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Acta Astronautica Vol. 31. pp. 313-317. 1995 Cowripht Q 1995 Elscvier Science Ltd

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CARDIOVASCULAR AND ORGAN RESPONSES AND ADAPTATION RESPONSES TO

HYPOGRAVITY IN AN EXPERIMENTAL ANIMAL MODEL BION’DI R*, CAPODICASA E**, TASS1 C *** MEZZASOMA L*, BENEDETTI C*. VALIANT M*,

MARCO& P*, ROSS1 R*.

* Department of Clinical Medicine, Pathology and Pharmacology, General Pathology and Immunology Section, University of Perugia (Italy). ** Internal Medicine and Oncological Sciences Institute, University of Perugia (Italy). *** Department of Experimental Medicine and Biochemical Sciences, University of Perugia (Italy).

Abstract The head-down suspension (i.e antiorthostatic hypokinesia) rat is used to simulate weightlessness. However, little is known about cardiovascular and organ adaptation responses which, over a long time, can become pathologically significant. The purpose of this study was therefore to evaluate regional changes in the hematology parameters. Endotheline- I (ET- I ) concentration and urinary excretion of N-acetyl-P-D- glucosaminidase (EC 321.30) (NAG) in an experimental antiorthostatic rat model. The data indicate signilicant variations in the plasma ET-l level in time, in the superior and inferior cava vessel blood of animals maintained for 10 days in hypogravity with respect to controls. These changes do not seem to be due to hemoconcentration. The increase in urinary NAG was observed during the first 24h of experiment, indicating renal stress, probably due to adverse blood flow variations within the organ. We conclude that the plasma ET-1 level changes could be responsible, overall for the blood flow variations in the kidney and renal stress could be the consequence of extended antiorthostatic hypokinesia. The ET-l behaviour and urinary NAG excretion in rats exposed to antiorthostatic hypokinetic hydynamia offer possibilities for understanding if these changes might be reversible or when they become pathological. This could give some relevant information about the effects of prolonged hypogravity during the space voyage.

Introduction The weightless environment of space flight has produced certain biomedical effects in humans lq2. Since the effects of weightlessness in orbital space flight cannot be studied under earth conditions various experimental animal models, simulating hypogravity conditions, have been devised 3,4. The least traumatic method proposed is with the animals in head-down (anteriorthostatic hypokinesia) position s. The principal aspects. evaluated in this model, were mineral metabolism and skeletal respon&‘, cardiovascular adaptation - limited to modifications in central arterial pressure 5 - hemopoiesis regulation ceil response 8

7, inflammatory

behaviour 9. and quantitative morph0 leukocytaric

Allowing for the limitations of the experimental model, carried out under earth conditions, antiorthostatic suspension simulates quite

well the effects of weightlessness at regional levels in ‘the down-sloping parts of the body. With reference to the cardiovascular system we studied, with the antiorthostatic suspension model, the adaptation computed as regards redistribution of the hematic flow 7. The first significant modifications and adaptation reactions observed concerned the vascular system 8. These alterations can create problems both during space flight and re-entry to earth atmosphere. Various experiments carried out during space flights have demonstrated that in weightless conditions there is a redistribution of the astronauts’ blood flow which comes about earlier in the lower extremities than in the more central and cephalic sections of the cardiovascular system and this determines adaptation reactions also involving secretions of natriuretic atrial hormones which condition the subsequent hypovolemia which in turn causes h otension on return to normal hypogravity conditions YB At present there are no data about the behaviour of endothelines in weightless conditions. However, it is thought that they play a fimdamental role in the cardiovascular response, both under normal and pathological conditions. Besides, vascular distribution and expansion of the hematic volume are among the physical stimuli determining endotheline incretion. In addition, endotheline, part of a complex neurohormonal mechanism, could act in different ways, e.g. probable increased secretion of natriuretic atria1 hormone (NUA), constriction of the tierent arterioles of the renal glomendi and so activation of the renine - angiotensine system. This activation of hormone systems, influencing blood flow and its distribution, could play an important role during orbital flight and re-entry to the earth. The marked renal coparticipation in cardiovascular adaptation reactions and the repercussions, connected with adaptation reactions, prompted us to evaluate the urine concentration of N-acetyl-P D-glucosaminidase (NAG) which is an early marker of renal parenchymal injury. Copyright cE 1993 by the International Astronautical Federation. A II rights reserved Prof Ruggero Rossi. Profe.uor of General Pathology. Ilniversi~ of Perqia. Italy.

