Life Sciences, Vol . 23, pp . 2307-2314 Printed in the U.S .A .
Pergamon Press
PHYSIOLOGICAL CHANGES IN RATS ERPOSED TO COLD/REST&4INT STRESS Doreen Beattie Institute of Medical Research, Wyeth Laboratories, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, U .R . (Received in final form October 13, 1978) Summary An investigation was made into the effects of cold/restraint stress in rats on renal function, plasma constituents, blood count and endocrine function . Cold/restraint caused an increased volume of urine output, with increased K+ and urea excretion, and decreased Na+ and C1 - excretion. There was a fall is plasma glucose, and a rise in plasma urea . A marked leucopenia was found, and the blood pH was significantly lowered. Cold/restraint was also found to cause marked increases in corticosterone and thyroxine levels, and a fall in the insulin level . Many methods are described is the literature for the induction of experimental gastric erosions in animals . Oae of the simplest and most effective methods is by exposing rats to a combination of cold and restraint (1) . Although a good deal is known about the changes which occur in the gastric mucoaa under conditions of stress (2, 3), little has been published on the general physiology of cold/restrained rata . Since this animal model is widely used to screen potential anti-ulcer agents, it is important to understand more fully the associated general physiological changes which occur in this condition, and to determine whether such changes contribute to, or reflect, those known to occur locally in the gastric mucosa during the development of gastric erosions . To this end, as investigation was made into the physiological changes which occur during the exposure of rata to cold/restraint stress . This paper describes the effects of cold/restraint upon renal function, plasma constituents, blood count and endocrine function, and attempts are made to assess the significance of any changes which were found to occur. METHODS The basic method used was similar to one described by Senay and Levine (1) . Male Charles River rats, weighing 80-1008, were fasted overnight, but allowed
water ad libitum. The rats were dosed orally with 0.3x carboxymethylcelluloae (CMC) in 0.9x saline, and divided into two groups . Rats in one group were inserted in aluminium open wire restraint cages, measuring 4 .2cm in diameter by 12 .7cm in length, and placed in a cold room (4° f loC) for a period of three hours ; a matched control group remained unrestrained in the laboratory for the three hour experimental period . In every experiment, after the three hour stress period the stomachs of each group of rata were examined, to confirm the presence of gastric erosions in the test group. 0300-9653/78/1204-230702 .00/0 Copyright (c) 1978 Pergamon Press
2308
Physiology of Cold/Restraint Stress
Vol . 23, No . 23, 1978
In the study of renal function during cold/restraint, both groups of rats were placed in metabolism cages during the experimental period, to allow collection of urine . After three hours, the urine was weighed, and analysed for electrolyte and urea content, as described below . In order to investigate the effect of cold/restraint on plasma constituents, both groups of rats were deeply anaesthetised immediately after the stress period and a blood sample taken directly from the versa cavâ of each rat by needle puncture through a cut in the diaphragm . The blood was collected in fluoride-oxalate tubes and estimation of plasma electrolyte composition was carried out using an EEL flame photometer and an EEL chloride meter . The BUN Auto-Analyzer method 16-b was used for estimation of plasma glucose and urea levels, and serum creatinine levels were estimated by Auto-Analyzer method N11-b . For haematology studies, the samples were collected in tubes containing EDTA (ethylenediaminetetra-acetic acid) . The following estimations were made :i) ü) iii) iv) v) vi)
Packed cell volume, by means of a micro-method using a Clay-Adams haematocrit centrifuge . Haemoglobin, using a cyanmethaemoglobin method. Red blood cell count, using a Coulter Counter (model A) method. Total white blood cell count, using a Coulter Counter (model A) method. Platelet count, using a Coulter Counter (model A) method" Differential white cell count (neutrophils and lymphocytes) using the May-Grunwold staining technique.
To determine the pH of blood of cold/restrained rats, blood samples were taken at the end of the experimental period, as described above, and placed in fluorideoxalate tubes . The pH of each sample was read to the nearest 0 .01 pH unit, using a Pye model 291 pH meter . Serum thyroxine levels were estimated by a radioimmunoassay technique using 125 1 , (Thyopac 4 kit, Radiochemical Centre, Amersham) . Serum insulin levels were estimated by a radioimmunoassay technique using 1251 . (Insulin kit ; Lepetit Pharmaceuticals) . Statistical analyses were made using Student's t-test (4) . In all tables, means f SEM are quoted . ** = P < 0 .01, * = P < 0 .05, N .S . = P > 0 .05 .
2309
Physiology of Cold/Restraint Stress
Vol . 23, No . 23, 1978
RESULTS 1.
