Red cell survival in heat-exposed hamsters

Comp. Biochem. Physiol., 1975, Vol. 50A,pp. 691 to 694. Pergamon Press. Printed in Great Britain

RED CELL SURVIVAL

IN HEAT-EXPOSED

HAMSTERS

N. MEYERSTEIN,R. YAGIL AND U. A. SOD-MORIAD University of the Negev, Research and Development Authority, Beer Sheva, Israel (Received 14 November 1973)

Abstract-l. Chronic heat exposure of hamsters has been found to affect the metabolism, osmotic fragility and membrane composition of the red cells. 2. Red cell survival was studied using slCr lahelling. 3. Half-life and mean cell life times were found to be reduced. It is suggested that heat exposure causes a mild, well-compensated, haemolytic process.

INTRODUCTION IN VIVOand in vitro exposure to heat is known to have various effects on the red blood cell (RBC). Exposure of these cells in vitro to temperatures over 45°C results in extensive morphological changes such as fragmentation and spherocytosis (Schultze, 1865; Ham et al., 1948). In addition, osmotic fragility has been found to increase (Isaac et al., 1925). Karle (1969a) has shown that a slight rise above normal body temperature, both in vivo and in vitro, augments osmotic fragility and reduces the survival of these cells on re-injection into the bloodstream. Chronic exposure of golden hamsters (Mesocricetus auratus) to heat does not induce hyperthermia (Meyerstein & Cassuto, 1970). It does, however, cause metabolic changes in the RBCs such as lowered ATP level, as well as increasing the osmotic fragility (Meyerstein & Cassuto, 1970), and changing the membrane composition (Kuiper et al., 1971). The cell morphology and specific density remain unaffected (Meyerstein & Cassuto, 1970). The reduction of RBC lifespan upon in vitro heating or hyperthermia suggests that the milder damage caused by chronic heat exposure of the organism-here the hamster-may shorten the survival time of these cells.

RBCs (Brock, 1960). This method of in vivo chromation is the method of choice for RBC survival studies in hamsters (Rigby et al., 1961). Serial heparinized blood samples, in aliquots of 0.142 ml, were taken by cardiac puncture from unanaesthetized animals, added to O-5 ml isotonic sodium chloride solution and then haemolyzed by saponine. The radioactivity was determined in a welltype scintillation counter (Auto-y-counter, Packard). Initially, samples were taken twice weekly, and thereafter once a week. There were longer intervals between taking the samples at the end of the experiment. The radioactivity in the samples was determined. Comparison with Wr standard was used to correct for Wr decay, and for slight variations in counting efficiency during the 2month period. This standard was prepared on the first day of the experiment, when the animals were injected. On the initiation of the experiment the samples were counted as “whole blood”, and were then centrifuged, plasma and cells being read separately. Haemoglobin and haematocrit were determined in each blood sample, and the radioactivity was expressed as counts/min per g Hb, according to Rigby et al. (1961). The results of RBC survival-time are given as “apparent life-span”, since no correction was made for elution. In the hamster, the daily elution of Wr from labelled RBCs has not been determined. However, it is less than three per cent (Brock, 1960).

RESULTS Haematological

MATBIUALS AND METHODS RBC survival studies Adult male hamsters were randomly divided into two groups, one heat-exposed (H), and the other control (C) (Meyerstein 8z Cassuto, 1970). The animals were kept individually in cages. The RBC survival in both groups was determined by in vivo labelling of RBCs with Wr. The Wr was supplied by Nuclear Research Centre, Negev, as sodium chromate in sterile isotonic sodium chloride solution. Specific activity was 3.9 mCi/mg Cr. Each hamster was injected intraperitoneally with 25 pCi, a dose known to be harmless to both the animal and the

data

Haemoglobin and haematocrit levels showed that no ill effect was induced by the repeated intracardiac sampling. The animals were not anaemic, and the values were similar to those reported previously (Meyerstein & Cassuto, 1970). The lack of ill effect was also confirmed by post-mortem examination. The heart showed no pathological changes. RBC survival studies Table 1 shows the decrease with time in RBC radioactivity in both experimental groups. These 691

