listed below:

Xylene

Xylene,
Absolute alcohol
Absolute alcohol
95% alcohol

80% alcohol

Tap water
Hematoxylin

Tap water

Acid alcohol

Tap water

Eosin

95% alcohol

95% alcohol
Absolute alcohol
Absolute alcohol
Absolute alcohol
Xylene

Xylene

Xylene
Drying‘period

Plastic coating

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2 l/2 minutes

2 1/2 minutes

2 1/2 minutes

2 1/2 minutes

few dips (in and out)

few dips (in and out)

two changes (one minute each)
five minutes

three changes (one minute each)
one to three rapid dips

two changes (one minute each)
one minute

several dips

several dips

one minute

one minute

one minute

two minutes

two minutes

two minutes

one minute on a paper towel

quantity sufficient to cover
the lower 7/8 of film.

The reels were removed from the liquid plastic and

supported over the staining dish for a period of one minute

to allow any excess plastic to fall back into the dish.

44
The reels were then placed on paper towels for five minutes
and any additional plastic allowed to drain. They were then
placed in a staining dish containing 20 cc of xylene which
cleared the plastic from the lower 1/8 of the film. This
allowed the film to be maneuvered freely during subsequent
microsc0pic evaluation.

The film strips were then removed from their
respective reels and one end attached to the laboratory
ceiling while the other end was allowed to hang freely.
After a twenty—four hour drying period, the film strips
were trimmed and spliced together sequentially in

preparation for microscopic evaluation.

Microscopic Evaluation

Before proceeding with the microscopic evaluation,
it was necessary to develop a method of stabilizing the film
on the microsc0pe stage. A film holder (Figure 6) was
constructed out of 1/8 inch glass and two strips of etched
1/8 inch plexiglass. The film holder is a modification of
one developed by Wilson and Pickett (1970). This device
kept the film secure as it passed under the objective. The
holder was firmly adhered to the movable stage on the
microscope and thus was easily maneuvered in horizontal and
vertical directions.

In order to make stereologic measurements, a
viewing plate was constructed in accordance with specifi—
cations developed by Weibel gt gt. (1966) (Figure 7). The

plate consisted of a round piece of 1/16 inch plexiglass

45

having a diameter of 3 1/2 inches. A metal etcher was used
to groove the plate at the proper points. The grooves were
then filled with black wax and the excess wiped off. The
circumference of the plate was covered with a strip of tape
to protect it from scratches.

The microscopic equipment used in section evaluation
was then assembled in the following manner (Figure 8). A
Nikon vertical monocular phototube (cat. no. 77745) was
secured atop a Nikon S-KE microscope stand equipped with
Koehler illumination and a movable stage. A 10X Nikon high
eyepoint compensating widefield eyepiece (cat. no. 77857)
was then placed in the photo tube. A projection head with
a 3 1/2 inch screen (cat. no. 76865) was attached, through
a universal adapter (cat. no. 77111), to the photo tube
through which all subsequent measurements were taken. The
equipment was assembled in such a manner that the micrometer
was projected onto the screen at all times and could be
rotated to measure any vessel in the field. The microscope
was equipped with 1.2x, 4X, 10X, and 42X objectives. The
Weibel viewing plate was then fixed to the front of the
viewing screen by means of small strips of tape placed on
its upper and lower surfaces. The complete unit (Figure 9)
allowed one to see the Weibel counting plate superimposed
over an ocular micrometer which was superimposed over the

tissue being evaluated.

46

Stereologic Megsurements

 

Utilization of stereologic techniques enables the
researcher to obtain three—dimensional information from
two-dimensional random section sampling. Sections were
evaluated by superimposing a stereologic grid on each sample
and noting the relationship of structures in the section to
points and lines on the grid. The present investigation is
concerned with length and volume measurements. According
to Weibel gt gt. (1966), length measurements require a
specific test area and volume measurements require a
uniform lattice of test points. A grid (Figure 7) was

designed to meet these specifications.

Volume Measurements

 

Weibel 23.21- (1966) proposed a method for
determining relative and absolute volumes of structures
enclosed within histologically sectioned tissue. He
suggested that a grid (Figure 7) be placed over the
serially sectioned tissue at regular intervals. This grid
contained forty-two evenly spaced crossbars. Each time one
of these crossbars fell partially or wholly within the outer
walls of the structure being measured it was counted as a
hit. This process was repeated on all subsequent tissue
samples. The relative volume was equal to the number of
hits recorded divided by the possible number of hits. Thus:

Relative volume = Number of hits recorded
Number of possible hits

 

47
The relative volume indicated the percent of the total
volume of the sample occupied by the structure being
measured.

The absolute volume of the structure could be
determined by taking the actual volume of the tissue sample
and multiplying it by the relative volume of the structure.
Thus:

Absolute volume = Actual volume of the tissue sample

Relative volume of the structure

The Relative Volume Occupied By
The Pulmonary Arteries,
ArteriBles, Veins, and
Venules

 

 

In order to insure an unbiased evaluation, it was
necessary to develop a method whereby all areas of the
tissue could be equally sampled. This was accomplished by
subjectively dividing each side of a section into three
equal parts and placing imaginary dots at these divisions.
Imaginary lines were then drawn to connect opposite dots.
The resultant figure was that of a tissue section divided
into nine units numbered as seen in Figure 10. The sections
were then sampled according to unit areas in the following
manner:

The first hilar section was brought into focus at
100x and the ti3sue maneuvered to unit area 1. The section
being observed had superimposed on it a movable ocular
micrometer and the Weibel stereologic viewing plate. Count-
ing proceeded as follows: When any portion of one of the

forty—two crossbars of the Weibel plate fell partially or

48
wholly within the lumen of an artery, arteriole, vein, or
venule, it was counted as a hit and the number recorded.
The same section was subsequently maneuvered to unit areas
two and three and counts again taken. The film strip was
then advanced six sections and the process repeated; this
time, counting the number of hits in unit areas four, five,
and six. The film strip was then advanced another six
sections and counts taken in unit areas seven, eight, and
nine. This method of sampling every sixth section with
rotating unit areas continued in the manner described above
until the stimulated and control lobes had been counted.
The data was placed in the previously stated relative
volumetric formula and the percent of the total volume of
the lobe occupied by the pulmonary arteries, arterioles,
veins, and venules calculated.
The Relative Volume Occupied

By The Ink Filled Pulmonary
Capillaries

 

 

 

At this point, a few comments on the rationale of
taking ink perfusion measurements should be included. Krahl
(1968) stated that the point of constriction during vagal
stimulation is the precapillary arteriolar site. One would
thus expect that during vagal stimulation, ink penetration
would be terminated, or significantly reduced, at this
constricted site. A comparison of the volume of ink
reaching the capillary beds of both stimulated and control
lobes would thus indicate the degree of effectiveness of

vagal stimulation on the precapillary anteriolar smooth

49
muscle. With this in mind, a measurement of the relative
volume occupied by the ink filled capillary beds was
undertaken.

The first hilar section was brought into focus at
420x and maneuvered to unit area 1. At this magnification,
pulmonary capillaries were easily seen and those with and
without ink penetration could readily be identified.
Counting proceeded as follows: An ink filled capillary
falling on any portion of one of the forty—two crossbars
of the Weibel plate was counted as a hit and that number
recorded. The same section was then moved to unit areas
two and three and the process repeated. The method of
choosing sample areas for capillary ink perfusion was the
same as that described earlier for the relative volume
occupied by the larger vessels. Again, every sixth section
was sampled using rotating unit areas until the stimulated
and control lobes had been counted. The data was then
placed in the relative volumetric formula and the percent
of the total volume of the lobe occupied by the ink filled
pulmonary capillaries calculated.

The Relative Volume Occupied By
The Pulmonary Airways Ranging

From Secondary Bronchi To
'Terminal Bronchioles

 

 

 

 

A study of the vagal effects on pulmonary circulation
would be incomplete without some mention of the vagal effects
on pulmonary airways. An increase or decrease in the patency

of these airways would obviously have a secondary effect on

50
the nearby vessels. With this in mind, a study was
undertaken to determine the relative volume occupied by the
pulmonary airways ranging in size from secondary bronchi to
terminal bronchioles.

The first hilar Section was brought into focus at
40X and maneuvered to unit area 1. Counting proceeded as
follows: Whenever one of the forty-two crossbars of the
Weibel viewing plate fell partially or wholly within the
lumen of the airway it was counted as a hit and the number
recorded. The same section was then maneuvered to unit
areas two and three and the process repeated. The method
for choosing sample areas for measuring the relative volume
occupied by the airways was the same as that described
earlier for the relative volume occupied by the larger
vessels. As before, every sixth section was sampled using
rotating unit areas until the stimulated and control lobes
had been counted. The data was then placed in the relative
volumetric formula and the percent of the total volume of
the lobe occupied by airways ranging in size from secondary

bronchi to terminal bronchioles calculated.

Absolute Volume

 

The absolute volume of a tissue component can be
determined by multiplying the relative volume by the
measured volume of the tissue sample. The volume of the
right and left middle lobes of three experimental dogs was
determined by the water displacement technique. The method

was carried out as follows: A 500 cc wide mouthed flask

51
was fitted with a double holed rubber stopper. A graduated
pipet was inserted through one hole and allowed to penetrate
the flask a distance of five cm. The other hole was fitted
with a glass tube which penetrated the flask a distance of
one cm. The outer end of the glass tubing was connected to
a 50 cc plastic syringe. A zero point was noted on the
pipet a distance of one cm above the rubber stopper. Using
the syringe the flask was filled with water until the water
level reached the zero point on the pipet. The volume of
water was then recorded and the flask emptied and dried.
The middle lobe in question was placed in the flask and the
rubber stopper secured. Again, the flask was filled with
water until the zero point was reached. The volume of
water was then recorded and the flask emptied. The
differences between the first and second volumes equaled the
volume of the lobe in question. This process was repeated
for all subsequent volumetric analyses.
The Length of Pulmonary:

Arteries, Arterioles,
' Veins, and Venules

 

 

Evaluation of vascular constriction or dilation in
histologic sections most often concerns itself solely with
changes in vessel diameter. This would seem to be only part
of the picture for Patel (1956) has shown that vessels can
and will change in their total length. His corrosion cast
studies of the rabbit pulmonary arterial tree revealed that
injection of norepinephrine (dosesgreater than 10 ug)

resulted in extensive knarling and twisting of the arterial

52
vessels. Rabbits who were not pre—injected with norepine-
phrine demonstrated smooth and straight arterial vessels.
Measuring the stimulated and control lobes for changes in
length of the pulmonary vessels would thus give a third
dimension to the evaluation of the stimulation effects.

Elias and Henning (1967) proposed that the length of
specific structures per unit volume could be determined by
stereologic evaluation of histologic sections. Their
suggested techniques were incorporated in the present
investigation to measure the combined lobar lengths of all
pulmonary vessels (larger than capillaries).

