The ionic and cellular feedback relationships operating to effect the vascular decompensatory modifications were examined to reveal procedures for implementing protective measures guarding against vascular collapse when returning from a weightless environment to that of the earth's gravity. The surgical procedures for preparing the rat cremaster, and the fixation methods are described. Abstracts of publications resulting from this research are included

Alexander, W. C.

Hutchins, P. M.

Kimzey, S. L.

English

NASA Technical Reports Server

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FINAL REPORT
CONTRACT NAS 9-11690
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
REPORT PERIOD: May 1, 1971 - Mar. 31, 1973
CASE FILE
COPY
CONTRACTOR: The Bowman Gray School
of Medicine of Wake
Forest University
March 30, 1973
https://ntrs.nasa.gov/search.jsp?R=19730014342 2020-03-23T05:13:38+00:00Z
Introduction
This contract was designed to investigate the basic
causal mechanisms responsible for the loss of vascular
tone during alterations in transmural distending pressure
consistent with that accompanying shifts to a no-gravity
environment. The ionic and cellular feedback relationships_
operating to effect the vascular decompensatory modifications
were examined in an effort to reveal procedures that could
be implemented as protective measures guarding against
vascular collapse when returning from a weightless en-
vironment to that of the earth's gravity. :
: Since the hydrodynamic stimulus for normal vascular
tone is transmural distending pressure, appropriate challanges
to a skeletal muscle vascular bed were effected by graded
reductions in perfusion pressure. The microvascular di-
mensions and responsivity were monitered during such changes
and subsequently the cellular constituents responsible for
the Vascular adjustments'were analyzed utilizing the electron
microprobe located in the Manned Spacecraft Center Clinical
Laboratories.
Method o f Approach • . ' . ' .
Surgical Procedures: The microcirculatory preparation
used was an extensive modification of the Baez preparation
of the. rat cremaster, a thin skeletal muscle (approximately
250 microns thick in a 100 gm rat) with two layers of
muscle fibers oriented obliquely to each other.
The rats were anesthetized intraperitoneally, with a
warm solution of 10$ urethane and 2% chloralose in physio-
logical saline. .This solution has been demonstrated by us
to provide for the best maintenance of vascular tone and
vasombtion, in contrast to other anesthetics.
The animals 'were placed upon a special mounting board
and a tracheostomy performed with.a tube which had in-
corporated into its wall a heated thermistor, serving as
a pneumotachograph when connected to the appropriate
 ;
electronic circuitry. Rectal temperature was monitored with
a thermistor probe and maintained at 38°C by an electrically
heated mat.
An incision was made along the ventral suface of the
scrotum and the cremaster muscle and intact testis were
microdissected of surrounding connective tissue. The
cremaster was incised along its ventral aspect with care
being taken not to damage the underlying, testis. The
testis was then freed from the cremaster and extirpated..
All surgery was performed utilizing a micro heat cautery
and a stereo dissecting microscope. The cremaster was .
spread over an electrically heated .pedestal, 1.8 cm
diameter, and maintained between 3*+ and 35 C. The in situ
temperature of the cremaster had previously been deter-
mined by passing a thermistor through the peritoneum,
down into the scrotum between the testis and cremaster.
A round 1.8 cm #2 microslide cover:slip was position-
ed atop the cremaster. In this manner, the cremaster was
not subjected to equilibration with environmental gases
or ionic disturbances resulting from continual washing with
exogenous bathing solutions. •
After the preparatory.surgery, .a waiting period,of
approximately one hour allowed the cremaster arterioles to
regain tone and vasomotion. .
The cremaster vasculature was observed at powers
from 50 to 600X with a microscope modified to accept
long working distance optics and a high intensity, fiber
optic illuminator containing no infrared arid only minimal
near infrared radiation. The phototube of the microscope
projected to the front surface of a vldicon tube in a lense-
less TV camera. The electronic video image was recorded on
a one-half inch helical scan video recorder and viewed on
a 23 inch television monitor.
 :
The carotid artery was cannulated for a direct
recording of systemic arterial pressure and heart rate. It
had previously been determined in this laboratory that,
after initial transient, cannulation of one carotid artery
does not produce an alteration in systemic hemodynamic
parameters. ' . '
Fixation Procedures: The intial method of fixing the
rat cremaster muscle in vivo was by perfusing or suffusing
with Karnovsky's; FG fixative.
The methods considered were: 1) unidirectional per-
fusion from beneath the cremaster muscle, preparation;
2) bidirectional perfusion .from the top and bottom.of the.
preparation; and 3) intra-arterial (i.a.) injection of the
