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Experimental production and control of stomach lesions in the rat
Journal of
Psychosomatic
Research,1960,Vol. 4, pp. 297to 300. PergamonPress Ltd. Printedin Northern Ireland
EXPERTMENTAL PRODUCTION AND CONTROL OF STOMACH LESIONS IN THE RAT J. 0. SINES* (Received 17 Novenzber1959) SEVERAL recent reports have indicated that gastric ulcers and severe stomach lesions can be produced in the rat by means of psychological stress (approach-avoidance conflict) (SAWREY and WEISZ, 1956; SAWREY, CONGER and TURRELL, 1956), and immobilization (SINES, 1959). One of these reports (SINES, 1959) further demonstrated that there is a significant genetic factor in the susceptibility to stress-induced stomach lesions. The physiological mechanisms underlying development of such lesions in the rat have not been examined, nor are there data relative to the pharmacologic control of lesion development in this species. One approach to the latter question involves the study of the effects of drugs known to be parasympathetic blocking agents. The present report deals with the incidence and severity of immobilization-produced stomach lesions in normal and lesion-susceptible rats after treatment with several anticholinergic compounds-i_ and a major tranquilizer. STUDY
I
Subjects Forty-eight male albino rats 80-90 days of age were used. All were descendants of Holtzman Sprague-Dawley albinos which had been born and raised in this laboratory. The group from which the animals in the first study were drawn was the F3 generation all of which developed stomach lesions following of lesion-susceptible animals, 24 hours of immobilization (SINES, 1959). Before this study all the subjects had been maintained on standard laboratory regime which involved group housing and nd lib food and water. Table I indicates the number of subjects in each sub-group in the first study. Stress technique, medication and stomach ecaluation Stomach lesions were produced by immobilization which consisted of wrapping each animal in a plaster of paris cocoon. After 24 hr of immobilization all animals were sacrificed with ether and the stomachs were inspected for lesions. Lesions were rated 0 to 4 depending upon their severity, with 0 indicating a normal stomach; “2” irregularity of the mucosal lining; “3” a clearly defined “1” small petechiae; defect which usually involved bleeding; and “4” the most severe lesion observed, being a palpable defect which almost invariably included considerable congealed blood in the base of the crater, * From the Washington University School of Medicine. t Drugs used were generously provided by: Pro-Banthine, Searle; Atrenyl, Ciba; Darbid, Smith and French. The brand names of the several drugs arc registered by the respective manufacturers. 297
Kline
298
J. 0.
SINES
One and one-half hr before, and 3 hr after, immobilization 0.45 mg/kg of Pro-Banthine, 0.33 mg/kg of Antrenyl and 10 mg/kg of chlorpromazine were administered to each animal in the appropriate groups. Three hours before and 6 hr after immobilization subjects in another group were given 0.16 mg/kg of Darbid. All drugs were given in 0.1 to 0.2 cc saline solutions. The anticholinergics were administered subcutaneously and chlorpromazine was given intraperitoneally before immobilization and by stomach tube after it. The doses of the anticholinergic drugs used were approximately equivalent to twice TABLE
1 .-hCIDENCE AND
AND
SEVERITY
CHLORPROMAZINE
,
Number of animals in each group
RATS
Probanthine 0.45 mg/kg
, Untreated I Severity
OF STOMACH
TREATED
N
1
N
LESIONS
24
Antrenyl 0.33 mgikg
I
N
I
14
IN UiiTREATED,
FOLLOWIKG
9
HOURS
ANTICHOLIP;ERGIC
TREATED
OF IMMOBILIZATI0N
1 Darbid / 0.16 mg/kg
I Chlorpromazine 1 10 “g/kg
1
(
pi
N
9
10
9.0
45
~
Anticholiner@c Drug vs.
‘6”” _
I I __-
Control
I
Anticholinergic Drug vs.