The measurements proceeded as follows: The first
hilar section was brought into focus at 100x and maneuvered
to unit area 1. At this magnification the superimposed
Weibel square covered an area of tissue .81 mmz. The
screen was then scanned counting any pulmonary vessel which
fell within the outer limits of the Weibel square. Vessels
which were intersected by the left and upper edges of the
square were counted. Vessels which were intersected by
the right and lower edges of the square were not counted.
The section was then maneuvered to unit areas two and three
and the process repeated. The method for choosing sample
areas for measuring the length of pulmonary vessels was the
same as that described for measuring the relative volume
occupied by the larger vessels. As before, every sixth
section was sampled using rotating unit areas until the

stimulated and control lobes had been counted.

53
Elias and Henning concluded that the length (L) of
linear structures within a unit volume (V) equals twice the
number of mean profiles (P) counted divided by the area (A)
of the test square. Thus:

LV=.2_1:
A

For example, if a sampling of 10 histologic sections resulted
in a total profile count of 20 vessels then:

L

(2) 20/10
V 2
0.81 mm

 

3

4.9 mm/mm
Thus in every cubic milimeter of pulmonary tissue vessels
larger than capillaries had a total length of 4.9 mm. In
order to compute the combined length of these vessels in

an entire lobe it was necessary to make the following
conversions:

4.9 mm/mm3 = 4900 mm/cm3 = 490 cm/cm3

If the actual volume of the lobe in question was 10 cc then
the combined lengths of all the measured pulmonary vessels

in that lobe was equal to:

(10 cc) (49o cm/cm3) = 4900 cm = 49 m

Statistical Analysis

 

Control and experimental data was evaluated
statistically by an analysis of covariance (Ostie, 1970),
and Stapelton's (1972) procedure for comparing individual
means.

Each lobe was divided into three equal parts, a

hilar, a middle, and a peripheral portion. The absolute

54
lobar values were then statistically analyzed as stated
above. If statistically significant differences at the
95 percent confidence level were observed between the lobar
values, the three pulmonary compartments were examined to
determine the specific anatomical regions most responsive

to vagal stimulation.

Control Dog

 

The surgical, histologic, microscopic, and
statistical evaluation procedures just described were
repeated on a dog whose specifications met those of the
experimental group. The animal did not undergo vagal
stimulation and therefore it was not necessary to administer
alpha and beta blocking agents prior to surgery. All other
procedures were identical to those previously described.

The volumes of right and left middle lobes of the control
dog were determined by the water displacement method and the
data used to compute the absolute volumes of the tissue

components being studied.

Experimental Rabbits

 

Five New Zealand white rabbits, each weighing
between three and five kg, were used in the second animal
group. The animals were intravenously anesthetized with
sodium pentobarbatal via the left marginal ear vein, and
the ventral cervical and thoracic regions shaved with
electric clippers. The animals were then restrained in a

supine position on the surgical table.

55

The anatomical orientation in the rabbit is such
that a mid-cervical stimulation of only vagal fibers is
quite feasible because, unlike the dog, the rabbit has a
separate cervical vagus nerve and sympathetic trunk. Both
are enclosed within the carotid sheath but can easily be
separated and identified since the vagus is consistently
larger than the sympathetic trunk. Therefore, it was not
necessary to administer sympathetic blocking agents prior
to the initiation of the surgical procedure.

The surgery was carried out in a manner similar to
that performed on the dog. The cannulations were also
similar save for the fact that a size 4F Lehman
ventriculography cardiac Catheter was used for the left
ventricle catheterization.

The parameters for vagal stimulation were within
the ranges of 2-10 volts/ 2.5-6.0 msec/10—30 cycles per
second and had a duration of one minute. Ink injection,
formalin fixation, histologic preparation, microscopic and
statiStical evaluation were all performed in a manner
similar to that described for the dog.

The volumes of the right and left middle lobes of
three experimental rabbits were determined by the water
displacement method. The average volumes were then used
to compute the absolute volumes of the tissue components

being studied.

56

Control Rabbits

 

The surgical, histologic, microscopic and statistical
evaluation procedures just described were repeated on a
rabbit whose specifications met those of the experimental
group. The animal did not undergo vagal stimulation. The
volumes of the right and left middle lobes of the control

rabbit were determined by the water displacement method.

Experimental Cats

 

Five mongrel cats, each weighing between three and
five kg, were used in the third group. The experimental
procedures followed the pattern described for the rabbit
with a few exceptions.

The vasculature of the cat is subject to intense
reflex vasoconstriction in vessels where catheters are
placed. This vasoconstriction often sealed the open end of
the size 4F cardiac catheter as it was being maneuvered down
the right common carotid artery. Blood trapped in the
catheter readily clotted. The subsequent decrease in the
catheter's patency impaired the successful recording of
left ventricular pressure. To counteract the clotting
mechanism the animals were heparinized prior to surgery.
This was accomplished by injecting heparin (350—500 units/
kg) via the cephalic vein just prior to the initial incision.

The method of anesthesia differed from the rabbit in
that the dosage was administered intraperitoneally. The
vagal and sympathetic fibers are in closer association than

in the rabbit and careful dissection was required to separate

57
the two without damage to the nerve tissue. The cervical
vagus nerve, as in the rabbit, is of larger diameter than
the cervical sympathetic trunk.

The stimulation parameters were within the ranges
described for the dog and were employed for a duration of
one minute. Ink injection, formalin fixation, lung removal,
histologic preparation, microscopic and statistical
evaluation were all performed in a manner similar to that
described for the dog.

The volumes of the right and left middle lobes of
three experimental cats were determined by the water
displacement technique. The average volumes were then
used to compute the absolute volumes of the tissue

components being studied.

Control Cat

The surgical, histologic, microscopic, and statistical
evaluation procedures just described were repeated on a cat
whose specifications met those of the experimental group.
The animal did not undergo vagal stimulation. The volumes
of the right and left middle lobes of the control cat were

determined by the water displacement technique.

58

Figure 1. Formalin inflation apparatus

Figure 2. Microtome with tissue strip

Figure 3. Film transport device with Kinderman
guide and develOping reel

59

 

Figure 2

 

Figure 3

60

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61

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62

Figure 8. Assembled microscopic equipment

Figure 9. Viewing screen with Weibel plate and ocular
micrometer superimposed over tissue sample
at 100X magnification

Figure 10. Tissue sample divided into unit areas

63

   

.
.
.
.
.
.
.

p

Figure 10

 

Figure 8

 

Figure 9

RESULTS AND DISCUSS ION

Perfusion—Percentage Results

 

Tables 1, 2, and 3 contain the interobserver
percentage averages of the distribution of ink on the
external surface of the lungs following left vagal stimu-
lation. The tables are listed by dog, rabbit, and cat
respectively. Black areas are considered ink-perfused and
pink areas non-perfused. Figures 11, 12, and 13 graphically
demonstrate the percentage of ink distribution on the
external surface of the lungs following left vagal stimu—
lation on a lobe basis. Table 4 contains the lobar
perfusion-percentages for the control animals. These
animals did not undergo vagal stimulation. As before, black

areas are considered ink-perfused.

Photographic Results

 

Figures 14, 15, and 16 contain photographic
reproductions of a representative experimental animal from
each species. They are listed by dog, rabbit, and cat
respectively. Pictures were taken of the right lateral,

left lateral, dorsal, and ventral surfaces of the lungs.

Stereologic And Volumetric Results

 

Table 5 contains the mean lobar volumes and the

vessel lengths of the experimental and control dogs,
64

65
rabbits, and cats. An asterisk was placed to the left of
the appropriate data when statistically significant
differences, at the 95 percent confidence level, were
observed in comparing lobes from the experimental animals,
or lobes from experimental to control animals.

Each lobe underwent dual analyzation. The mean
lobar values were statistically analyzed to determine any
significant lobar differences. The data from each lobe was
then divided into a hilar, middle, and peripheral portion.
If statistically significant differences were observed in
the mean lobar values, the data from the three pulmonary
compartments was examined to determine the anatomical site
most responsible for the observed differences. The
original data used to compute actual volume, relative
volume, absolute volume, and length per unit volume on a
lobar basis and in the pulmonary compartments is contained

within Appendix B.

66

Table l.--Lobar perfusion-percentages for the dog.
Percent presented: black - pink

 

 

Lobe Studied Dog Number

 

Left Vagal Stimulation

Left lung Dog-1 Dog-2 Dog-3
cranial lobe 98%- 2% 92%- 8% 100%-
middle lobe 100 - 0 88 - 12 100 -
caudal lobe _ 98 - 2 85 - 15 98 -

Right lung
cranial lobe 100 - 0 43 — 57 95 -
middle lobe 95 - 5 28 - 72 95 -
caudal lobe 100 - 0 95 - 5 98 -
intermed. lobe 100 - 0 45 - 55 100 -

Left lung Dog-4 Dog-5
cranial lobe 98 - 2 100 - 0
middle lobe 100 - 0 95 - 5
caudal lobe 100 - 0 100 - 0

Right lung
cranial lobe 100 - 0 100 - 0
middle lobe 100 - 0 95 - 5
caudal lobe 100 — 0 100 - O
intermed. lobe 98 - 2 100 - 0

N00
60

OU'IU'IU'I

 

67

Table 2.-—Lobar perfusion-percentages for the rabbit.
Percent presented: black - pink

 

 

Lobe Studied Rabbit Number

 

Left Vagal Stimulation

Left lung Rabbit-l_ Rabbit-2 Rabbit-3
cranial lobe 60%- 40% 93%- 7% 100%- 0%
middle lobe 55 - 45 67 — 33 70 - 30
caudal lobe 93 - 7 75 - 25 100 - 0

Right lung
cranial lobe 48 - 52 78 - 22 100 - 0
middle lobe 10 - 90 75 - 25 98 - 2
caudal lobe 48 - 52 88 - 12 98 - 2
intermed. lobe 30 - 70 88 - 12 93 - 7

Left lung Rabbit-4 Rabbit-5
cranial lobe 80 - 20 95 - 5
middle lobe 70 - 30 75 - 25
caudal lobe 90 - 10 95 - 5

Right lung
cranial lobe 95 — 5 90 - 10
middle lobe 70 - 30 75 - 25
caudal lobe 60 - 4O 80 - 20

intermed. lobe 85 - 15 80 - 20

 

68

Table 3.-—Lobar perfusion-percentages for the cat.

Percent presented: black — pink

 

 

Lobe Studied Cat Number

 

Left Vagal Stimulation

Left lung Cat-l Cat-2
cranial lobe 100%-' 0% 100%-. 0%
middle lobe 75 - 25 100 - 0
caudal lobe 100 - 0 100 - 0

Right lung
cranial lobe 100 — 0 100 - 0
middle lobe 75 — 25 100 - 0
caudal lobe 100 - 0 100 - 0
intermed. lobe 100 - 0 100 - 0

Left lung Cat-4 Cat-5
cranial lobe 100 - 0 95 - 5
middle lobe 100 - 0 98 - 2
caudal lobe 100 - 0 90 - 10

Right lung
cranial lobe 100 - O 95 - 5
middle lobe 100 - 0 93 - 7
caudal lobe 100 - 0 75 - 25
intermed. lobe 100 - 0 67 - 33

Cat—3

100%-
100 -
100

100 -
100
100 -
100

OOOO

 

69

Figure ll.--Average lobar perfusion-percentages for the dog.