N. MEYERSTEIN, R. YAGIL AND U. A. SOD-MORIAH

692

Table I. Wr in red cells, in heat-exposed animals and controls* Day I

4

8

I2

I5

I9

22

Heat-exposed

100

86.3 kIO.3

60.8 &II.1

43.4 &II.7

41.2 + 3.3

30.2 * 12.0

26,3 -+ 7.7

Control

100

91.3 + 5.8

72.7 +11.1

57 +_16.4

50.8 + 9.9

(not determined)

f

34.0 5.9

28

36 ______ 15.0 8.0 + 5.5 k3.4

5.8 t1.0

0

20.9 * 3.5

9.2 k3.1

0

14.9 + 3.1

49

67

* Percentage of radioactivity on day 1, corrected by the decay of the standard. Data not corrected for elution. data are given as a percentage of 51Cr radioactivity in the RBCs, after correction for the standard. The fourth day after the i.p. injection was defined as day 1 or 100 per cent radioactivity, as established by Rigby et al. (1961). From the fifth day, radioactivity was confined to the RBCs, in accordance with the findings of Rigby et al. (1961). Our data show that on each day of the experiment there were fewer labelled RBCs in the heat-exposed hamsters than in the controls, a difference which is statistically significant (PC 0.05). The heat-exposed animals showed only an 11-day half-life, a 27 per cent reduction on the known 15-day half-life in the controls (Fig. 1). The mean cell life span (MCL) of the entire RBC population was deduced, according to Dacie & Lewis (1970), by extrapolation of a tangent at the initial slope to the

Fig. 1. Red cell survival curves in heat-exposed (O-O) and control hamsters (O-O). Mean cell life-span (MCL) is deduced by extrapolation of a tangent at the initial slope to the abscissa (- - - -).

abscissa (see Fig. 1). This value was 27 days in the controls and 18 days in the heat-exposed animals (33 per cent reduction). No radioactivity was detectable on the sixty-seventh day of the experiment in both groups.

DISCUSSION Chronic exposure of golden hamsters to a hot environment induces substantial changes in the red blood cells during circulation. Although the morphology remains unchanged, the ATP level decreases by 20 per cent, the osmotic fragility is increased and there are changes in the lipid composition of the cell membrane. A known correlation exists between all these changes, and some of them have been shown to shorten the life span of these cells. When red cells are stored, a progressive deterioration takes place in several major parameters. This shortens post-transfusion survival time, i.e. survival of the cells after re-injection into the recipient (Valeri et al., 1965). The correlation between intracellular ATP level and osmotic fragility is known, but only a marked reduction in ATP content, to 20 per cent of its initial value, augments osmotic fragility extensively (Nakao, 1960). The slight decrease in ATP in the heatexposed hamsters, as such, does not seem to increase fragility, nor to reduce survival. Increased fragility caused by hyperthermia or in vitro heating reduces survival in man and rabbit (Karle, 1969a). Hyperthermia induced in rabbits, either by pyrogens or by external heating, reduces survival time of the circulating red cells by 14 per cent, as shown by Wr and aeFe studies (Karle, 1969b). In vitro heating at 42°C has been also found to enhance membrane permeability and cell rigidity in human and rabbit RBCs (Karle & Hansen, 1971). This was considered as being due to changes in the structure of the membrane. The effect of heat on the membrane is also supported by the observations of Danon et al. (1972), while exposing whole blood to temperatures in the heat stroke range. Heat exposure, as mentioned, does cause changes in the lipid composition of the red cell membrane

Red cell survival in heat-exposed hamsters (Kuiper et al., 1971). This is probably the cause of the increase in osmotic fragility in heat-exposed hamsters (Livne et al., 1972). The body temperature of the heat-acclimated hamster is not elevated by continuous exposure to 35°C. Nevertheless, the red cells are changed by this exposure, possibly owing to heating (during circulation in superficial vessels). As shown in this study, they are prematurely destroyed. Our findings of a half-life of 15 days in the control group is similar to that of 165+7*8 days (mean f2 S.D.) reported by Rigby et al. (1961). A halflife of 15 days was also demonstrated by Brock (1960). In our study, we found that chronic heatexposure induces a 27 per cent reduction in half-life and a 33 per cent reduction in mean cell life-span (MCL), while the complete disappearance of labelled RBCs from the circulation was observed concurrently in both groups. To clarify the apparently conflicting data we compared the times of disappearance of 90-95 per cent of the radioactivity, and not the 100 per cent (Horwitt et al., 1963). This showed that 90 per cent of the radioactivity had disappeared on the fortyeighth day in the control, as compared with the thirty-fifth day in the heat-exposed group, i.e. again a 27 per cent reduction. The mathematical model presented by Marvin (1963) may also apply here. He plotted the percentage survival as a function of time and found that different curves, differing in their exponential coefficient, and in their 50 per cent (i.e. “half-life”), converge towards the moment of complete disappearance. As the half-life, the mean cell-life and the 90 per cent disappearance are all more accurate in our experimental condition it may feasibly be deduced that RRC survival is indeed reduced by heat exposure. Despite this reduction in survival, the heatexposed hamsters are not anaemic. The percentage of young cells, as expressed by both the reticulocyte count, and by D.D.C. (Density Distribution of Cells) @anon & Marikovsky, 1964), does not increase. On moderate shortening of RBC life-span in man, anaemia is avoided by increased erythropoiesis. This is known as a compensated haemolytic process. The number of reticulocytes in the peripheral blood is usually higher, but this is not essential. When the cell life-span is one-seventh of the normal (in man-less than 15 to 20 days’ survival) the maximum effective erythropoietic effect of the bone marrow can no longer compensate, and “anaemia” appears (Crosby & Akeroyd,l952). The slight increase in destruction rate of the RBC’s, caused by heat exposure, is usually well compensated, and the hamster maintains cell populations, the number and age distribution of which are unchanged, when estimated by standard methods. Direct interspecies correlation exists between basal metabolic rate and red cell survival (Rodnan et al., 1957). However, in a given species the basal

