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Stress ulcer in normotensive and spontaneously hypertensive rats
Physiology & Behavior, Vol. 36, pp. 699-705. Copyright©PergamonPress Ltd., 1986.Printedin the U.S.A.
0031-9384/86$3.00 + .00
Stress Ulcer in Normotensive and Spontaneously Hypertensive Rats W I L L I A M P. PARI~ A N D G R E G G T. S C H I M M E L
V e t e r a n s A d m i n i s t r a t i o n M e d i c a l C e n t e r (15R), P e r r y P o i n t , M D 21902 R e c e i v e d 11 M a r c h 1985 PARE, W. P, AND G, T. SCH|MMEL. Stress ulcer in normotensive and spontaneously hypertensive rats. PHYSIOL BEHAV 36(4) 699-705, 1986.--Spontaneously hypertensive rats (SHR) and their normotensive progenitors, the WistarKyoto (WKY) rats, were tested in the open-field arena and subsequently exposed to either cold-restraint stress or activity-stress. SHR rats were more active and judged less fearful in the open-field test. Changes in core body temperature, and adrenal and thymus weights did not differentiate between SHR and WKY rats in the cold-restraint procedure. A significant adrenal hypertrophy was observed for SHR rats in the activity-stress procedure. WKY rats were more susceptible to stress ulcer in both the cold-restraint and the activity-stress procedures. While running-wheel activity had been considered an important etiological variable for activity-stress ulcer, the lower activity demonstrated by the ulcer-prone WKY rats suggested that genetic variables might be more relevant to stress ulcer disease. Activity-stress Stress ulcer Cold restraint Core body temperature Adrenal Thymus
Spontaneously hypertensive
SOME investigators have emphasized the importance of genetic factors in the etiology of stress ulcer. For instance, Martin, Martin, Andre and Lambert [20] described differential susceptibility to restraint-induced stomach ulcers in two distinct strains of Wistar rats. In a series of studies, Sines [38-41] reported his procedure of selectively breeding Sprague-Dawley rats which were susceptible to restraint ulcer. This procedure yielded a Sprague-Dawley strain with a high incidence of stress ulcer. Another approach to this problem would involve studying animal strains with characteristics which predispose to ulcer development. Such a strain is the spontaneously hypertensive rat (SHR) and its normotensive progenitor, the Wistar-Kyoto (WKY) control which were developed by Okamoto and his co-workers [30]. The SHR rats are characterized as being more reactive since these animals demonstrate higher levels of epinephrine (EP) and norepinephrine (NE) in response to footshock [21-24], and when subjected to restraint stress [19,26]. SHR rats are also more active behaviorally when tested in the open-field arena [16, 17, 28, 45] or exposed to a novel environment [24]. Since the SHR rat is considered hyperresponsive to stress [26], the hypothesis might be advanced that the SHR rat is more susceptible to stress ulcer. Unfortunately, investigators who have studied this problem have produced conflicting results. Goldenberg [10,11] exposed male Sprague-Dawley, Wistar, and SHR rats to restraint plus cold for 3 hr and reported that a greater percentage of SHR rats had gastric lesions as compared to Wistars. Ulcer incidence, however, was highest in the SpragueDawley rats. Hallback, Magnusson and Weiss [12] failed to observe any differences in ulcer incidence between SHR and normotensive control Wistar Kyoto (WKY) rats. In their study, SHR and WKY rats were restrained for 12 hr but not subjected to the additional cold stressor. More recently
SHR
Open-field
Athey and Iams [4] reported a higher incidence of stress ulcer in WKY rats exposed to restraint plus cold as compared to SHR rats. Thus, none of the investigations which have studied ulcer susceptibility of SHR and WKY rats have produced comparable results. The aim of the present work was to further study stress ulcer susceptibility in SHR and WKY rats, but in this instance the investigation was more rigorous since rats were subjected to two ulcerogenic procedures: restraint plus cold [44] and activity-stress [31-35]. Since SHR rats are considered more reactive, it was hypothesized that SHR rats would manifest more ulcers as a result of exposure to restraint plus cold. SHR's were also considered more vulnerable to the activity-stress procedure since SHR rats are behaviorally more active than WKY rats in the open-field test and excessive running activity apparently contributes to the ulcerogenic process in the activity-stress procedure [31-35]. In order to confirm that SHR's were more reactive to a novel environment, all rats were tested in an open-field arena before being subjected to one of the ulcerogenic procedures. In addition since control of core body temperature is considered to be a factor in the development of restraint ulcer [3, 5, 8, 27, 42], and since WKY rats are reported to have difficulty in maintaining body temperature [45], core body temperature was recorded from all rats exposed to restraint plus cold. METHOD
Animals Thirty SHR and 30 WKY male rats were secured from Taconic Farms, Germantown, NY. These animals were 50-60 days old. Animals were housed in group cages with no more than 4 rats per cage. Group housing continued for two weeks, after which all animals were housed in single cages. Food and water were always available.