% black % pink

Cr --- cranial lobe Ca --- caudal lobe

 

M --- middle lobe I --- intermediate lobe

Figure 12.--Average lobar perfusion-percentages for the

 

rabbit.
% black % pink
Cr --- cranial lobe Ca ——- caudal lobe
M —-- middle lobe I -—- intermediate lobe

Figure 13.--Average lobar perfusion-percentages for the cat.

”Q“ % black I I % pink

Cr --— cranial lobe Ca --- caudal lobe

 

M -—- middle lobe I --- intermediate lobe

7O

      
  
    
 
 

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Mean %

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71

Table 4.--Lobar perfusion-percentages for the control animals

 

 

Percent black

 

Cranial Middle Caudal Intermediate
L R L R L R R
Dog # l 100% 95% 100% 100% 95% 95% 90%
Dog # 2 95 95 95 95 100 100 100
Rabbit # 1 100 100 100 100 100 100 100
Rabbit # 2 95 90 90 90 90 90 80
Cat # 1 95 100 95 95 90 100 90

Cat # 2 80 100 95 95 95 9O 9O

 

72

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UOHmmH <Hm£

wwmswm Hm wmocwd

 

Home fimflmHmH <Hm€

 

<mSnHmH <Hm£

74

 

UOHmmH <Hm£

wwmcnm Hm nmd

 

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75

Table 5.--Absolute lobar volumes and total vessel lengths

 

 

 

 

 

 

Experimental Control
Left Right Left Right
(23cc) (33cc) (25cc) (35cc)
Dog Large vessel volume 1.96cc 3.20cc 1.65cc 3.92cc
Capillary volume 5.69 7.72 7.85 9.17
Airway volume 0.71 0.76 1.20 1.86
Vessel length 156m 189m 164m 241m
(9cc) (12cc) (9cc) (11cc)
Rabbit Large vessel volume 0.62cc 1.01cc 0.93cc 1.17cc
*Capillary volume 2.44 3.49 2.96 3.92
Airway volume 0.39 0.55 0.36 0.47
*Vessel length 74m 97m 96m 129m
(8cc) (10cc) (9cc) (11cc)
Cat Large vessel volume 0.53cc 0.56cc 0.75cc 0.99cc
*Capillary volume 1.31 1.77 2.30 2.67
Airway volume 0.21 0.16 0.36 0.48
*Vessel length 49m 60m 83m 109m
* =

Actual lobar volumes shown in parenthesis

Differences significant--P is less than 0.05

76

Subjective evaluation of the stereologic data from
right and left middle lobes of both experimental and control
animals revealed the following:

(1) In all species, the calculated absolute volume occupied
by pulmonary vessels (larger than capillaries) was
greater in the right middle lobes than in the left.

(2) In all species, the calculated absolute volume occupied

- by ink filled pulmonary capillaries was greater in the
right middle lobes than in the left.

(3) In all animals, with the exception of the experimental
cat, the calculated absolute volume occupied by airways
(larger than respiratory bronchioles) was greater in
the right middle lobes than in the left.

(4) In all species, the calculated total length of the
pulmonary vessels (larger than capillaries) in meters
per lobe was greater in the right middle lobes than
in the left.

Actual lobar volumes determined by the water
displacement technique revealed that the right middle lobes
of all species were larger than their left counterparts.
Rahn and Ross (1957) determined the percent weights of the
total canine lung for its individual lobes. The right
middle lobe proved to be 8.9 percent and the left middle
lobe 5.8 percent of the total lung weight. An increase in
the actual volume and weight of pulmonary tissue would
logically be accompanied by a greater number of blood
vessels and airways. This numerical increase accounts for
the consistantly larger stereological values observed in the
right middle lobes.

Weibel (1964) stereologically determined the percent

volume of the lung occupied by the alveoli, the alveolar

capillary networks, alveolar ducts and sacs, and reSpiratory

77
bronchioles. He concluded that these components occupied
90 percent of the total lung volume. The remaining 10 per-
cent was occupied by the larger blood vessels and airways.
The stereologically determined percent volumes and vessel
lengths in the present investigation (Tables 21-26) closely
coincide with those determined by Weibel. Morphometric
analysis revealed that 7 to 13 percent of the pulmonary
tissue was occupied by blood vessels larger than capillaries
and airways larger than respiratory bronchioles. The
remaining 87 to 93 percent of the pulmonary tissue was
occupied by the alveoli, the alveolar capillary networks,
alveolar ducts and sacs, and respiratory bronchioles.

The combined lengths of pulmonary vessels (larger
than capillaries) were calculated in meters per lobe. In
all instances the total vessel length in the right middle
lobe was greater than that in the left. The combined
lengths in the control rabbit left middle lobe was approxi-
mately 96 m. According to Rahn and Ross (1957) this lobe
occupies 5.8 percent of the total canine lung weight. Thus

the projected vessel lengths for the entire canine lung is:

96 m = X m
5.8% 100%
X = 1,655 m

A review of the literature has revealed no other similar
stereologic studies of intrapulmonary vessel lengths other
than Weibel's (1964) determination of pulmonary capillary

lengths.

78

According to Daly and Hebb (1966f), the pulmonary
artery accompanies the bronchi as far as the respiratory
bronchioles. At this point an artery is given off to each
alveolar duct which in turn gives off a number of small
arteries supplying the alveolar capillary networks.

Weibel (1964) has calculated the combined length of
the branches of the adult human respiratory tree down to the
level of the terminal bronchiole. This length approaches
257 m. The fact that the present investigation measured both
pulmonary arteries and veins down to the alveolar capillary
level accounts for the greater vessel length.

Weibel (1964) has also determined the combined
length of the pulmonary capillary networks. .He concluded
that:

The total number of capillary segments per alveolus = 1800

The mean length of each capillary segment = 10,3°10’4 cm

The total number of alveoli = 300'106
Therefore the combined lengths of all pulmonary capillary
segments is equal to:
(1800)(10.3'10_4 cm)(300'106) = 5,562,000 m
Thus the calculated length of pulmonary vessels larger than
capillaries appears to be realistic.

The stereologic data was then statistically evaluated
by an analysis of covariance (Ostie, 1970), and Stapelton's
(1972) proCedure for comparing individual means. This

technique eliminated the inherent anatomical variations

between the right and left lobes and gave an accurate

79
evaluation of the stimulation effects. The results of that
analysis are incorporated in the following discussion.

Essentially, there are three primary avenues through
which vagal stimulation may influence pulmonary circulation:
(1) indirectly through vagally induced bronchomotion and its
mechanical effects on nearby vessels, (2) indirectly through
a vagally induced decrease in cardiac output, and (3)
directly by either constricting or relaxing smooth muscle in
the pulmonary blood vessels.

Neither isolated perfused nor tg gttt techniques
have been able to eliminate the simultaneous bronchomotor
and vasomotor responses to electrical or chemical stimuli.
Aviado (1965) discussed the interrelationship of the
pulmonary airway and arterial vascular trees. He suggested
that, because of their close anatomical relationship, some
of the vascular responses observed during parasympathetic
stimulation may be due to purely mechanical effects on the
vessels from associated changes in the caliber of pulmOnary
airways. Constriction of the airways would increase the
extra-vascular tension on nearby vessels resulting in an
increase in pulmonary vascular resistance. Relaxation of
the airways would have the opposite effect.

Daly and Hebb (1966g) summarized the passive effects
of acetylcholine on the pulmonary circulation. They noted
that in the majority of studies changes in pulmonary
arterial pressure (vasomotion) were accompanied by changes

in tidal volume (bronchomotion). The likelihood of these

80
secondary effects made it difficult to authoritatively state
that variations in pulmonary arterial pressure seen during
parasympathetic stimulation were solely due to direct
pulmonary vasomotion.

Several researchers have been able to demonstrate
significant vasomotor responses without concomittant
bronchomotor activity. Dale and Narayana (1935) studied
isolated perfused guinea-pig lungs under positive pressure
intratracheal inflation. Injection of acetylcholine
(5-50 ug) into the pulmonary artery resulted in a decrease
in outflow volume (vasoconstriction). The majority of
animals also demonstrated a decrease in tidal volume
(bronchoconstriction). In three preparations, a significant
vasoconstriction was observed unaccompanied by bronchomotor
activity. Dale and Narayana concluded that the increase in
pulmonary vascular resistance seen during parasympathetic
stimulation was a result of the combination of two factors:
(1) the direct vasomotor activity of the pulmonary blood
vessels, and (2) the mechanical force exerted on the
pulmonary vessels due to changes in airway caliber.

Alcock, Berry, and Daly (1935) studied the effects
of acetylcholine injection (200 ug-l.0 mg) on isolated
perfused canine lungs in which volume inflow and tidal air
was recorded. Smaller doses of acetylcholine resulted in an
increase in volume inflow (fall in pulmonary arterial
.pressure) while larger doses resulted in a decrease in

volume inflOw (rise in pulmonary arterial pressure). Both

81
these responses were accompanied by a decrease in tidal air
(bronchoconstriction). They concluded that a situation
could exist where pulmonary vasomotion would be partially
or solely independent of bronchomotor secondary effects.

In the present investigation, stereologically
determined absolute lobar volumes occupied by pulmonary
airways larger than respiratory bronchioles (Tables 21—26)
were statistically unaltered during the vagal stimulation.

The observed differences were due solely to the normal

variations in actual lobar volumes. The fact that active

bronchomotion was absent during vagal stimulation negates i
the possibility of airway constriction secondarily

influencing pulmonary vasomotion.

With the elimination of secondary bronchomotor
effects on pulmonary vasculature we now concern ourselves
with the possibility of cardiac output passively influencing
pulmonary circulation.

According to Folkow and Neil (1971), vagal
stimulation results in a decrease in cardiac rate, a decrease
in the contractility of atrial muscle, and no significant
alterations in the contractility of ventricular musculature.
The ultimate outcome of the combination of these three
effects is a decrease in cardiac output proportionate to the
degree of vagal stimulation.

Bell (1956) stated that the decrease in cardiac
output during vagal stimulation would be partially

compensated for by an increased stroke volume. He concluded

82
that the prolonged diastolic ventricular filling period and
its resultant increase in ventricular volume would stimulate
ventricular musculature thus increasing stroke volume.

The present investigation approached the evaluation
of passive cardiac output influences on pulmonary circulation
through an examination of the perfusion percentage results.
If variations in ink perfusion patterns were solely due to
changes in cardiac output then one would expect these
changes to be observed bilaterally on the surface of the
lungs.

Krahl (1968) studying the rabbit noted a marked
difference in ink perfusion between vagally stimulated and
unstimulated lobes. He found that vagally stimulated lobes
were extremely underperfused while the non-stimulated lobes
demonstrated the usual mosaic pattern of ink perfusion.
Krahl concluded that parasympathetic stimulation results in
active pulmonary vasoconstriction.

DeFouw (1970) in similar studies on the dog, rabbit,
and cat was unable to demonstrate extreme differences in
ink perfusion following either right or left vagal stimu-
lation. Contrary to Krahl, DeFouw concluded that vagal
stimulation results in a mild active pulmonary vasodilation
in the rabbit and cat, and no significant changes in the
perfusion of the canine lung.