Effeçt of cold/restraint on renal function
Cold/restraint resulted in a statistically significant increase in the weight of urine produced, with marked decreases in the concentrations of Na+, IC+, C1' and urea (see Table 1) . However, when the total molar quantities of electrolytes and urea excreted during the three hour period were calculated, it was found that cold/restraint produced as increase in total potassium and urea excretion, and decreased sodium and chloride excretion . TABLE 1 Effect of Cold/Restraint on We1Aht and Concentration of Urine Wt(g)
Na+(mmol/1>
x+(mmol/1)
cl'(mmol/1)
urea (g/looms)
Control
0 .51010 .18
275±20
79 .3±9 .2
111±7 .7
2 .04±0.23
Cold/ restraint
1 .42f0.28
55 .2±5 .6
44 .8f8 .7
13 .Tt3 .2
1 .21±0.25
t~2.748*
t=10 .700**
t~2 .736*
t=11 .549**
t~2 .462*
2.
Effect of cold/restraint on plasma electrolytes
It can be seen from Table 2 that a three hour period of cold/restraint had no effect upon plasma electrolytes . TABLE 2 Effect of Cold/Restraint oa Plasma Electrolyte Levels Na+(mmol/1)
x+(mmol/1)
ca~(mg/looml)
cl'(mmol/1>
Control
143±0 .82
4.94=0.19
6 .59*0 .37
108±0.95
Cold/restraint
144±0 .79
5.03±0 .23
5.87?'0 .39
109±0.83
t=0.782N.S .
t=0 .460N.S .
ts1 .354N .S .
t=0.950N .S .
3.
Effect of cold/restraint oa plasma~lucose, plasma urea and serum creatinine
Cold/restraint produced a statistically significant fall in blood glucose level (Table 3) although it should be noted that the control levels were also lower than normal owing to overnight fasting. A marked rise in plasma urea was also evident, but cold/restraint did not affect serum creatinine levels .
2310
Physiology of Cold/Restraint Stress
Vol . 23, No . 23, 1978
TABLE 3 Effect of Cold/Restraint on Plasma Glucose, Plasma Urea and Serum Creatinine Levels Plasma glucose (mg/looms)
Plasma urea (mg/looms)
Serum creatinine (mg/looms)
Control
71 .8±2 .7
32 .8f4 .8
1 .800 .065
Cold/restraint
56 .0±4 .9
52 .8±6 .2
1 .51±0.14
t=2 .838*
t=2.543*
t=1 .818N.S .
There was .a highly significant positive correlation between the ulcer score and urea level of individual rats in this experiment, with a correlation coefficient Following this finding, a retrospective examination was of 0.8237 (~0.001~) . made of all experiments in which plasma urea levels and ulcer scores had been The data from all these experiments were pooled (n~38) and a obtained . correlation coefficient of 0.6485 (
Pergamon Press
PHYSIOLOGICAL CHANGES IN RATS ERPOSED TO COLD/REST&4INT STRESS Doreen Beattie Institute of Medical Research, Wyeth Laboratories, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, U .R . (Received in final form October 13, 1978) Summary An investigation was made into the effects of cold/restraint stress in rats on renal function, plasma constituents, blood count and endocrine function . Cold/restraint caused an increased volume of urine output, with increased K+ and urea excretion, and decreased Na+ and C1 - excretion. There was a fall is plasma glucose, and a rise in plasma urea . A marked leucopenia was found, and the blood pH was significantly lowered. Cold/restraint was also found to cause marked increases in corticosterone and thyroxine levels, and a fall in the insulin level . Many methods are described is the literature for the induction of experimental gastric erosions in animals . Oae of the simplest and most effective methods is by exposing rats to a combination of cold and restraint (1) . Although a good deal is known about the changes which occur in the gastric mucoaa under conditions of stress (2, 3), little has been published on the general physiology of cold/restrained rata . Since this animal model is widely used to screen potential anti-ulcer agents, it is important to understand more fully the associated general physiological changes which occur in this condition, and to determine whether such changes contribute to, or reflect, those known to occur locally in the gastric mucosa during the development of gastric erosions . To this end, as investigation was made into the physiological changes which occur during the exposure of rata to cold/restraint stress . This paper describes the effects of cold/restraint upon renal function, plasma constituents, blood count and endocrine function, and attempts are made to assess the significance of any changes which were found to occur. METHODS The basic method used was similar to one described by Senay and Levine (1) . Male Charles River rats, weighing 80-1008, were fasted overnight, but allowed
water ad libitum. The rats were dosed orally with 0.3x carboxymethylcelluloae (CMC) in 0.9x saline, and divided into two groups . Rats in one group were inserted in aluminium open wire restraint cages, measuring 4 .2cm in diameter by 12 .7cm in length, and placed in a cold room (4° f loC) for a period of three hours ; a matched control group remained unrestrained in the laboratory for the three hour experimental period . In every experiment, after the three hour stress period the stomachs of each group of rata were examined, to confirm the presence of gastric erosions in the test group. 0300-9653/78/1204-230702 .00/0 Copyright (c) 1978 Pergamon Press
2308
Physiology of Cold/Restraint Stress
Vol . 23, No . 23, 1978
In the study of renal function during cold/restraint, both groups of rats were placed in metabolism cages during the experimental period, to allow collection of urine . After three hours, the urine was weighed, and analysed for electrolyte and urea content, as described below . In order to investigate the effect of cold/restraint on plasma constituents, both groups of rats were deeply anaesthetised immediately after the stress period and a blood sample taken directly from the versa cavâ of each rat by needle puncture through a cut in the diaphragm . The blood was collected in fluoride-oxalate tubes and estimation of plasma electrolyte composition was carried out using an EEL flame photometer and an EEL chloride meter . The BUN Auto-Analyzer method 16-b was used for estimation of plasma glucose and urea levels, and serum creatinine levels were estimated by Auto-Analyzer method N11-b . For haematology studies, the samples were collected in tubes containing EDTA (ethylenediaminetetra-acetic acid) . The following estimations were made :i) ü) iii) iv) v) vi)
Packed cell volume, by means of a micro-method using a Clay-Adams haematocrit centrifuge . Haemoglobin, using a cyanmethaemoglobin method. Red blood cell count, using a Coulter Counter (model A) method. Total white blood cell count, using a Coulter Counter (model A) method. Platelet count, using a Coulter Counter (model A) method" Differential white cell count (neutrophils and lymphocytes) using the May-Grunwold staining technique.