I
Chlorpromazine
p
_
“U” _ I_______-p _ 1
I
1.0 __~ 0.001
0.001
0
0
15
0.001
0.001
0.01
/
0.05
the recommended initial doses with humans, prorated on the basis of each animal’s body weight; the injection times reflected the effective periods noted for each drug in the manufacturers’ literature. The dose level of chlorpromazine was one which in our experience significantly reduced both activity and learning rate and further, produced gross behavioural changes lasting for approximately 4 hr. RESULTS Table 1 summarizes the data regarding incidence and severity of lesions in the various groups in the first study. The statistic used was the Mann-Whitney “U” and each anticholinergic treated group was compared separately to the control and chlorThese results indicate that treatment with the antipromazine treated animals. cholinergic compounds employed tended to reduce severity and incidence of immobilization-produced stomach lesions although not all of the drugs or doses employed were
Experimental
production
and control
of stomach
lesions
in the rat
299
chlorpromazine appeared to increase equally effective in this respect. Furthermore, incidence and severity of immobilization-produced stomach lesions if, indeed, it had any effect at all. This last finding led to the second study, designed to examine the effect of chlorpromazine on immobilization-produced stomach lesions in a more lesion-resistant group of animals. STUDY
Stress, medication
I1
aud stomach eualuatiotl
In view of the unexpected effect of chlorpromazine on severity of lesions noted in lesion-susceptible rats following 24 hr of plaster-cast immobilization, 2 groups of stock TABLE ~.-~~KI~ENCL
AND
SEVERITY OF STOMACH
RELATIVEIMMOBILIZATIONFOR
Lesion severity 4
I
Nonmedicated
I
I
x2 = 5.85 (calculated
I
I
8 Number of animals
one-tailed
~
0 0 0 2
3 2
1 0
LESIONS IN SPRAGUE-DAWLEY
12 HR WITH AND WITHOUT
10
from 2 x 2 table,
( lesion,
RATS SUBJECTED TO
CHLORPROMAZINE
Chlorpromazine treated 0 3 4 4
5 Number of animals
16
no lesion cs drug, no drug);
P = 0.01.
animals were subjected to a less severe stress experience. This second stress situation involved relative immobilization for 12 hr in a confining wire enclosure which prevented the animals from moving in any direction but was not as closely form-fitting as the plaster of paris cocoons. We had previously found that only a small p’srcentage of our Sprague-Dawley stock animals developed lesions, usually mild, after 12 hr in the wire mesh enclosure. Two groups of 80-90 days old male Sprague-Dawley rats, one group non-medicated (10 animals) and another treated with 10 mg/kg of chlorpromazine intraperitoneally (16 animals) 13 hr before and 3 hr after immobilization were placed in the wire mesh containers for 12 hr, after which all animals were sacrificed and the stomachs were evaluated as previously described. RESULTS
The results of the second study, summarized in Table 2, indicate that two 10 mg/kg doses of chlorpromazine, administered 44 hr apart, are related to development of stomach lesions in a significantly greater proportion of Sprague-Dawley rats than is found in a group of animals not so treated but subjected to an identical amount of stress. CONCLUSION
Immobilization-produced stomach lesions in susceptible rats can be controlled by means of several anticholinergic compounds but larger doses of chlorpromazine tend to increase both severity and incidence of such lesions. The effect of the anticholinergic s
.
300
J. 0. SINES
agents suggests that stomach lesion development in the rat is at least in part dependent upon parasympathetic hyperactivity. Rats which are highly susceptible to stomach lesion development following stress would thus seem to be convenient and valid subjects for the screening of anticholinergic compounds. Although an atropine-like effect on volume of gastric secretion in man has been noted by HAVERBACK, STEVENSON and SJOERDSMA(1955), HALEY, MCCORMICK and MCCULLOH (1955) have found that the effect of chlorpromazine in prolonging survival time in irradiated mice occurred at a ZOWdose 1eaeZonly (5 mg/kg). The present findings relative to the effect of chlorpromazine (two 10 mg/kg doses within 5 hr) on stomach lesion development may thus reflect the detrimental influence of higher doses of the drug on another type of stress tolerance. REFERENCES HALEY T. J., MCCORMICK W. G., and MCCULLO~I EVE F. (1955), Effect of chlorpromazine on survival time in irradiated mice. Proc. Sec. exp. Biol. Med. 88, 475. HAVERBACK B. J., STEVENSON T. D. and SJOERDSMA A. (1955), The effect of rescrpine and chlorpromazine on gastric secretion. Amer. J. Med. Sci. 230, 601. SAWREY W. L. and WEISZ J. D. (1956), An experimental method of producing gastric ulcers. J. camp. physiol. Psychol. 49, 269. SAWREY W. L., CONGER J. J. and TURRELL E. S., (1956), An experimental investigation of the role of psychological factors in the production of gastric ulcers in rats. J. camp. physiol. Psycho/. 49,457. SINES J. O., Selective breeding for development of stomach lesions following stress in the rat. (1959), J. camp. physiol. Psychol. 52, 615.