313

374 45th IAF Congress

Methods Head-Down Susnension

Male Sprague-Dawley rats (Charles River) average

wt. 170 g were suspended by “tail traction” technique

10 induce antiorthostatic hypokinesia. Briefly the rat

was placed in a head-down position at the lwel

corresponding to an angle of approximately 30“ in a

‘specially designed plexiglass ca e modeled after those

described by Shellock et B al. The animals were

maintained in this model for a 10 d. during which food

consumation and body weight were monitored daily.

The control rats were housed individually and pair-fed

3according to the food consumed by the suspended

rats. 4The room, in which the animals were housed,

was maintained at 24’ C with a 12 h light-dark cycle.

Collection of blood

For a 10 d period, every day, suspended rats and

synchronous controls were anesthetized with ether and

after laparatomy the cava vessel was clamped at the 2

cm level of the liver upper surface. The inferior cava

vessel was cannuled by the heparinized 17 G venflon 2, immediately the chest was opened and the heparinized

same canula was inserted into the superior cava vessel

up to the clamp. The blood samples, taken from these

two different sites, were called ICVB and SCVB

respectively

Hematolo@y

The total leukocyte, plated counts and hematocrit

values were determined using Coulter STKR on the

blood collected in vacutainer tubes containing EDTA

(1.5 mg/ml).

Extraction of olasma for RIA

Blood samples, collected in vacuitainer tubes

containing EDTA (I .5 mg/ml), 100 ~1 each of aprotinis

(200 Kallicrein inhibitor units/ml) and soybean trypsin

inhibitor (20 Units/ml), were centrifuged at 3000 rpm

at 4” C. To extract ET from plasma, we followed the

protocol recommended using Amersham’s Amprep 500

mg C2 columns.

RIA for ET- I

The ET RIA was performed by Endotheline - 1.2 112s

assay system (Amersham). This assay is based on the

competition between unlabelled ET and a fixed

quantity of 1l25 labelled ET for a limited number of

binding sites on an ET specific antibody. Although this

RIA method does not distinguish ET-I from ET-2 only

the plasma level of ET-I was measured in the rat as

ET-2 has geen found only in monkey kidney cell

cullures lo

samples Urine

Urine specimens. collected from rats, were centrifuged

at I 500 s g for 5 min. and stored at 4’ C. NAG

activity was performed wilhin 24 h of collection and

therefore no preservatives were used The variation in

the concentration of the samples was eliminated as far

as possible by dividing the enzyme activity by

creatinine concentration of the samples 1 I, Enzvme assay

Enzyme activity of NAG in 1 20 diluted urine samples

was measured as previously described I2 using 4-

Methylumbelliferyl-2-acetamido-2-deoxy+D-

glucopyranoside (4MUGINAc. Sigma Chemical Co.,

St Louis, MO, USA) as substrate in 0.1 M citrate/O.2

M phosphate buffer (pH 4 5). The sensitivity of the

fluorogenic substrate is such that urine may be diluted

prior to the assay. This dilution eliminates the effects

of any inhibitors present and the necessity to dialyse

the urine prior to the assay. Fluorescence of the

liberated 4-methylumbelliferone was measured on a

Perkin Elmer LS-3 fluorimeter. with excitation at 360

nm and emission at 446 nm The fluorimeter was

calibrated with 4-methylumbelliferone solution in 0.2

M glycine buffer (pH 10 6) One unit was defined as

the amount of the enzyme which converts I nmol

substrate per min into 4-methylumbelliferone at 37’C

Urinary activity was expressed as unitsimg of urinary

creatinine.