The present study is in partial agreement with the
work of DeFouw. Comparison of the perfusion-percentages

(Tables 1-4) of the stimulated and unstimulated lobes from

83
the eXperimental animals demonstrates no significant
alterations in all three species. Examination of the
photographs (Figures 14-16) taken of the right and left
lungs of the experimental animals readily reveals that vagal
stimulation did not produce the marked differences in ink
perfusion as earlier reported by Krahl. Comparison of the
perfusion-percentages of the eXperimental to the control
animals demonstrates no significant variations in the dOg
and the cat but a bilateral decrease in perfusion in the
experimental rabbit. The bilateral nature of this decrease
indicates that it was most probably due to a passive reaction
to the vagally induced decrease in cardiac output. All other
perfusion-percentage variations were extremely small and
when one takes into consideration the subjective nature of
the data collection these variations become functionally
insignificant.

Stereologically determined absolute lobar volumes
occupied by pulmonary vessels (larger than capillaries) were
statistically unaltered during the vagal stimulation and
subsequent ink injection. The analysis of covariance
revealed that the differences observed between left and
right middle lobes were in accordance with that expected due
to the normal variations in lobe size. These results negate
the possibility of direct parasympathetic vasomotor activity
influencing the volume occupied by the larger pulmonary
vessels. They also suggest that cardiac output was not
significantly altered during the vagal stimulation and

subsequent ink injection.

84
Statistical evaluation of the volume occupied by ink
filled pulmonary capillary beds, and the length of larger
pulmonary vessels gave negative results in the dog and
statistically significant differences in the rabbit and cat.
These differences are discussed according to species. In
each case four comparisons were made.

Left control lobe to right control lobe

 

Left stimulated lobe to right stimulated lobe

 

Left control lobe to left stimulated lobe

 

Right control lobe to right stimulated lobe

 

Interpretations of the results of these comparisons are

incorporated in the following discussion.

Rabbit
Statistical evaluation of the capillary ink perfusion
in the rabbit middle lobes revealed the following:

Left control volume was less than right control volume

 

Left stimulated volume was less than right stimulated volume

 

In both the control and stimulated left lobes, the volume
occupied by ink filled pulmonary capillaries was less than

in their right counterparts. The decrease in volume was over
and above that which was expected due to the normal vari-
ations in total lobar volume. The fact that both the
stimulated and control lobes were affected indicates that

the differences were not due to vagal stimulation. The data
suggests the presence of an inherent variation in the
pulmonary capillary perfusion of the rabbit allowing greater

perfusion to the right middle lobe.

85
Left control volume was not different than left stimulated

volume. Right control volume was not different than right

 

stimulated volume.

 

The fact that no differences were observed between unilateral
stimulated and control lobes suggests that vagal stimulation
had neither a passive nor an active effect on the rabbit
pulmonary capillary ink perfusion.

1 Statistical evaluation of the length of pulmonary
vessels in the rabbit revealed the following:

Left control vessels were shorter than right control vessels

 

Left stimulated vessels were shorter than right stimulated
vessels

Again, in the rabbit, a situation exists in which both the
stimulated and control vessels of the left lobes were
shorter than their right counterparts. This was over and
above that expected due to the normal variations in total
lobar volume. It suggests the presence of an inherent
variation in the rabbit pulmonary tissue in which vessels
in the left middle lobe are significantly shorter than
those in the right.

Left control vessels were longer than left stimulated

 

vessels. Right control vessels were longer than right

 

 

stimulated vessels.

This data indicates that there was a bilateral decrease in
'the length of the pulmonary vessels in the stimulated
rabbit. The decrease in vessel length cannot be directly

attributed to an active pulmonary vasomotor response because

86
it was observed in both the right and left lobes of the
stimulated animals. A vagally induced decrease in cardiac
output could result in a decrease in vessel length but the
volume occupied by these vessels was not significantly

altered during the stimulation and subsequent ink injection.

9.5113.

Statistical evaluation of the volume occupied by ink
filled capillaries in the cat middle lobes revealed the
following:

Left control was not different than right control

 

Left stimulated was not different thgn right stimulated
Thus, the numerical differences in the volume occupied by
ink filled pulmonary capillaries between left and right
lobes of both experimental and control cats were due to the
normal variations in total lobar volume.

Left control volume was‘greater than left stimulated volume

Right control volume was greater than right stimulated volume

 

The bilateral nature of the increase suggests that it was
passively mediated. The fact that large vessel volume was
unaltered during the stimulation indicates that pulmonary
capillaries may be the first vessels affected by a decrease
in right ventricular outflow.

Statistical evaluation of the length of pulmonary
vessels in the cat revealed the following:

Left control was not different than right control

 

Left stimulated was not different than right stimulated

 

87
The numerical differences observed in the length of pulmonary
vessels between the right and left stimulated and control
lobes weredue solely to the normal variations in total
lobar volume.

Left control vessels were not different than left stimulated

 

vessels. Right control vessels were longer than right

 

stimulated vessels.

A reevaluation of the statistical data showed that the

.4-—-“_ fl!

vessels in both the left and right stimulated lobes were
shorter than those in their control counterparts. The ‘
differences between left stimulated and left control were
not large enough to be statistically significant. The
observed decrease in vessel length could be attributed to
a decrease in cardiac output, but the volume occupied by
the same vessels was not significantly altered during the
stimulation.
By far, the most perplexing results were obtained in
the stereologic data from the rabbit. Both the volume
occupied by the ink filled capillaries and the lobar vessel
lengths were significantly less in the left middle lobes
than in the right. These differences were observed in both
the stimulated and control animals and were larger than that
expected due to the normal variations in total lobar volume.
One can only conclude that there exists an inherent difference
in the pulmonary tissue of the rabbit which accounts for the

observed variations.

88
The present investigation has found no evidence
indicating that parasympathetic stimulation results in a
direct vasomotor response by the pulmonary circulation.

Evaluation Of The Experimental
Techniqtg

 

 

As with any experiment the more complicated the
technique the greater the probability of error. It is
therefore advantageous to discuss the areas most likely
subject to variability. Examination of the present
technique reveals several areas which require further
clarification.

Does the effect of vagal stimulation under sodium
pentobarbital anesthesia differ from that in a physio-
logically nOrmal state? One must concede that any anesthesia
alters the physiologic norm. The researcher must therefore
choose the type of anesthesia which produces the least
significant adverse effects. The barbiturate sodium
pentobarbital, was the anesthetic of choice in the present
investigation. Unfortunately there have been very few
definitive studies on the effects of pentobarbital on the
pulmonary circulation. Goldberg (1968) studying the effects
of barbiturates on the dog concluded that sodium pentobarbital
insignificantly altered the pulmonary circulation. It did
produce tachycardia and decreased cardiac stroke volume.

Carbachol, used in terminating the animal, is an
extremely potent parasympathomemitic drug. Should it reach

the lungs via the bronchial circulation would its effect

89
overshadow that of the vagal stimulation? If immediate
cardiac arrest does not follow the left ventricular carbachol
injection there is every likelihood that a small percentage
of the drug will reach the pulmonary circulation via
bronchial-pulmonary anastomoses. Carbachol entering the
lungs in this manner would be distributed equally to both
the stimulated and unstimulated lobes, making evaluation of
the vagal effects extremely difficult. The animals chosen
for histologic study were those which demonstrated the
most rapid cardiac arrest following injection of carbachol.
The drug's effect on the pulmonary circulation of these
animals was assumed to be minimal. Evaluation of the ink
perfusion-percentages revealed no significant differences
between animals who demonstrated immediate cardiac arrest
and those where the heart gave several functional beats
after left ventricular carbachol injection.

Is the vagal effect on pulmonary airways disrupted
by the constant pressure formalin inflation technique used
during the histOlogic preparation? The possibility does
exist that the constant pressure formalin inflation technique
employed for a twenty-four hour period may partially over-
shadow a vagally induced bronchoconstriction. Further
studies are required to accurately interpret the signi-
ficance of this proposed variability.

The technique of arranging the main artery and
bronchus in the center of each lobe sample was the method

employed to subjectively decrease sample variability.

90
Future attempts to stereologically evaluate pulmonary tissue
on a lobe basis would be enhanced by utilization of the
70 mm film strip technique developed by Wilson and Pickett
(1970). This would allow the entire lobe to be histologically
prepared and mounted on the larger film strip. It would
thus eliminate all questions concerning the reliability of
samples on a lobe basis.

The reliability of stereologic data could also be
increased by premeasuring the actual volumes of the right
and left histologically sectioned lobes by the water
displacement technique. Thus the volume of each lobe would
be directly related to the tissue being sectioned.

Section evaluation need not be carried out at the
sixth section interval. Data in Appendix A indicates that
sampling every twenty-first section would yield reliable
results. Only every twenty-first section from the strip
of serial sections need be mounted on the film. Thus, for
example, the number of mounted sections in the rabbit
experimental left middle lobe would decrease from 844 to 40.
This would enable the researcher to increase the number of

lobes being sampled and thus increase the data reliability.

SUMMARY AND CONCLUSIONS

Electric mid-cervical stimulation of the distal end
of the severed left vagus nerve of dogs, rabbits, and cats
was followed by ink injection and tg gttt formalin fixation.
The middle lobes from both stimulated (left) and control
(right) lungs were evaluated grossly for the degree of
peripheral ink perfusion, and histologically by sectioning
from the hilus to the periphery.

Histologic serial sections were stereologically
evaluated and the following calculated for both stimulated
and control lobes: (l) the relative volume occupied by the
pulmonary arteries, arterioles, veins, and venules, (2) the
relative volume occupied by the ink filled pulmonary
capillaries, (3) the relative volume occupied by the
pulmonary airways ranging in size from secondary bronchi to
terminal bronchioles, and (4) the length per unit volume of
the pulmonary arteries, arterioles, veins, and venules.

The above procedures were repeated on control animals which
did not undergo vagal stimulation.

Statistical analysis of the stereologic data as well

as subjective analysis of ink perfusion-percentages revealed

the following:

91

92

Dog. Data from ink perfusion-percentages and stereologic
section evaluation revealed that the vagal stimu—
lation and subsequent ink injection had neither a
passive nor an active effect on the canine
pulmonary circulation and airway volume.

Rabbit. Data from ink perfusion percentages and total vessel
length suggested the presence of a passively induced
pulmonary vasoconstriction during vagal stimulation.
The vessel length and volume occupied by ink filled
capillaries of both the stimulated and control left
lobes were significantly smaller than their right
counterparts. These observations suggest the
presence of inherent variations in rabbit pulmonary
tissue.

The remainder of the data indicated neither an
active nor a passive effect.

Cat. Data from the volume occupied by the ink filled
capillaries and vessel lengths suggests the
presence of a passively induced pulmonary
vasoconstriction during the vagal stimulation.
The remainder of the data indicated neither an
active nor a passive effect.

Statistical and subjective evaluations of the
eXperimental data, in all species, revealed no evidence
indicating that parasympathetic stimulation results in
active pulmonary vasomotion.

The possibility exists that during the vagal
stimulation small muscular pulmonary arteries constricted
and larger elastic arteries dilated. In such a situation,
the net effect on the total volume occupied by the larger
vessels would remain unaltered. This could account for the
concomitant bilateral decrease in capillary ink volume in

the stimulated rabbit and cat and the unaltered large vessel

volume.