Psychosomatic
Research,1960,Vol. 4, pp. 297to 300. PergamonPress Ltd. Printedin Northern Ireland
EXPERTMENTAL PRODUCTION AND CONTROL OF STOMACH LESIONS IN THE RAT J. 0. SINES* (Received 17 Novenzber1959) SEVERAL recent reports have indicated that gastric ulcers and severe stomach lesions can be produced in the rat by means of psychological stress (approach-avoidance conflict) (SAWREY and WEISZ, 1956; SAWREY, CONGER and TURRELL, 1956), and immobilization (SINES, 1959). One of these reports (SINES, 1959) further demonstrated that there is a significant genetic factor in the susceptibility to stress-induced stomach lesions. The physiological mechanisms underlying development of such lesions in the rat have not been examined, nor are there data relative to the pharmacologic control of lesion development in this species. One approach to the latter question involves the study of the effects of drugs known to be parasympathetic blocking agents. The present report deals with the incidence and severity of immobilization-produced stomach lesions in normal and lesion-susceptible rats after treatment with several anticholinergic compounds-i_ and a major tranquilizer. STUDY
I
Subjects Forty-eight male albino rats 80-90 days of age were used. All were descendants of Holtzman Sprague-Dawley albinos which had been born and raised in this laboratory. The group from which the animals in the first study were drawn was the F3 generation all of which developed stomach lesions following of lesion-susceptible animals, 24 hours of immobilization (SINES, 1959). Before this study all the subjects had been maintained on standard laboratory regime which involved group housing and nd lib food and water. Table I indicates the number of subjects in each sub-group in the first study. Stress technique, medication and stomach ecaluation Stomach lesions were produced by immobilization which consisted of wrapping each animal in a plaster of paris cocoon. After 24 hr of immobilization all animals were sacrificed with ether and the stomachs were inspected for lesions. Lesions were rated 0 to 4 depending upon their severity, with 0 indicating a normal stomach; “2” irregularity of the mucosal lining; “3” a clearly defined “1” small petechiae; defect which usually involved bleeding; and “4” the most severe lesion observed, being a palpable defect which almost invariably included considerable congealed blood in the base of the crater, * From the Washington University School of Medicine. t Drugs used were generously provided by: Pro-Banthine, Searle; Atrenyl, Ciba; Darbid, Smith and French. The brand names of the several drugs arc registered by the respective manufacturers. 297
Kline
298
J. 0.
SINES
One and one-half hr before, and 3 hr after, immobilization 0.45 mg/kg of Pro-Banthine, 0.33 mg/kg of Antrenyl and 10 mg/kg of chlorpromazine were administered to each animal in the appropriate groups. Three hours before and 6 hr after immobilization subjects in another group were given 0.16 mg/kg of Darbid. All drugs were given in 0.1 to 0.2 cc saline solutions. The anticholinergics were administered subcutaneously and chlorpromazine was given intraperitoneally before immobilization and by stomach tube after it. The doses of the anticholinergic drugs used were approximately equivalent to twice TABLE
1 .-hCIDENCE AND
AND
SEVERITY
CHLORPROMAZINE
,
Number of animals in each group
RATS
Probanthine 0.45 mg/kg
, Untreated I Severity
OF STOMACH
TREATED
N
1
N
LESIONS
24
Antrenyl 0.33 mgikg
I
N
I
14
IN UiiTREATED,
FOLLOWIKG
9
HOURS
ANTICHOLIP;ERGIC
TREATED
OF IMMOBILIZATI0N
1 Darbid / 0.16 mg/kg
I Chlorpromazine 1 10 “g/kg
1
(
pi
N
9
10
9.0
45
~
Anticholiner@c Drug vs.
‘6”” _
I I __-
Control
I
Anticholinergic Drug vs.