699
700
P*\RI:~ AN I) S{ 'HI M M t':l TABLE l
G R O U P M E A N S (-+SE) O F O P E N F I E L D M E A S U R E S F O R SPONTANEOUSLY HYPERTENSIVE (SHR) AND NORMOTENSIVE (WKY) RATS
Ill
Trial
AMBULATION k~ Trial
1
Z
W
Rat Strains Open Field Measures
SHR Rats (n=30)
WKY Rats (n=30)
p*
¢a~ 10 I-Z iii
2
SHR R a t s
'O~,J J "\ "o - -o
• ~/-
WKY
o
Rats
"0
W
Latency-see Rearings-number Grooming-number Grooming, duration-see Boluses-number
2.10 21.70 0.66 11.13 1.93
+_ 0.39 + 2.57 +_ 0.15 +_ 3.01 _+_0.30
3.73 1.36 0.26 2.33 2.06
+ 0.48 +_ 0.26 _+ 0.09 + 1.22 + 0.34
0.05 0.01 0.05 0.01 NS
*Statistical significance of t-test comparing SHR and WKY groups.
Apparatus The open-field arena was designed after the unit described by Broadhurst [6]. The arena was round with a diameter of 82 cm. The round wall was 30 cm high and was constructed of aluminum sheeting. The arena was situated on a p l y w o o d floor. The floor and wall were painted with black enamel paint. The arena was divided by three concentric circles. The smallest inner circle had a diameter of 20 cm; the second circle had a d i a m e t e r of 50 cm and the outside circle was defined by the arena wall. Each circle was divided into essentially equal areas. The number o f areas in the inner, middle and outer circles were one, six, and twelve, respectively. A ceiling was situated 132 cm a b o v e the arena floor. Five 100-W bulbs were mounted in the ceiling and served as the illumination for the arena. Cheese cloth was draped from the ceiling and dropped outside the arena wall. The cloth served to diffuse the light and functioned as a one-way vision screen. The immobilization device consisted of a 23 x 31 cm plastic board with cleats at each corner. The cleats were used to secure the braided umbilical tape which was used to tie the rat's four limbs. The cages for the activity-stress ulcer procedure consisted of standard running-wheel activity cages (Wahmann Manufacturing Co., Baltimore, MD). Each activity wheel was equipped with an adjoining cage measuring 25 x 15 x13 cm. Wheel revolutions were recorded on digital counters. Daylight conditions were artificially maintained in all housing and testing areas b e t w e e n 6 a.m. and 6 p.m.
Procedure The open-field testing phase was initiated after all rats were placed in single cages. The single cage, with the rat inside, was transported to the open-field testing room. Rats were placed in the inner circle. Five behaviors were measured: latency (in sec) to leave the inner circle, n u m b e r of field segments entered, n u m b e r of rearings, n u m b e r of groomings, and number of fecal boluses. Open-field testing consisted of two trials. The first trial lasted 5 min after which the rat was placed in its transport cage for l0 rain. Data for the four behaviors was recorded and the arena was cleaned with a damp sponge. Finally, the rat was returned to the arena for a second 5-min trial. After the second trial, the data for the same five behav-
1
2
3
4
.b
16
17
18
19
20
MINUTES
FIG. I. Mean open-field activity scores, in terms of segments entered for SHR rats (n=30) and WKY rats (n=30). Data are presented for the first 5 min during Trial 1 and for Trial 2 when rats were returned 10 rain later to the open-field for another 5 rain.