93

Vagal stimulation did not result in the massive
constriction of precapillary muscular sphincters as earlier
reported by Krahl (1968). The evidence suggests that the
primary function of pulmonary vagal fibers is to carry
afferent signals from the pulmonary tissue to the central
nervous system. This is in agreement with Hirsch's (1969)
conclusions that the majority of pulmonary vagal fibers are
structurally and functionally afferent.

The close correlation between the present stereologic
data and that retrieved by Weibel (1964) attests to its
validity. The inherent variations observed in the rabbit
pulmonary tissue demonstrates the need for further

quantitative morphologic studies of the pulmonary circulation.

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98

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99

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100

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APPENDICES

APPENDIX A

APPENDIX A. Original data collected from the rabbit left
middle lobe.

Tables 6-9 contain the original data collected during
the histologic evaluation of the rabbit left middle lobe. A
survey of literature revealed that the optimum sample size
for this type of experiment had not been established. An
assumption was made that sampling every third section would
give extremely reliable data.’ The results of sampling every
third section were then tested for reliability at sixth,
twelfth, fifteenth, twenty—first, and thirtieth section
intervals. The data from the reliability tests were applied
to all subsequent section sampling.

Section sampling proceeded according to unit areas.
Three unit areas were sampled in each section large enough
to accomodate them without overlap. Obviously sections
close to the hilus and the periphery were too small to allow
three unit area samples. These sections necessitated
sampling of only one or two unit areas and are listed indi-
vidually on the tables. When section size permitted three
unit area samples, the counts of all three areas were

totaled and appear in the table as a sum.

101

 

 

102
Table 6.-—Rabbit--Left Middle Lobe
The original data indicating the number of times any portion
of one of the forty-two cross bars of the Weibel viewing
plate fell partially or wholly within the lumen of an artery,

arteriole, vein, or venule.

Sampling every third section beginning with section one at
the periphery and proceeding to the hilus.

A = Unit Area

Section # A-l A-2 A—3 A—4 A-5 A-6 A-7 A-8 A-9

 

1. 0
4. 0
7. 1
10. 1
l3. 0
16. 2
l9. 4
22. 4
25. 2
28. 0
31. 0
34. 3
37. 0
40. 4
43. 2
46. 4
49. 3
52. 1
55. 1
58. 3
61. 0
64. 3
67. 5
70. 5
73. 3
76. 3
79. 2
82. 1
85. 3
88. 1
91. 3
94. 1 6
97.; l 2
100. 4 4
103. 1 2
106. l 0
109. 0 1
112. l 5
115. 2 1
118. 2 4

Table 6.--(cont'd.)

103

 

 

Section # A-l A-Z A-3 A-4 A-5 A-6 A-7 A-8 A-9
121. 1 4
124. O 2
127. 1 2
130. 0 3
133. 1 1
136. 1 6
139. l 3
142. 10 4
145. l l
148. 0 l
151. 2 2
154. 3 l
157. 2 0
160. 0 0
163. 0 3
166. 0 3
169. 4 2
172. 5 l
175. 7 1
178. 0 3
181. 4 l
184. 1 l
187. 2 0
A A A
Section # 1-2-3 Section # 4—5-6 Section # 7-8-9
193. 2 196. 6 190. 10
202. 20 205. 3 199. 8
211. 4 214. 8 208. 7
220. 5 223. 4 217. 6
229. 6 232. 8 226. 2
238. 4 241. 6 235. 11
247. 8 250. 3 244. 12
256. 3 259. 7 253. 8
265. 8 268. 7 262. 5
274. 18 277. 9 271. 7
283. 17 286. 17 280. 15
292. 12 295. 16 289. 7
301. 14 304. 17 298. 14
310. 7 313. 4 307. 6
319. 24 322. 5 316. 9
328. 4 331. 11 325. 7
337. 19 340. 12 334. 13
346. 8 349. 5 343. 3
355. 15 358. 9 352. 7
364. 17 367. 5 361. 15
373. 5 376. 7 370. 17

Table 6.-—(cont'd.)

104

 

 

A A A
Section # 2-3 Section # 4-5—6 Section # 7-8-9
382. 9 385. 8 379. 3
391. 10 394. 10 388. 12
400. 6 403. 8 397. 9
409. 5 412. 10 406. 2
418. 3 421. 19 415. 9
427. 9 430. 10 424. 5
436. 21 439. 13 433. 5
445. 5 448. 14 442. 12
454. 12 457. 5 451. 11
463. 11 466. 11 460. 9
472. 6 475. 9 469. 8
481. 5 484. 10 478. 7
490. 7 493. 13 487. 3
499. 5 502. 17 496. 15
508. 15 511. 25 505. 8
517. 6 520. 13 514. 9
526. 4 529. 13 523. 3
535. 5 538. 10 532. 9
544. 8 547. 12 541. 7
553. 556. 7 550. 7
562. 565. 9 559.. 3
571. 574. 8 568. 6
580. 583. 7 577. 12
589. 592. 10 586. 5
598. 601. 7 595. 8
607. 610. 6 604. 8
616. 6 619. 11 613. 12
625. 13 628. 11 622. ’ 5
634. 6 637. 19 631. 3
643. 5 646. 8 640. 8
652. 3 655. 10 649. 4
661. 5 664. 5 658. 4
670. 9 673. 23 667. 8
679. 5 682. 5 676. 3
688. 8 691. 21 685. 13
697. 6 700. 7 694. 18
706. 8 709. 28 703. 11
715. 7» 718. 5 712. 3
Section # A-l A-4 A-7 A-8 A-9
721. 2
724. 0
727.
730. l
733.
736. 1 2

105

Table 6.--(Cont'd.)

Section # A-l A-2 A-3 ‘A-4 A-S A-6 A—7 A-8 A-9

 

739. l 2

742. l 2

745. 6 8

748. l 0

751. 3 0
754. 1 1

757. 3 2

760. 0 3

763. 0 9
766. 1 2

769. 4 2

772. 2 14

775. 1 3

778. 1 0
781. 2 2

784. 2 4

787. 2 O

790. l 2
793. 0 17

796. 1 8

799. 0 l

802. 0 0

805. 2 2
808. 0 12

811. 1 2

814. 1 3

817. 0 4
820. 7 3

823. 2 3

826. 12 2

829. 1 0

832. l 9
835. 2 0

838. 0 2

841. 0 4

844. 2 4

106

Table 7.--Rabbit—-Left Middle Lobe

The original data indicating the number of times any portion
of one of the forty-two cross bars of the Weibel viewing
plate fell on an ink filled capillary.

Sampling every third section beginning with section one at
the periphery and proceeding to the hilus.

A = Unit Area

Section # A-l A—2 A-3 A-4 A—5 A—6 A-7 A-8 A-9

 

1. 13

4. 0

7. 0

10. 1

13. 13

16. 15

19.’ 16

22. 15

25. 12
28. 7

31. 7

34. 16

37. 1

40. 18

43. 0

46. 17

49. 2

52. 13
55. 16

58. . 18

61. 5

64. 18

67. 14

70. 19

73. 10

76. 12

79. 0
82. 13

85. 16

88. 15

91. 17

94. 13 9

97. 12 10

100. 19 20
103. 8 11

106. 12 6

109. 3 12

112. 14 11
115. . 4 17
118. 8 12
121. 6 13

124. 6 2

Table 7.-—(Cont'd.)

107

 

 

Section # A-l A-2 A-3 A-4 A-S A-6 A-7 A-8 A-9
127. 8 16
130. ll 18
133. 13 13
136. 3 14
139. 15 11
142. 3 9
145. 19 11
148. 13 14
151. 0 12
154. 18 16
157. 19 17
160. 4 10
163. 22 21
166. 10 O
169. 0 7
172. 18 14
175. 12 15
178. O 4
181. 0 17
184. 21 12
187. 11 12

A A A

Section # 1-2-3 Section # 4-5—6 Section # 7-8-9
193. 51 196. 12 190. 38
202. 35 205. 14 199. 25
211. 46 214. 30 208. 43
220; 30 223. 17 217. 34
229. 44 232. 10 226. 37
238. 43 241. 26 235. 42
247. 21 250. 15 244. 48
256. 36 259. 16 253. 42
265. 16 268. 11 262. 51
274. 36 277. 15 271. 26
283. 24 286. 24 280. 30
292. 27 295. 30 289. 52
301. 44 304. 31 298. 40
310. 38 313. 23 307. 35
319. 30 322. 30 316. 32
328. 50 331. 21 325. 42
337. 22 340. 37 334. 36
346. 55 349. 19 343. 47
355. 49 358. 40 352. 58
364. 32 367. 23 361. 26
373. 33 376. 32 370. 45
382. 30 385. 25 379.. 43
391. 47 394. 40 388. 35

108

Table 7.—-(cont'd.)

 

A A A
Section # 1-2-3 Section # 4-5—6 Section # 7-8—9

400. 52 403. 23 397. 52
409. 26 412. 23 406. 48
418. 21 421. 22 415. 58
427.‘ 43 430. 22 424. 48
436. 30 439. 14 433. 55
445. 53 448. 35 442. 26
454. 45 457. 35 451. 48
463. 44 466. 27 460. 43
472. 31 475. 42 469. 40
481. 52 484. 33 478. 48
490. 41 493. 48 487. 47
499. 46 502. 24 496. 38
508. 37 511. 32 505. 33
517. 15 . 520. 27 514. 42
526. 32 529. 36 523. 19
535. 38 538. 46 532. 47
544. 32 547. 23 541. 46
553. 17 556. 30 550. 31
562. 46 565. 23 559. 46
571. 16 574. 35 568. 51
580. 41 583. 57 577. 48
589. 28 592. 26 586. 45
598. 39 601. 19 595. 55
607. 30 610. 35 604. 37
616. 25 619. 22 613. 39
625. 31 628. 30 622. 42
634. 25 637. 32 631. 45
643. 48 646. 46 640. 47
652. 39 655. 33 649. 47
661. 46 664. 37 658. 43
670. 27 673. 24 667. 44
679. 24 682. 37 676. 40
688. 26 691. 24 685. 36
697. 37 700. 28 694. 37
706. 22 709. 19 703. 47
715. 35 718. 17 712. 36

Section # A-l A—2 A—3 A-4 A-Sv A-6 A-7 A-8 A-9

 

721. 12 13

724. 3 9
727. 19 O

730. 20 13

733. 14 12

736. 15 17
739 ll 11

742. 17 10

Table 7.--(cont'd.)

Section #

745.
748.
751.
754.
757.
760.
763.
766.
769.
772.
775.
778.
781.
784.
787.
790.
793.
796.
799.
802.
805.
808.
811.
814.
817.
820.
823.
826.
829.
832.
835.
838.
841.
844.

A-l

11

20

l7

l9

A-Z

ll

18

17

12

16

11

21

17

12

19

14

14

109

17

15

ll

19

17

13

16

19

l6

14

21

18

14

12

10

14

10

16

13

19

12

14

16

15

10

26

16

15

10

22

18

11

17

20

19

18

19

110
Table 8.—-Rabbit—-Left Middle Lobe

The original data indicating the number of times pulmonary
arteries, arterioles, veins, and venules fell within the
outer limits of the Weibel square. Vessels intersected by
the left and upper edges of the square were counted while
those intersected by the right and lower edges were not.