fixative through the femoral artery. .
In the unidirectional perfusion technique the
crerhaster was covered with a microscope slide cover s.lip
1+
(1.8 cm - diameter). The fixative was pumped up through
holes (OA mm in diameter) in the top of the pedestal.
Holes of this size are occluded easily by extravasated
erythrocytes. It was found in the initial studies that
some areas of the cremaster began to fix before others,
due to insufficient numbers of holes. The exhisting holes
were enlarged to 0.6 mm diameter arid others of the same
size were drilled in the pedestal top such that no part of
the cremaster was more than 1 mm distant from a hole.
Using the cessation of blood flow in the arterioles
under observation as a rough .index of tissue fixation, the
fixation.time for the unidirectional perfusion varied from
2 to 6.minutes. These time intervals for fixation were a
function of the geometrical relationships between the
tissue arid pedestal top and the hydraulics of the perfusion
system. Air pockets forming at the top of the pedestal
reservoir prevented areas above the air pockets from being
fixed at the same rate as areas not overlying air pockets.
More air escape holes were drilled in the outer portion of
the pedestal. This alleviated the problem to some degree
but air pockets were still observed occasionally.
When holes clogged with erythrocytes were observed
they were usually surrounded by other occluded holes.
Here again vessels overlying the group of clogged holes
would present longer fixation times. In future experi-
ments this problem should be corrected by covering the
pedestal with Saran Wrap until the muscle is mounted atop
the pedestal and by employing repetitive irrigation
techniques. It is possible that the pedestal top could
be covered with a membrane permeable for the fixation but
impermeable to erythrocytes.
In.early trials it.was, observed, utilizing the >
unidirectional perfusion method, that as increasing .amounts
of fixative were perfused, the cremaster was distended up-
ward, thus possibly upsetting any homeostatic equilibrium
that had been obtained. This problem was s'olved in later
experiments by decreasing the perfusion rate (to .07 ml/mm)
resulting :in no apparent shift in tissue position.
 :
In an attempt to shorten the fixation time it was
decided to employ a .preparation with no cover.slip and
to perfuse from both sides of'the cremaster. Fixative was
forced up from the reservoir thru the pedestal holes and
at the same time suffused onto the cremaster via irriga-
tion tubes. Fixation times using this technique ranged
from 1 to 2 minutes, an improvement, time-wise, over the
unidirectional perfusion. Air pockets, holes clogged with
erythrocytes and distention of the cremaster were also less
of a problem using this technique. The overlying layer of
fixative presented some small problems in.focusing, and
in general decreased the resolution, but not below tolerable
limits.
A third technique - intra-arterial injection of the
fixative upstream into the femoral artery - was then
attempted. In these experiments blood flow usually stopped
prior to a one minute mark after a 15 second i.a. infusion
of fixative at approximately 0.07 ml/min. It is believed
'
that this method fixes only the. blood vessel walls and
contents. Cremaster muscles fixed by this procedure were
generally more flexible in relation to cremasters fixed by
the two preceding methods, thus indicating insufficient
fixation of.the skeletal muscle by this method. Since the
vessel walls are fixed internally preserving their ionic
environment, the skeletal muscles surrounding the blood
vessels may be post-fixed., to allow proper sectioning for
the microprobe. It is unlikely that the pressure in the
cremaster arteries and arterioles was increased by this
method, in view of the small perfusion rate.
. .7
Cremasters fixed via the above described techniques
were flown to Houston and analyzed with the microprobe.
Contrary to prior consultative advice and reports in the
literature, it was found that Karnovsky's FG fixative did
not maintain the in vivo ionic concentrations but rather
rendered the cell membrane extremely porous,_it^particular, .
with respect to potassium. It seemed likely that any
fixation procedure based upon chemical binding would pro-
duce the same result. For this reason we then considered
methods of physically preserving the assay tissue.
: An asbestos and metal animal support board for the
preparation and quick-freezing of the cremaster by ; liquid
nitrogen application was fabricated and evaluated. The:
support of the experimental animal, including all structures
and tissues except the cremaster muscle, was fabricated from
asbestos in order that minimal central reflexes and respons-
es would be elicited daring the rapid fixation procedure.
A hollow stainless steel cylinder (1.8 cm diameter, 2.5 cm
height) supported the cremaster and connected via a .6 cm
I.D. stainless steel tube to a stainless steel reservoir.
The rapid fixation was initiated by pouring liquid nitrogen
into the reservoir which in turn immediately filled the
cremaster support cylinder. The support cylinder was vented