Results Head-down susoension

All of the rats in this study appeared to tolerate head-

down suspension well and were observed drinking and

eating during the IO d period of exposure to

antiorthostasis Rats subjected to tail suspension

gained weight at a rate comparable 10 that of the pair-

fed control group. At the end of the 10 d period of

simulated weightlessness the weights of tail-suspended

and control rats were not significantly different

Hematoloey

Table I shows that no significant changes were noted

in the absolute values of some hematological

parameters in the hypogravity with respect to the

controls and the sites from which the blood was taken

Table I - Some hematological parameters from

antiorthosratically suspended rats

Control Experimental

ICVB SCVB ICVB SCVB

HtC 34.04 32 05 32 II 32 95 + + + +

2.09 1.72 1 79 2.60

WBC I2 I7 12 32 1080 I3 26 ( 106/ml) z + + +

2 97 0:26 060 2-50

PTL 440 382 313 386 (106/ml) + + + +

98 159 So 6

Plasma ET- 1 level The behaviour of plasma ET-I level detected in the samples collected From SCVB as ICVB in hypogravity experimental rats and synchronous controls are represented in Fig. 1

Fig. I - Changes in plasma ET-I concentrations during 10 d of suspension (C-SCVB, C-ICVB and SCVB- ICVB represent control and experimental rats respectively).

1 3 5 7 Q 10

3m &YS

--P--G5CvE--t--Sam

--.A ClCvB + lCv5

There were no statistically significant differences for non-suspended controls among the SCVB and 1CVB (the value range was between 11.93 f 0.23 pg/ml and 13.86 f 0.30 pg/ml The plasma ET-1 level was followed during the 10 d period of suspension. On day 1 of suspension the ET-l concentrations were 9.33 f 0.22 pg/ml in ICVB and 18.02 f 0. I3 in SCVB respectively, which decreased progressively reaching the lower values at day 3 of suspension (7.39 f 0.32 pgml in ICVB) and at day 5-7 (I I .20 f 0.42 pg/ml in SCVB). This last value was not statistically different from the control. For the remainder of the suspension period the behaviour of plasma ET-I levels became inverted in both samples and the values increased reaching that of the controls at day 7 of tail suspension in the ICVB and in the SCVB. The plasma ET-I levels continued to increase reaching the maximum values at the last day of suspension in the ICVB The same increase was observed in the SCVE3 with a ET-I level of 15.38 f 0.36 pg/ml, a lower value with respect to the ICVB samples (18.80 * OS3 pg/ml ). Urine NAG activity Fig. 2 shows the urine NAG activity values normalized to the urine creatinine concentration (Ulmg of creatinine).

Fig. 2 - Urinary NAG activity from rats during IO d of suspension

04 : : : : : : : : : : 0 1 2 3 4 5 6 7 8 9 10

Days aitcr antlorthoatatic suspendon

At the start of suspension there were no significant changes in the urine NAG activity between the experimental animal group and that of controls (17.5 + 2.5 U/mg. creatinine). On the 2nd d of antiorthostatic hypokinesia the urine NAG concentration started to increase reaching a value 3 times that found in the controls at the 3rd d of suspension. For the following 4 d of the tail traction, the urine NAG activity showed a “plateau” like behaviour with a tendency to decrease on the last days of head-down suspension (10 d).

Permanence in a weightless ambient has significant repercussions on the organism and causes a whole series of short, medium and long-term adaptation responses. The adaptation responses may be insufficient and cause. if intense and protracted in time. damage to organs and create difficulties on return to gravity conditions. The limited number of space flights so far completed, the technical difficulties, involved in studying the effects and the reactions with permanence in weightless conditions and the intention to proceed with numerous other bold space missions makes it still more urgent to evaluate the adaptation modifications set in action and to what extent they are physiologically and pathologically significant. The cardiovascular system is without doubt the most influenced by variations in gravity. The antiorthostatic suspension model is considered the most suitable for studying variations in blood flow and pressure under hypogravity conditions, especially as regards the distal extremities of the body. Little is known about the behaviour of the hormone mediators which regulate cardiovascular reactions in these conditions and there are no data about the behaviour of endotheline. The aim of this study was to evaluate various hematologic parameters and the variations in ET-1 at regional levels (SCVB and ICVB) in this experimental model. Since endotheiines affect the kidney. the repercussions