Sampling every third section beginning with section one at
the periphery and proceeding to the hilus.

A = Unit Area

Section # A-l A-2 A—3 A-4 A-S A—6 A-7 A-8 A—9

 

1. 4

4. 1

7. 6

10. 7

13. 3

16. 3

19. 7

22. 6

25. 3
28. 3

31. 1

34. 8

37. 2

4o. 6

43. 3

46. 8

49. 5

52. 5
55. 2

58. 4

61. 1

64. 5

67. 6

7o. 4

73. 3

76. _ 4

79. 1
82. 5

85. 5

88. 2

91. 5

94. 1 3

97. 1 5
100. 7 2
103. 3 6
106. 2 o
109. ‘ 2 4
112. , 2 7
115. 5 5

Table 8.——(cont'd.)

111

 

 

Section # A—l A-2 A-3 A-4 A—5 A-6 A-7 A-8 A—9
118. 4 4
121. 2 7
124. 1 2
127. 1 4
130. l 4
133. 2 5
136. 2 2
139. 3 9
142. 2 4
145. 1 4
148. 1 2
151. 4 6
154. 3 2
157. 1 1
160. 1 1
163. 1 3
166. 1 1
169. 2 5
172. 2 3
175. 2 1
178. O 1
181. 5 l
184. 1 3
187. 5 6
A A A
Section # 1-2—3 Section # 4-5-6 Section # 7-8-9
193. 6 196. 8 190. 8
202. 5 205. 5 199. 6
211. 10 214. 10 208. 11
220. 10 223. 8 217. 11
229. 13 232. 11 226. 4
238. 10 241. 11 235. 7
247. 12 250. 7 244. 9
256. 10 259. 14 253. 9
265. 9 268. 13 262. 9
274. 8 277. 8 271. 9
283. 8 286. 9 280. 4
292. 6 295. 10 289. 8
301. 11 304. 6 298. 10
310. 9 313. 4 307. 14
319. 10 322. 5 316. 13
328. 8 331. 12 325. 6
337. 11 340. 9 334. 10
346. 5 349. 8 343. 6
355. 11 358. 18 352. 12
364. 8 367. 10 361. 7

Table 8.—-(cont'd.)

112

 

 

A A A
Section # 1—2-3 Section # 4—5-6 Section # 7-8-9
373. 7 376. 12 370. 7
382. 8 385. 12 379. 9
391. 8 394. 19 388. 17
400. 12 403. 12 397. 7
409. 9 412. 12 406. 10
418. 10 421. 13 415. 9
427. 10 430. 8 424. 11
436. 7 439. 9 433. 10
445.‘ 9 448. 14 442. 10
454. 11 457. 13 451. 9
463. 13 466. 16 460. 9
472. 7 475. 12 469. 11
481. 12 484. 11 478. 14
490. 16 493. 8 487. 7
499. 11 502. 6 496. 13
508. 11 511. 10 505. 15
517. 11 520. 11 514. 12
526. 10- 529. 19 523. 4
535. 11 538. 13 532. 6
544. 14 547. 9 541. 11
553. 8 556. 17 550. 12
562. 15 565. 14 559. 10
571. 12 574. 13 568. 14
580. 6 583. 12 577. 18
589. 7 592. 12 586. 17
598. 10 601. 16 595. 14
607. 8 610. 13 604. 20
616. 9 619. 11 613. 14
625. 7 628. 10 622. 17
634. 9 637. 11 631. 11
643. 10 646. 13 640. 14
652. 8 655. 8 649. 14
661. 11 664. 12 658. 10
670. 11 673. 6 667. 13
679. 10 682. 7 676. 15
688. 7 691. 11 685. 11
697. 9 700. 11 694. 17
706. 6 709. 8 703. 14
715. 14 718. 18 712. 16
Section # A—l A-2 A-3 A—4 A-S A~6 A-7 A-8 A—9

721. 3
724. 2 1
727. 3 3
730. 4 3
733.
736. 5 4
739. 5 5

113

Table 8.~~(cont'd.)

Section # A-l A-2 A-3 A—4 A-5 A-6 A-7 A-8 A-9

 

742. 4 3

745. 2 3

748. 3 2

751. 2 . O
754. 6 l

757. 5 2

760. 0 8

763. 2 2
766. 7 4 '
769. ' 3 4

772. 4 2

775. 3 5

778. O 4
781. 2 5

784. l 5

787. 3 0

790. 2 2
793. 0 2

796. 2 3

799. 3 ' 3

802. 1 2

805. 4 2
808. l 3

811. l 3

814. 4 4

817. 0 2
820. 4 2

823. 2 3

826. 3 5

829. l 2

832. 2 2
835. 7 3

838. 5 6

841. O 5

844. 4 8

114

Table 9.—-Rabbit--Left Middle Lobe

The original data indicating the number of times any portion
of one of the forty-two cross bars of the Weibel viewing

plate fell partially or wholly within the lumen of an airway
ranging in size from secondary bronchi to terminal bronchioles.

Sampling every third section beginning with section one at
the periphery and proceeding to the hilus.

A = Unit Area
Section # A-l A-2 A-3 A-4 A-S . A-6 A—7 A-8 A-9

l. 0
4. 0
7. 0
10. 0
l3. 0
16. 0
19. 0
22. 0
25. 0
28. 0
31. O
34. 0
37. 0'
40. O
43. 0
46. O
49. 0
52. 0
55. O
58. 0
61. l
64. O
67. 0
70. 0
73. O
76. 1
79. 0
82. 0
85. 1
88. O
91. 0
94. O l
97. l 0
100. 3 0
103. 0 0
106. 2 1
109. 0 0
112. 1 0
115. 0 1
118. 1 2

115

Table 9.-~(cont'd.)

A-6 A-7 A-8

 

 

Section # A-l A-2 A-3 A—4 A—5 A—9
121. l l
124. 3 1
127. 0 0
130. 3 2
133. 1 O
136. 0 5
139. l 2
142. O l
145. 0 3
148. 2 2
151. O l
154. 5 2
157. l l
160. O O
163. 1 0
166. l 4
169. 0 l
172. 2 4
175. O 3
178. 0 0
181. 4 l
184. 0 0
187. 0 2
A A A
Section # 1-2-3 Section # 4—5-6 Section # 7-8-9
193. 2 196. 3 190. 10
202. 2 205. 6 199. 7
211. 10 214. 3 208. 9
220. 3 223. 2 217. 2
229. 2 232. 3 226. 4
238. l 241. l 235. 5
247. 5 250. 3 244. 10
256. 2 259. 1 253. 2
265. 4 268. 3 262. 12
274. 2 277. 4 271. 11
283. 6 286. 7 280. 7
292. 4 295. 2 289. 3
301. 8 304. 11 298. 6
310. 3 313. 6 307. l
319. 4 322. 11 316. 3
328. 0 331. 2 325. 3
337. 10 340. 8 334. 10
346. 7 349. O 343. 2
355. l 358. 4 352. 3
364. 7 367. 1 361. 1
373. 9 376. 3 370. O

116

Table 9.--(cont'd.)

 

 

A A A
Section # 1—2-3 Section # 4—5—6 Section # 7-8—9
382. 11 385. 2 379. l
391. 11 394. 1 388. 1
400. 14 403. 0 397. 2
409. 8 412. 5 406. 2
418. 2 421. -2 415. 9
427. 6 430. 9 424. 8
436. 8 439. 3 433. 0
445. 16 448. 5 442. 7
454. 3 457. l 451. 5
463. 5 466. 2 460. 9
472. 2 475. 2 469. 2
481. 9 484. 1 478. 5
490. 3 493. 14 487. 6
499. 1 502. 20 496. 1
508. 4 511. l 505. O
517. 3 520. 1 514. 2
526. 2 529. 3 523. 27
535. l 538. 4 532. 22
544. 3 547. l 541. 2
553. 29 556. 1 550. 0
562. 15‘ 565. 1 559. 2
571. 26 574. 4 568. 11
580. 14 583. 13 577. 3
589. 15 592. 3 586. 4
598. 21 601. 4 595. 0
607. 14 610. 7 604. 5
616. 24 619. 1 613. 4
625. 8 628. 2 622. 3
634. 17 637. 2 631. 4
643. 7 646. ,4 640. l
652. 10 655. 6 649. 2
661. 5 664. 0 658. 5
670. 16 673. 4 667. 4
679. 12 682. l 676. 5
688. 13 691. 2 685. 2
697. 9 700. 4 694. 6
706. 4 709. 1 703. l
715. 11 718. 2 712. 1
Section # A-l A—2 A—3 A-4 A-5 A-6 A-7 A-8 A-9
721. 0 0
724. 0 2
727. 2 O
730. 0
733. 13 O
736. l 1
739. 0 0

117 _

Table 9.--(cont'd.)

Section # A—l ‘A-2 A-3 A-4 A-S A-6

742. l 1

745. O O
748.

751. l

754. l O

757. 1 2

760. 13
763.

766. O 6

769. 0 2

772. O 1
775.

778. 12

781. 0 O

784. 1 1

787. 0
790.

793. 14 O

796. 1 l

799. 2 3
802.

805. 3

808. 1 1

811. 2 3

814. l
817.

820. 2 l

823. 3 l

826. 0 0
829.

832. l

835. O 0

838. O 4

841. 13
844.

ll

12

10

118

Totals of the original data indicating the number of
times any portion of one of the forty-two cross bars
of the Weibel viewing plate fell partially or wholly
within the lumen of an artery, arteriole, vein, or
venule.

Totals of the original data indicating the number of

_ times one of the forty-two cross bars of the Weibel

viewing plate fell on an ink filled capillary.

Totals of the original data indicating the number of
times pulmonary arteries, arterioles, veins, and
venules fell within the outer limits of the Weibel
square. Vessels intersected by the left and upper
edges of the square were counted while those
intersected by the right and lower edges were not.

Totals of the original data indicating the number of
times any portion of one of the forty-two cross bars
of the Weibel viewing plate fell partially or wholly
within the lumen of an airway ranging in size from
secondary bronchi to terminal bronchioles.

Table 10.—-Rabbit——Left Middle Lobe

119

 

Section Intervals 3 6 12 15 21 30
1.

Total hits 2054 1010’ 528 407 307 219

Unit areas sampled 710 354 178 142 103 71

Mean value 2.89 2.85 2.96 2.86 2.98 3.08
2.

Total hits 8288 4069 2016 1607 1164 768

Unit areas sampled 710 354 178 142 103 71

Mean value 11.67 11.49 11.32 11.31 11.30 10.81
3.

Total hits 2418 1180 578 493 367 244

Unit areas sampled 710 354 178 142 103 71

Mean value 3.40 3.30 3.80 3.47 3.56 3.43
4.

Total hits 1248 585 296 256 194 144

Unit areas sampled 710 354 178 142 103 71

Mean value 1.75 1.65 1.66 1.80 1.88 2.02

APPENDIX B

APPENDIX B. Original data from lobar volumetric analyses
and stereologic data measurements.