. . ' • • • ' ' . - • • • . ' . 3
to atmosphere via a .15 cm I.D. stainless steel tube
extending 2 cm above the cremaster level, thus allowing
air trapped in the cylinder to escape. This technique
allows for an almost instantaneous application of liquid
nitrogen to the thin stainless steel in direct contact with
the cremaster. .
Once the liquid nitrogen was applied and the con-
comitant fixation of the cremaster tissue observed micro-
scopically, the cremaster. was severed: from .the . animal.
The cremaster was removed from the support pedestal and
immediately placed on dry ice where it remained during
shipping to the analysis site at NASA-Houston.
Four samples of cremaster muscle, two controls and
two animals under simulated werlghlessness (reduced perfusion
pressure), were prepared by the above methods. The samples
were flown to Houston.where they underwent analysis.
The frozen muscles were cut in 8^u sections, and
placed on either glass slides for H & E staining or on
silicon discs for microanalysis.
The silicon disc sections were coated with Pd-Au
o o(200 A) and C (bOO A) and analyzed with the microprobe
9in the scanning secondary electron mode. The sections were
not protected sufficiently by the coatings used and it
was noted that future analyses would be performed with
thicker coatings. . -
Additional frozen sections were left at the Manned
Spacecraft Center for future analysis. -•• . ."
Efforts then were turned to perfecting of a method
for rapid fixation of the cremaster tissue by microwave
irradiation. Late in the period one Litton Model #500
microwave oven was received by the Contractor from NASA-MSC,
The laboratory housing the oven has necessarily been re-
wired to accomodate the unit with its specialized power
connections. No modification of the microwave oven has
been necessary. .
An animal support board, suitable for use with •.•
microwave irradiation has been constructed. This board,
which has been tested in several preliminary experiments
does not interfere with the irradiation of the tissue,
nor does it absorb sufficient energy to render itself a
thermal source in the fixation of the tissue.
The cremaster, after being surgically prepared, was
j .
( 10
Initially fixed, in a Litton Model Number 500. microwave
oven to preserve the biochemical and ionic balance.
The cremaster muscle was then removed from the animal
and placed in Ringer's solution at room temperature. The
muscle was cut into halves to facilitate.handling and
sectioning. - —
The muscle tissue was placed in an Ames Lab-Tek Model
Number D cryostat/microtome at -30°C. The tissue was later
embedded in Ames O.C.T. compound on a brass tissue block
\
and placed in the microtome holder for sectioning.
It was determined that sections of 8.0/i thickness
would be the most suitable size for electron microprobe
analysis. '
Initially, the first few sections cut at 8.0/1 were
mounted on a glass slide. Mounting was accomplished by
merely touching room temperature slides to the cut tissue
sections, where they firmly adhered. These sections were
then examined under a light microscope at low power with
dark-field illumination to insure that the current sections
being cut included portions of the vascular system. .When
It was determined that the sections on the glass slide con-
tained parts of the vascular system, the next sections
11
cut from the tissue were placed on polished metal discs
cooled to -30 C in the cryostat. These discs with the
sections remained .in the cryostat to prevent thawing of the
tissue sections. Subsequent sectioning of the muscle at
different levels and observation at low power under the
light microscope continued until 3 good tissue sections
were obtained which contained large portions of the vascular
network.
.The polished discs were then quickly placed :in a
Mikros (Varian) VE 10 vacuum evaporator before the tissue
sections had sufficient time to thaw out and thereby destroy
the vascular morphology, especially the vessel walls. The
ice crystals were then rapidly sublimed at a pressure of
0.1 to 0.05 u for 30 minutes to insure that all ice was re-
moved. In this manner, liquid water could not form in the
cells, which upon exposure to room temperature, would eventu-
ally evaporate and cause destruction of the vascular structures
After the vacuum evaporation, the discs and sections
could be exposed to .room temperatures without damage to
the cellular contents.
The sections were packed in plastic petri dishes with
layers of lens tissue paper between the' discs and shipped
12
to NASA for examination with the electron microprobe.
This method of rapid freeze-drying seems, to offer
the most promising technique for preservation of mor-
i - • .
phological and biochemical characteristics of the. vascular
network.
These sections also are awaiting analysis at the
Manned Spacecraft Center. .
Pressure Measuring System: The Biome.dical Engineering
Department has designed and constructed several devices
for use in measuring pulsatile pressures in rhicrovessels.
The pressure measuring system constructed is an extensive
modification of that reported by Dr. Curt A. Wiederhelm