I
Chlorpromazine
p
_
“U” _ I_______-p _ 1
I
1.0 __~ 0.001
0.001
0
0
15
0.001
0.001
0.01
/
0.05
the recommended initial doses with humans, prorated on the basis of each animal’s body weight; the injection times reflected the effective periods noted for each drug in the manufacturers’ literature. The dose level of chlorpromazine was one which in our experience significantly reduced both activity and learning rate and further, produced gross behavioural changes lasting for approximately 4 hr. RESULTS Table 1 summarizes the data regarding incidence and severity of lesions in the various groups in the first study. The statistic used was the Mann-Whitney “U” and each anticholinergic treated group was compared separately to the control and chlorThese results indicate that treatment with the antipromazine treated animals. cholinergic compounds employed tended to reduce severity and incidence of immobilization-produced stomach lesions although not all of the drugs or doses employed were
Experimental
production
and control
of stomach
lesions
in the rat
299
chlorpromazine appeared to increase equally effective in this respect. Furthermore, incidence and severity of immobilization-produced stomach lesions if, indeed, it had any effect at all. This last finding led to the second study, designed to examine the effect of chlorpromazine on immobilization-produced stomach lesions in a more lesion-resistant group of animals. STUDY
Stress, medication
I1
aud stomach eualuatiotl
In view of the unexpected effect of chlorpromazine on severity of lesions noted in lesion-susceptible rats following 24 hr of plaster-cast immobilization, 2 groups of stock TABLE ~.-~~KI~ENCL
AND
SEVERITY OF STOMACH
RELATIVEIMMOBILIZATIONFOR
Lesion severity 4
I
Nonmedicated
I
I
x2 = 5.85 (calculated
I
I
8 Number of animals
one-tailed
~
0 0 0 2
3 2
1 0
LESIONS IN SPRAGUE-DAWLEY
12 HR WITH AND WITHOUT
10
from 2 x 2 table,
( lesion,
RATS SUBJECTED TO
CHLORPROMAZINE
Chlorpromazine treated 0 3 4 4
5 Number of animals
16
no lesion cs drug, no drug);
P = 0.01.
animals were subjected to a less severe stress experience. This second stress situation involved relative immobilization for 12 hr in a confining wire enclosure which prevented the animals from moving in any direction but was not as closely form-fitting as the plaster of paris cocoons. We had previously found that only a small p’srcentage of our Sprague-Dawley stock animals developed lesions, usually mild, after 12 hr in the wire mesh enclosure. Two groups of 80-90 days old male Sprague-Dawley rats, one group non-medicated (10 animals) and another treated with 10 mg/kg of chlorpromazine intraperitoneally (16 animals) 13 hr before and 3 hr after immobilization were placed in the wire mesh containers for 12 hr, after which all animals were sacrificed and the stomachs were evaluated as previously described. RESULTS
The results of the second study, summarized in Table 2, indicate that two 10 mg/kg doses of chlorpromazine, administered 44 hr apart, are related to development of stomach lesions in a significantly greater proportion of Sprague-Dawley rats than is found in a group of animals not so treated but subjected to an identical amount of stress. CONCLUSION
Immobilization-produced stomach lesions in susceptible rats can be controlled by means of several anticholinergic compounds but larger doses of chlorpromazine tend to increase both severity and incidence of such lesions. The effect of the anticholinergic s
.
300
J. 0. SINES
agents suggests that stomach lesion development in the rat is at least in part dependent upon parasympathetic hyperactivity. Rats which are highly susceptible to stomach lesion development following stress would thus seem to be convenient and valid subjects for the screening of anticholinergic compounds. Although an atropine-like effect on volume of gastric secretion in man has been noted by HAVERBACK, STEVENSON and SJOERDSMA(1955), HALEY, MCCORMICK and MCCULLOH (1955) have found that the effect of chlorpromazine in prolonging survival time in irradiated mice occurred at a ZOWdose 1eaeZonly (5 mg/kg). The present findings relative to the effect of chlorpromazine (two 10 mg/kg doses within 5 hr) on stomach lesion development may thus reflect the detrimental influence of higher doses of the drug on another type of stress tolerance. REFERENCES HALEY T. J., MCCORMICK W. G., and MCCULLO~I EVE F. (1955), Effect of chlorpromazine on survival time in irradiated mice. Proc. Sec. exp. Biol. Med. 88, 475. HAVERBACK B. J., STEVENSON T. D. and SJOERDSMA A. (1955), The effect of rescrpine and chlorpromazine on gastric secretion. Amer. J. Med. Sci. 230, 601. SAWREY W. L. and WEISZ J. D. (1956), An experimental method of producing gastric ulcers. J. camp. physiol. Psychol. 49, 269. SAWREY W. L., CONGER J. J. and TURRELL E. S., (1956), An experimental investigation of the role of psychological factors in the production of gastric ulcers in rats. J. camp. physiol. Psycho/. 49,457. SINES J. O., Selective breeding for development of stomach lesions following stress in the rat. (1959), J. camp. physiol. Psychol. 52, 615.