376 45th IAF Congress

determined from experimental conditiops in this organ,

on concentration of urine N.4G, an early marker of

renal. damage, have been evalued No significant

modifications were observed in the various

hematologic parameters e.g. hematocrit, leukocyte and

platelet counts, between SCVB and ICVJ3 in the

experimental model compared to controls. These data,

agree with that of Dunn et a1.8 and indicate that, at

least under experimental conditions, there are no

significant variations in the hematologic parameters

Besides, no regional variations which could determine

a different leukocytaric division were observed The

lack of variations in hematocrit values suggests that

under these experimental conditions there is no

hematoconcentration in the districts examined This

suggests that eventual variations of analytical

parameters do not depend on hemoconcentration With

reference to the ET-1 concentrations no significant

differences between the SCVB and ICVB values of’

controls was observed. This apparently surprising

result, considering the different capacity for synthesis

and release of endotheline by different organs and

apparatuses could be interpreted as an effective

compensation mechanism acting above and below the

diaphragm. Contrary to what was observed in controls in the hypogravity experimental animals, significnt

differences were found in ET-I concentrations

between SCVB and ICVB. During the first days we

observed higher levels of ET-I in the SCVC and lower

levels in the JVCS compared to values observed in the

controls. There was an initial phase of decrease in ET-

1, in both SCVB and ICVJ3 on day I From the 5th day there was instead an increase in the concentration in

both districts examined, up to the last days of the

experiment when the levels reached were significantly

higher compared to controls in both SCVB and ICVB

On the basis of present knowledge about endothelines,

different modalities could underline variations in ET- I such as:

a) variations in the production and release of ET- 1

b) variations in the locoregional hematic flow

c) variations in the transformation of big ET- 1 to ET- I

(the activity of the endotheline conversion enzyme)

d) different clearance velocity

With reference to biosynthesis modifications, It IS

known that ET are not deposited in the cells but are

continually synthesised and excreted The biosynthesis process involves, first of all the synthesis of a

preproendotheline. subsequently secreted by the cells

as proetiotheline or big endotheline. This can be

converted lo ET by the so-called converting enzyme

(ECE) 1 3, I43 1 5 Endotheline cells only produce ET-1 Among the factors which increase synthesis of prepro

ET, ET-l are thrombine, ILl, TGFP, phorbole esters,

calcioionophores, adrenaline end PGDF ’ 6 I 7- ] 8 which sem to mediate cytosolic reactions of Car+

induced by phosphatidylinositole. Vasopressine

arginine, angiotensine II, phorbole esters, wateT

deprivation, adrenaline and other factors lg.20,21.22

augment the release of ET-I Some of these factors

could actually be operating in our model and would

contribute, at least in part. to modifying the concentrations of endotheline observed It is possible

that variations in the flow and vasa parietal tension and

modifications in different biohormone systems (arginine-vasopressine, angiotensine II. adrenaline etc ), due to stress and forced position could operate in

this situation as they would in weightlessness when

there is for example the so-called fluid shift from the

lower limbs towards the cephalic extremities In

relation to changes in other parameters it could be that

changes in receptor density or ECE activity plays a

role in the variations in plasma ET-1 concentration

during the last three experiment days It is possible that

such a pattern is connected with the reduced hematic

volume and flow, usually computed in time. In fact.

simply reducing the hematic flow could cause an

Increase in local venous endotheline concentration.

even though local production remains unvaried 23 We

also noted a significant increase in the urinary NAG

concentrations in hypogravity animals compared to

controls during the experimental period This enzyme,

localised in tubular cell lysosymes, is considered one of

rhe most sensitive markers of renal damage I2 In our

model we noticed a slight increase from the first day

which became staristically significant at the 3rd day and

maintained an almost constant level up to the end of

the experiment when there was a tendency to decrease

The reason for this renal stress could be an adverse

modification in the renal hematic flow which in itself

could produce and alter this parameter. due to stress and enforced position. It is well known that very high

infUsions of ET are necessary to produce a

vasoconstrictor effect It is also known that there is no

correlation between the plasma concentration and the

vasoconstrictor response Therefore other factors. such

as different receptor capacity and internalization of

endotheline together with its receptor could play a

role. This might sustain vasoconstriction even with

relatively low ET- I plasma concentrations The special

renal vasa sensitivity for ET-I also supports this

hypothesis. The tendency for a minor increase in NAG.

observed in the last experimental days could be a

reflection of already realised adaptation or the

beginning of the relative ET-l tollerance which occurs

in experimental conditions

.4cknowledements

This work was supported by Space Agency (ASI)

Contract number 92-RS-130 The authors are grateful

for the eccelent editor assistance of C Bennett and the

secretarial assistance of S Cocco Special thanks to

Dr F Ussia and Mr S Pagnotta for their assistance

and guidance with the rat model of simulated

weightlessness

45th XAF Congress 311

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