Tables 11, 12, and 13 contain the original data from
the volumetric analyses. In each case, three experimental
animals of each species were used to determine the mean
lobar volume. The previously described water displacement
technique was employed for all measurements. Table 14
contains the original data from the volumetric analyses of
the control animals. Only one representative animal of each
species was used in this study.

The extremely large quantity of numerical data
prohibited individual lobar evaluations similar to that
seen in Appendix A. The original data was summarized by
evaluating each lobe as a whole and then dividing the data
into three anatomical portions; a hilar, a middle, and a
peripheral. Tables 15-20 contain the summarized results.
Tables 21-26 contain the calculated absolute volumes and
lobar vessel lengths for both the experimental and control
animals. The abbreviations appearing in Tables 15-26 are
listed as follows:

(1) = Volume occupied by pulmonary vessels larger than
capillaries.

(2) 2 Volume occupied by ink filled pulmonary capillaries.

(3) = Volume occupied by pulmonary airways larger than
respiratory bronchioles.

120

121

(4)

Length of pulmonary vessels larger than capillaries.

T. = Total number of hits

P. = Possible number of hits

R.V. = Relative volume

P.C. = Total number of profiles counted
U.A. = Total number of unit areas sampled
M. = Mean value

S.A. = Area of Weibel square

A.V. = Actual volume

Ab.V. = Absolute volume

122

Table ll.--Volumetric analysis by water displacement
technique

Dog

Experimental animals-Middle lobes

 

Left Right
Dog # l Flask volume 2120 cc 2120 cc
Flask volume 2095 2085
with lobe
Lobe volume 25 35
Dog # 2 Flask volume 2120 2120
Flask volume 2095 2085
with lobe
Lobe volume 25 35
Dog # 3 Flask volume 2120 2120
Flask volume 2100 2090
with lobe
Lobe volume 20 '30
Mean volume 23 33

 

123

Table 12.-—Volumetric analysis by water displacement

technique

Rabbit

Experimental animals-Middle lobes

 

Rabbit # l Flask volume

Flask volume
with lobe

Lobe volume

”Rabbit # 2 Flask volume

Flask volume
with lobe

Lobe volume

Rabbit #3 Flask volume

Flask volume
with lobe

Lobe volume

Mean volume

Left

510 cc

501

510

501

510

501

Right
510 cc

498

12

510

499

11

510

498

12

12

 

124

Table 13.-~Volumetric analysis by water displacement
technique

Cat

Experimental animals—Middle lobes

 

Left Right
Cat # 1 Flask volume 485 cc 485 cc
Flask volume 477 474
with lobe
Lobe volume 8 11
Cat # 2 Flask volume 485 485
Flask volume 478 475
with lobe
Lobe volume 7 10
Cat # 3 Flask volume 485 485
Flask volume 477 475
with lobe
Lobe volume 8 10

Mean volume 8 10

 

125

Table l4.-—Volumetric analysis by water displacement
technique

Control animals——-Middle lobes

 

Left Right
Dog # l Flask volume 2130 cc 2130 cc
Flask volume 2105 2095
with lobe
Lobe volume 25 35
Rabbit # l Flask volume 530 530
Flask volume 521 519
with lobe
Lobe volume 9 11
Cat # l Flask volume 530 530
Flask volume 521 519
with lobe

Lobe volume 9 11

 

Table 15.--Experimental Dog

(1)

(2)

(3)

(4)

Hilar
Middle
Periph

Me an

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

126

 

 

907
1164
474

2545

1959

3040

2396

7395

237
443
200

880

P.C.

 

608
786
549

1943

 

 

 

 

 

 

 

Left Right
P. R.V. T. P. R.V
8,190 .1107 1639 10,206 .1605
12,474 .0933 872 11,340 .0768
8,988 .0527 471 9,492 .0496
29,652 .0856 2982 31,038 .0956
8,190 .2391 1951 10,206 .1911
12,474 .2437 2941 11,340 .2593
8,988 .2665 2281 9,492 .2403
29,652 .2498 7173 31,038 .2302
8,190 .0289 557 10,206 .0238
12,474 .0355 324 11,340 .0285
8,988 .0222 164 9,492 .0172
29,652 .0289 1045 31,038 .0232
.A. S.A. p.c U.A. S.A.
195 3.117 .81mm 568 243 2.337 .81mm
297 2.646 .81mm 641 270 2.374 .81mm
214. 2.565 .81mm 485 226 2.146 .81mm
706 2.776 .81mm 1694 739 2.286 .81mm

Table l6.--Control Dog

(1)

(2)

(3)

(4)

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

127

 

 

 

 

 

 

 

 

 

 

 

Left _ Right __
T. P. R.V. T. P. R.V.
405 8,862 .0457 1480 8,526 .1811
733 9,324 .0786 715 8,568 .0833
665 9,198 .0723 606 8,442 .0719
1803 27,384 .0657 2801 25,284 .1107
2270 8,862 .2562 1610 8,526 .1967
2582 9,324 .2769 2453 8,568 .2863
3450 9,198 .3750 2554 8,442 .3025
8302 27,384 .3027 6617 25,284 .2618
395 8,862 .0443 547 8,526 .0669
694 9,324 .0745 516 8,568 .0602
279 9,198 .0303 268 8,442 .0317
1368 27,384 .0476 1331 25,284 .0529
P.C. U.A. M. S.A. P.C. U.A. M. S.A.
376 211 1.782 .81mm 551 212 2.714 .81mm
574 222 2.585 .81mm2 584 204 2.714 .81mm
793 219 3.621 .81mm2 552 186 2.746 .81mm2
1743 652 2.673 .81mm 1684 602 2.784 .81mm2

Table l7.-—Experimenta1 Rabbit

(l)

(2)

(3)

(4)

Hilar
Middle
Periph

Mean

Hilar
Middle

Periph

'Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

128

 

 

 

 

Left
T. P. R.V.
411 5,040 .0815
474 5,922 .0800
182 3,906 .0466
1067 14,868 .0693
1446 5,040 .2869
1688 5,922 .2850
946 3,906 .2422
4080 14,868 .2713
260 5,040 .0515
233 5,922 .0393
92 3,906 .0235
585 14,868 .0381
P.C. .A. M. S.A.
408 120 3.400 .81mm
464 141 3.291 .81mm
307 93 3.236 .81mm
1179 354 3.309 .81mm

 

 

 

 

Right
T. P. R.V.
663 7,434 .0892
868 8,316 .1044
275 6,174 .0445
1806 21,924 .0793
2109 7,434 .2836
2581 8,316 .3103
1837 6,174 .2971
6527 21,924 .2971
529 7,434 .0711
377 8,316 .0453
126 6,174 .0204
1032 21,924 .0463
P.C. .A. S.A.
624 177 3.525 .81mm
632 198 3.192 .81mm
453 147 3.081 .81mm
1709 522 3.266 .81mm

Table 18.--Control Rabbit

(l)

(2)

(3)

(4)

Hilar
Middle
Periph

Mean

Hilar

'Middle

Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

129

 

 

 

 

 

 

 

 

 

 

Left Right
T. P. R.V. T. P. R.V.
590 4,494 .1312 471 5,124 .0919
480 4,914 .0956 687 5,166 .1329
392 4,788 .0818 474 5,166 .0917
1462 14,196 .1029 1632 15,456 .1054
1451 4,494 .3228 1711 5,124 .3339
1506 4,914 .3064 1864 5,166 .3564
1708 4,788 .3567 1953 5,166 .3780
4685 14,196 .3286 5528 15,456 .3563
590 4,494 .0518 471 5,124 .0772
480 4,914 .0403 687 5,166 .0230
392 4,788 .0273 474 5,166 .0284
572 14,196 .0403 662 15,456 .0429
P.C. U.A. M S.A. P.C. U.A. M S.A.
438 107 4.093 .81mm 585 122 4.795 .81mm
467 117 3.991 .81mm 583 123 4.739 .81mm
557 114 4.886 .81mm 586 123 4.763 .81mm
1462 338 4.323 .81mm 1754 368 4.765 .81mm

Table l9.--Experimental Cat

(1)

(2)

(3)

(4)

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Me an

130

 

 

 

 

 

 

 

Left Right
T. P. R.V. T. P. R.V.
368 5,418 .0679 372 6,930 .0536
525 6,048 .0868 629 7,686 .0818
202 4,830 .0418 195 6,468 .0301
1095 16,296 .0655 1196 21,084 .0552
986 5,418 .1819 1350 6,930 .1948
941 6,048 .1555 1262 7,686 .1641
727 , 4,830 .1505 1090 6,468 .1685
2654 16,296 .1626 3702 21,084 .1758
88 5,418 .0162 121 6,930 .0174
238 6,048 .0393 166 7,686 .0215
97 4,830 .0200 65 6,468 .0100
423 16,296 .0252 352 21,084 .0163
.c. U.A. M S.A. p.c. U.A. M. S.A.
321 129 2,488 .81mm 489 165 2.964 .81mm
339 144 2.354 .81mm 437 183 2.278 .81mm2
294 115 2.556 .81mm 309 154 2.006 .81mm
954 388 2.466 .81mm 1235 502 2.416 .81mm2

 

 

 

 

Table 20.—-Control Cat

(1)

(2)

(3)

(4)

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

Hilar
Middle
Periph

Mean

131

 

 

 

 

 

 

 

 

 

 

Left Right
T. P. R.V. T. P. R.V.
532 6,216 .0855 563 7,812 .0721
649 6,174 .1051 1010 7,686 .1314
355 6,048 .0587 525 7,812 .0672
1536 18,438 .0831 2098 23,310 .0902
1480 6,216 .2218 1699 7,812 .2174
1588 6,174 .2572 1916 7,686 .2492
1645 6,048 .2719 2046 7,812 .2619
4713 18,438 .2556 5661 23,310 .2428
361 6,216 .0581 510 7,812 .0653
292 6,174 .0473 296 7,686 .0385
91 6,048 .0148 223 7,812 .0285
744 18,438 .0403 1029 23,310 .0441
P.C. U.A. M. S.A. P.C. U.A. M. S.A.
454 148 3.067 .81mm2 640 186 3.660 .81mm2
521 147 3.612 .81mm2 681 183 3.716 .81mm2
654 144 4.541 .81mm2 865 186 4.650 .81mm2
1629 439 3.740 .81mm2 2186 555 4.008 .81mm2

132

Table 21.-—Experimental Dog

 

 

 

Left Lobe Right Lobe
Large vessel volume
R.V. A.V. Ab.V. Ab.V. A.V. R.V.
.1107(23cc) = 2.550c -------- Hilar ------- 5.31cc = (33cc).1605
.0933(23cc) = 2.14cc -------- Middle ------ 2.54cc = (33cc).0768
.0527(23cc) = 1.22cc -------- Periph ------ 1.65cc = (33cc).0496
.0848(23cc) = = (33cc).0972

1.96cc -------- Mean -------- 3.200c

Capillary volume

.2391(230c)
.2437(23cc)
.2665(23cc)
.2585(23cc)

Airway volume

.0289(23cc)
.0355(23cc)
.0222(23CC)
.0306(23CC)

5.31cc -------- Hilar ------- 6.30cc
5.6lcc -------- Middle ------ 8.5500
6.14cc -------- Periph ------ 7.92cc
5.69cc -------- Mean -------- 7.72cc

0.67cc -------- Hilar ------- 0.79cc
0.83cc -------- Middle ------ 0.96cc
0.51cc -------- Periph ------ 0.56cc
0.7lcc -------- Mean -------- 0.76cc

Large vessel length

.4...