To determine the pH of blood of cold/restrained rats, blood samples were taken at the end of the experimental period, as described above, and placed in fluorideoxalate tubes . The pH of each sample was read to the nearest 0 .01 pH unit, using a Pye model 291 pH meter . Serum thyroxine levels were estimated by a radioimmunoassay technique using 125 1 , (Thyopac 4 kit, Radiochemical Centre, Amersham) . Serum insulin levels were estimated by a radioimmunoassay technique using 1251 . (Insulin kit ; Lepetit Pharmaceuticals) . Statistical analyses were made using Student's t-test (4) . In all tables, means f SEM are quoted . ** = P < 0 .01, * = P < 0 .05, N .S . = P > 0 .05 .
2309
Physiology of Cold/Restraint Stress
Vol . 23, No . 23, 1978
RESULTS 1.
Effeçt of cold/restraint on renal function
Cold/restraint resulted in a statistically significant increase in the weight of urine produced, with marked decreases in the concentrations of Na+, IC+, C1' and urea (see Table 1) . However, when the total molar quantities of electrolytes and urea excreted during the three hour period were calculated, it was found that cold/restraint produced as increase in total potassium and urea excretion, and decreased sodium and chloride excretion . TABLE 1 Effect of Cold/Restraint on We1Aht and Concentration of Urine Wt(g)
Na+(mmol/1>
x+(mmol/1)
cl'(mmol/1)
urea (g/looms)
Control
0 .51010 .18
275±20
79 .3±9 .2
111±7 .7
2 .04±0.23
Cold/ restraint
1 .42f0.28
55 .2±5 .6
44 .8f8 .7
13 .Tt3 .2
1 .21±0.25
t~2.748*
t=10 .700**
t~2 .736*
t=11 .549**
t~2 .462*
2.
Effect of cold/restraint on plasma electrolytes
It can be seen from Table 2 that a three hour period of cold/restraint had no effect upon plasma electrolytes . TABLE 2 Effect of Cold/Restraint oa Plasma Electrolyte Levels Na+(mmol/1)
x+(mmol/1)
ca~(mg/looml)
cl'(mmol/1>
Control
143±0 .82
4.94=0.19
6 .59*0 .37
108±0.95
Cold/restraint
144±0 .79
5.03±0 .23
5.87?'0 .39
109±0.83
t=0.782N.S .
t=0 .460N.S .
ts1 .354N .S .
t=0.950N .S .
3.
Effect of cold/restraint oa plasma~lucose, plasma urea and serum creatinine
Cold/restraint produced a statistically significant fall in blood glucose level (Table 3) although it should be noted that the control levels were also lower than normal owing to overnight fasting. A marked rise in plasma urea was also evident, but cold/restraint did not affect serum creatinine levels .
2310
Physiology of Cold/Restraint Stress
Vol . 23, No . 23, 1978
TABLE 3 Effect of Cold/Restraint on Plasma Glucose, Plasma Urea and Serum Creatinine Levels Plasma glucose (mg/looms)
Plasma urea (mg/looms)
Serum creatinine (mg/looms)
Control
71 .8±2 .7
32 .8f4 .8
1 .800 .065
Cold/restraint
56 .0±4 .9
52 .8±6 .2
1 .51±0.14
t=2 .838*
t=2.543*
t=1 .818N.S .
There was .a highly significant positive correlation between the ulcer score and urea level of individual rats in this experiment, with a correlation coefficient Following this finding, a retrospective examination was of 0.8237 (~0.001~) . made of all experiments in which plasma urea levels and ulcer scores had been The data from all these experiments were pooled (n~38) and a obtained . correlation coefficient of 0.6485 (