(Amer. J. Physiol 207:173, 196'+). Component devices. con-
structed for this system include:
; (1) 1800 Hz Carrier Oscillator
: (2) . Micropipet Resistance Bridge . . • '.
(3) FET-Input Carrier Amplifier . :
Phase Detector
(5) Micropipet Resistance Calibration
Tank
In addition, considerable experimentation was under-
taken In developing and perfecting an "electromagnetic
13
pump" capable of supplying the necessary active element
for the pipette system.
Recommendations:
We have been informed by the Manned Spacecraft Center
that due to the time limitations imposed by the Skylab
project, the microprobe will not be available for use on
this contract in the near forseeable future. Due to this
fact and the difficulties encountered in the preservation
of intracellular ionic profiles, it is our recommendation
that this project be terminated at the present stage.
Publications:
A copy of a publication resulting from this work is
attached to this report. Future publications will give
credit to NASA and will likewise be forwarded to the
appropriate office. .
MiCKOVASCUI.AR KUStAKCH 4, 319-332 (1972)
Twentieth Annual Meeting of The Microcirculatory
Society, Atlantic City .
April 8-9, 1972
Abstracts
Considerations for the Functional Preservation of Vascular Tissue. W. C. ALEXANDER, P. M. HUTCHINS,
AND S. L. KIMZEY, NASA, Manned Spacecraft Center, Houston, TX 77058; Bowman Cray School
or Medicine, Winston-Salem, NC.
Classical techniques Tor the fixation of tissue have often assumed accurate preservation of the morpho-
melry and functional dimensions of the microvasculalure. Whereas these techniques may serve the
purpose for purely morphological studies, it is suggested that these methods do not preserve the func-
tional integrity of the microcirculation. In studies designed to elucidate the relationship between the
extra- and intracellular ionic constituency of the vascular smooth muscle cell and its response to
hemodynamic alteration, it has been noted that chemical fixatives are inadequate for this purpose. In
a series of-experiments designed to study such relationships in vivo it has been shown that fixation
involving chemical stabilization of the cellular structure renders the cell membrane extremely porous,
and fails to maintain normal electrolyte distribution. It is known that osmotic changes in the vessel
grossly influence the dimensions of the vascular space. Theoretically, the ideal fixative must maintain
those features representing the normal anatomical and physiological behaviour of the tissue. For these
reasons, our laboratories are currently involved in investigations designed to explore alternative methods
of tissue preservation. Although locally applied liquid nitrogen offers a means of accurate and rapid
stabilization of tissue structures, this technique is costly, hazardous and extremely inconvenient to
apply to specific areas without disruption of the adjacent elements. An alternative approach utilizes
focused microwave irradiation to stabilize the cell membrane while maintaining the integrity of vascular
tissue. ... ' .
Comparative Studies on Alpha-Adrenergic Receptors in Rat Blood Vessels. BURTON M. AJ.TURA,
Departments of Aneslhesiology and Physiology, Albert Einstein Col. Med., Bronx, N.Y.
Recent work on mouse and .cat visceral smooth muscle indicates that there may be differences
between the a-adrenergic receptors in the splenic smooth muscle of the two species. To our knowledge
no such comparative study has been made on blood vessels. Studies were therefore undertaken with
female and male rats using 15 catecholamine analogs and the a-antagonist pheniolamine. Rat mesehteric
arterioles (MA) were.examined in vivo using the image-splitter television microscopy system of Baez.
Rat aortas (A) and portal veins (PV) were examined in vitro under conventional isometric recording
conditions. Complete dose-response curves (DRC) for the various catecholamine agonists and analogs
were determined on all 3 vessel types in the presence and absence of phentolamine. From these DRC
we determined the relative maximum contractile responses (intrinsic activities, i.a.). affinities (ED50)
and the apparent dissociation constant (KH) of the antagonist for the receptor-agonist complexes.
Although the i.a. for E and NE - 1.0 on the A and PV, the i.a. for NE on the MA •= 0.8. Data indicate
that not only do the relative i.a. in a series of 15 catecholamine agonists differ from one type of rat blood
vessel to another but that the affinities relative to E and NE also vastly differ. Furthermore, the
apparent Kn values of the antagonist for the various receptor-agonist complexes also differ from one
vessel type lo another. Overall, the data strongly suggest: (i) the existence of different n-receptor types
in different blond vessels within a single mammalian species, and (2) that the structure-activity relation-
ships for a series of «drcnerxic agonists also differ from one vessel type to another.
Copyright fj 1972 by Acmtemic I'reM, Inc. oiq
All liehlv "I leptiicluclion in uny liirm reserved. ''


Functional preservation of vascular smooth muscle tissue

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