2(3.1l7)(23cc)/.81mm
2(2.646)(23cc)/.81mm
2(2.565)(23cc)/.81mm
2(2.752)(23CC)/.81mm

A.V.

(33cc).19ll
(33CC).2593
(33cc).2403
(33cc).2403

(33cc).0238
(33cc).0285
(33cc).0172
(33cc).0232

A.V. S.A.

 

§iéi fl;
g=177m--Hilar---l90m=2(2-337)(33cc)/~31mm
2=150m--Midd1e--193m=2(2.374)(33cc)/-81mm
2=146m—-periph—-175m=2(2.146)(33cc)/-81mm

=156m--Mean----189m=2(2.314)(33cc)/.81mm

 

NNNN

133

Table 22.-~Control Dog

 

 

 

Left Lobe Right Lobe
Large vessel volume
R.V. A.V. Ab.V. Ab.V. A.V. R.V.
.0457(25cc) = 1.15cc -------- Hilar ------- 6.34cc = (35cc).1811
.0786(25cc) = 1.98cc -------- Middle ------ 2.9lcc = (35cc).0833
.0723(25cc) = 1.80cc -------- Periph ------ 2.52cc = (35cc).0719
.0655(25cc) = 1.65cc -------- Mean -------- 3.92cc = (35cc).1121

Capillary volume

.2562(25cc)
.2769(25cc)
.3750(25cc)
.3027(25cc)

Airway volume

.0443(250c)
.0745(25cc)
.0303(25cc)
.0476(25cc)

6.40cc -------- Hilar ------- 6.90cc
6.93cc -------- Middle ------ 10.0lcc=
9.38cc -------- Periph ------ 10.6lcc=
7.58cc -------- Mean -------- 9.17cc
1.10cc -------- Hilar ------- 2.35cc
1.88cc -------- Middle ------ 2-10cc
0.75cc -------- Periph ------ 1.12cc
1.20cc -------- Mean -------- 1.86cc

Large vessel length

14:.

2(1.782)(25cc)/.81mm
2(2.585)(25cc)/.8lmm
2(3.612)(25cc)/.81mm
2(2.652)(25cc)/.81mm

A.V.

 

(35cc).1967
(35cc).2863
(35cc).3025
(35cc).2618

(3500).0669
(35cc).0602
(35cc).0317
(35cc).0529

A.V. S.A.

 

S.A. g4_._
§=llom-—Hilar---235m=2(2.714)(35cc)/.81mm
2=160m--Middle--247m=2(2.862)(35cc)/.81mm
2=224m——Periph—--237m=2(2.746)(35cc)/.81mm

=164m--Mean-——-241m=2(2.784)(35cc)/.81mm

 

 

NNNN

134

Table 23.-~Experimenta1 Rabbit

Left Lobe Right Lobe

 

Large vessel volume

 

 

R.V. A.V. Ab.V. Ab.V. A.V. R.V.
.0815(9cc) = 0.74cc -------- Hilar -------- 1.07cc = (12cc).0892
.0800(9cc) = 0.72cc -------- Middle ------- 1.25cc = (12cc).1044
.0466(9cc) = 0.42cc -------- Periph ——————— 0.54cc = (12cc).0445.
.0686(9cc) = 0.62cc -------- Mean --------- 1.10cc = (12cc).084l

Capillary volume

.2896(9cc) = 2.58cc -------- Hilar -------- 3.4lcc = (12cc).2836
.2850(9cc) = 2.57cc -------- Middle ------- 3.72cc = (12cc).3103
.2422(9cc) = 2.18cc -------- Periph ------- 3.68cc = (12cc).2975
.2815(9cc) = 2.44cc -------- Mean --------- 3.49cc = (12cc).2908

Airway volume

.0515(9cc) = 0.47cc -------- Hilar -------- 0.85cc = (12cc).07ll
.0393(9cc) = 0.35cc -------- Middle ------- 0.54cc = (12cc).0453
.0235(9cc) = 0.22cc -------- Periph ------- 0.24cc = (12cc).0204
.0428(9cc) = = (12cc).0456

0.39cc -------- Mean --------- 0.55cc

Large vessel length

M-‘O A.V. S.A. M. A.V. S.A.

 

 

 

3.400)(9cc)/.81mm

( 6m--Hilar--—104m= 2(3.525)(12cc)/.81mm
(3.292)(9cc)/.81mm

(

(

7

73m--Middle---95m= 2(3.192)(12cc)/.81mm
3.236)(9cc)/.81mm 7
3.309)(9cc)/.81mm 7

NNNN
NNNN

2
2
2 2m--Periph---91m= 2(3.08l)(12cc)/.81mm
2 4m-—Mean ----- 97m= 2(3.266)(12cc)/.8lmm

135

Table 24.—-Control Rabbit

Left Lobe Right Lobe

 

Large vessel volume

 

 

R.V. A.V. Ab.V. Ab.V.

.1312(9cc) = 1.18cc -------- Hilar -------- 1.01cc
.0956(9cc) = 0.86cc -------- Middle ------- 1.46cc
.0818(9cc) = 0.74cc -------- Periph ------- 1.01cc
.1029(9cc) = 0.93cc -------- Mean ————————— 1.17cc

Capillary volume

.3228(9cc) = 2.19cc ———————— Hilar -------- 3.67cc
.3064(9cc) = 2.75cc -------- Middle ------- 3.93cc
.3567(9cc) = 3.21cc -------- Periph-e ----- 4.16cc
.3286(9cc) = 2.96cc -------- Mean --------- 3.92cc
Airway volume

.0518(9cc) = 0.47cc -------- Hilar -------- 0.85cc
.0403(9cc) = 0.36cc -------- Middle ------- 0.25cc
.0273(9cc) = 0.24cc -------- Periph ------- 0.31cc
.0389(9cc) = 0.36cc -------- Mean ————————— 0.47cc

Large vessel length

pg; A.V. S.A. 1_4__._
2(4.093)(9cc)/.81mm
2(3.99l)(9cc)/.81mm
2(4.886)(9cc)/.81mm
2(4.323)(9cc)/.81mm

NNNN

A.V. R.V.

 

 

(llcc).09l9
(llCC).1329
(llcc).09l7
(llCC).1055

(llCC).3339
(llCC).3569
(11cc).3780
(llcc).0426

(llcc).0772
(llcc).0230
(llcc).0284
(llcc).0426

A.V. S.A.

 

91m—-Hilar---130m= 2(4.795)(1lcc)/.81mm
89m--Middle--129m= 2(4.739)(11cc)/.8lmm
108m--Periph--129m= 2(4.763)(1lcc)/.81mm
96m--Mean----129m= 2(4.765)(1lcc)/.81mm

NNNN

136

Table 25.--Experimental Cat

Left Lobe Right Lobe

 

Large vessel volume

 

 

R.V. A.V. Ab.V. Ab.V. A.V. R.V.
.0679(8cc) = 0.54cc -------- Hilar -------- 0.54cc = (lOcc).0536
.0868(8cc) = 0.70cc -------- Middle ------- 0.82cc = (lOcc).0818
.0418(8cc) = 0.34cc -------- Periph ------- 0.30cc = (lOcc).0301
.0664(8cc) = 0.53cc -------- Mean --------- 0.56cc = (10cc).0564

Capillary volume

.1819(8cc) = 1.46cc -------- Hilar -------- 1.95cc = (10cc).1948
.1555(8cc) = l.25cc---—----Midd1e ------- 1.64cc = (10cc).164l
.1505(8cc) = 1.21cc -------- Periph ------- 1.69cc = (10cc).1655
.1642(8cc) = 1.31cc ----- *--Mean --------- 1.77cc = (10cc).l772

Airway volume

.0162(8cc) = 0.13cc -------- Hilar -------- 0.17cc = (10cc).0174
.0393(8cc) = 0.31cc -------- Middle ------- 0.22cc = (10cc).0215
.0200(8cc) = 0.16cc -------- Periph ------- 0.10cc = (10cc).0100
.0261(8cc) = 0.210c -------- Mean --------- 0.16cc = (10cc).0163

Large vessel length

M. A.V. S.A. M. A.V. S.A.

2(2.488)(8cc)/.81mm§
2(2.354)(8cc)/.81mm2
2

 

 

 

49m—-Hilar---73m= 2(2.964)(10cc)/.81mm
46m--Middle--56m= 2(2.278)(10cc)/.81mm
50m-—Periph--50m= 2(2.006)(10cc)/.8lmm
49m--Mean----60m= 2(2.450)(10cc)/.81mm

2(2.556)(8cc)/.81mm
2(2.465)(8cc)/.81mm

NNNN

137

Table 26.--Control Cat

Left Lobe

Right Lobe

 

Large vessel

R.V. A.V.

 

.0855(9cc)
.1051(9cc)
.0587(9cc)
.083l(9cc)

 

 

Capillary volume

.2218(9CC)
.2572(9CC)
.2719(9cc)
.2553(9cc)

Airway volume

.0581(9cc)
.0473(9cc)
.0148(9cc)
.0401(9cc)

Large vessel

4..

A.V.

volume

Ab.V. Ab.V.
0.77cc -------- Hilar -------- 0.79cc
0.95cc -------- Middle ------- 1.34cc
0.53cc -------- Periph ------- 0.74cc
0.75cc -------- Mean --------- 0.99cc
2.00cc -------- Hilar -------- 2.39cc
2.27cc -------- Middle ------- 2.74cc
2.45cc -------- Periph ------- 2.88cc
2.30cc -------- Mean --------- 2.67cc
0.52cc -------- Hilar -------- 0.72cc
0.42cc -------- Middle ------- 0.43cc
0.14cc -------- Periph ------- 0.32cc
0.36cc -------- Mean --------- 0.480c
length

S.A. M;

 

2(3.067)(9cc)/.81mm
2(3.612)(9cc)/.81mm
2(4.541)(9cc)/.81mm
2(3.740)(9cc)/.81mm

68m--Hi1ar--—99m=
- 80m--Middle-101m=
=101m--Periph-126m=
83m--Mean---109m=

NNNN

A.V. R.V.

 

(llcc).0721
(llCC).1314
(llcc).0672
(llcc).0902

(11CC).2174
(11CC).2492
(11cc).26l9
(11CC).2428

(llcc).0653
(llcc).0385
(llcc).0285
(llcc).044l

A.V. S.A.

 

2(3.660)(llcc)/.81mm
2(3.716)(llcc)/.81mm
2(4.650)(11cc)/.81mm
2(4.008)(llcc)/.81mm

 

NNNN

APPENDIX C

138

 

 

 

 

 

 

 

 

 

 

 

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22

 

 

SARASO’I'A, FLORIDA

 

 

NNNNN

 

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93