693

metabolic rate is one of the factors which controls the red cell mass by its effect on erythropoiesis and not on red cell destruction (Berlin, 1964). Hyperthyroidism in dogs leads to polycythemia not by prolongation of red cell survival, but by increased erythropoiesis (Waldman et al., 1962). Accordingly, the hypothyroid dog exhibits a reduced total red cell mass, again as a result of changed erythropoiesisthis time by reduced cell production. Red-cell survival time remains unchanged (Cline & Berlin, 1963). In general, thyroid hormone deficiency does not impair the production of the haemoglobin and red cells, but readjusts the level of erythropoiesis to accord with the lower oxygen demand and supply (Harris et al., 1970). The hypofunction of the thyroid indicated in heat-exposed hamsters (Cassuto, 1968) could lead to decreased erythropoiesis. Thermal damage could also lead to decreased cell mass, by reducing survival time of the cells. Our findings suggest a decrease in red-cell survival time, but Berlin’s strictures on Brock’s data on prolonged survival in hibernating hamsters also apply here (Berlin, 1964) and a definite statement cannot be made. What is the primary effect on the RBC of chronic heat exposure of the whole organisms? We hypothesize that, as a result of heat exposure, the permeability of the membrane is changed which leads to a series of secondary phenomena. A change in membrane properties is suggested by the following observations: (a) Increased osmotic fragility, which has been demonstrated in these cells (Meyerstein t Cassuto, 1970) in the face of unchanged osmotic pressure in the cells (Livne et al., 1972). An additional indication is the reduction in V,, critical haemolytic volume, in heat exposure (Livne et al., 1972). This could suggest membrane changes, as drug-induced increases in V, have been shown to correlate with membrane expansion. (Seeman et al., 1969). (b) The demonstration of changes in membrane composition (Kuiper et al., 1971) which are known to correlate with increased fragility (Walker & Kummerow, 1964). (c) The reports in the literature, which suggests that in vitro or hyperthermic heat exposure affects the cell membrane (Karle & Hansen, 1971, Danon et al., 1972). (d) Thermal damage in erythrocytes was accompanied by morphological changes in the cell membrane, such as general coarseness and areas of pitting (Baar & Arrowsmith, 1970). It should be noted, however, that these changes were demonstrated after burning injury. The damage caused by heat exposure is much milder; no morphological changes are observed during light microscopy and phase microscopy (Meyerstein & Cassuto, 1970) and the mechanism is not necessarily the same. Hence it is suggested that prolonged exposure of hamsterstohightemperaturesincreasescellmembrane

694

N. MEYERSTEIN, R. YAGIL AND U. A. SOD-MORIAH

permeability and leads to increased sodium influx and potassium efflux. This enhances the activity of the sodium-potassium pump and results in over-utilization of ATP. The over-utilization of ATP may be reflected by the reduced intracellular ATP content reported, and in reduced levels of other glycolytic intermediates such as DPG and PEP (phosphoenolpyruvate) demonstrated in these cells (Meyerstein & Cassuto, 1972; unpublished data). This hypothesis has yet to be confirmed. These changes in the membrane may lead to decreased deformability, which may in turn lead to increased sequestration in the spleen and early destruction. Acknowledgements-This study was partly supported by Grant No. BDPEC-OH-ISR-7 from the United States Department of Health, Education and Welfare, and also by the National Council for Research and Development. The authors are indebted to Professor Rami Rahamimoff and to Dr. Asher Bard for helpful discussion, and to Mrs. Cynthia Bellon for editing the manuscript. REFERENCES

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golden hamsters;