iors was recorded and a total score was derived for the two trials. The ambulation measure (i,e., segments entered) was r e c o r d e d for each min of both trials. After the animal was returned to the colony room, the arena was washed with soap and water. Each rat was exposed to only one open-field test session. S H R rats were rank ordered on the basis of their ambulation scores and subsequently assigned to one of four treatment conditions. These consisted of cold-restraint stress (n= 10), cold-restraint control (n=5), activity-stress experimental (n---10) and activity-stress control (n=5). These four groups were equated on the basis of the ambulation scores. The W K Y rats were also rank ordered and assigned to treatment groups in the same fashion. Coht-restraint. Mean body weights for S H R and W K Y rats, assigned to cold-restraint, were 279 and 307 g respectively. T h e s e rats had continuous access to food and water until 24 hr prior to the start of the stress session, at which time food was r e m o v e d . W a t e r was always available during the prerestraint period. Cold-restraint was accomplished by restraining the rat in a supine position on the plastic boards [43]. Each leg was e x t e n d e d at a 45 ° angle from the body midline and secured with a loop of braided nylon tape. Rats were then placed in a ventilated unilluminated refrigerator with an ambient temperature of 5°C. The restraint treatment lasted 3 hr. Core body temperature was measured at the beginning and end of the 3-hr restraint period with a Model 43TA Yellow Spring t h e r m o m e t e r and a No. 423 flexible nylon rectal temperature probe. S H R and W K Y rats in the cold-restraint control groups were food-deprived for 24 hr but were n e v e r restrained. Following the 3-hr restraint period, all experimental and control rats were sacrificed. The stomach was r e m o v e d immediately and cut along the greater curvature. It was rinsed with water, spread and pinned on a flat surface and c o v e r e d with 10% formalin to fix the stomach in a flat attitude. The stomach was e x a m i n e d with a binocular microscope. One e y e p i e c e of the scope was fitted with a reticle permitting ulcers to be m e a s u r e d in terms of millimeters of abnormal tissue. The n u m b e r and size of lesions were recorded. All stomach inspections w e r e c o n d u c t e d by a technician w h o was unaware of the rats' treatment condition. In addition, adrenal and thymus glands were r e m o v e d and wet weights were obtained. Weights w e r e expressed as milligrams per gram of body weight. Activity-stress. S H R and W K Y rats assigned to the exper-
STRESS U L C E R IN H Y P E R T E N S I V E RATS
701
TABLE 2 THE EFFECT OF COLD-STRESSON BODYTEMPERATURE,ADRENALGLAND AND THYMUS GLAND WEIGHT (MEANS --- SE)
n
Body Weight
Change in Body Temperature, %
Relative* Adrenal Weight
Relative* Thymus Weight
SHR Rats Experimental Control
10 5
279 ± 4.04 298 _+ 3.11
-12.9 ± 0.81 0
0.18 ± 0.01 0.19 ± 0.01
0.74 ± 0.03 0.73 ± 0.05
WKY Rats Experimental Control
10 5
307 ± 5.29 306 ± 4.91
-12.4 ± 1.44 0
0.19 ± 0.01 0.17 ± 0.01
0.73 ± 0.02 0.72 ± 0.04
Treatment Groups
*Expressed as milligrams per gram of body weight.
TABLE 3 SUMMARYOF STOMACHCONDITIONSFOR RATS EXPOSED TO COLD-RESTRAINT Treatment Groups SHR Rats Experimental Control WKY Rats Experimental Control
n
10 5 10 5
Number of Rats with Ulcers
5 0 9 0
Mean (-SE) Number Lesions/Rat
0.70 ± 0.26 0 10.00 ± 1.43 0
Mean (-SE) Cumulative Length (mn)
1.80 + 0.71 0 19.62± 3.09 0
imental activity-stress group were individually housed in running-wheel activity cages for a 4-day habituation period during which food (granular Purina Rat Chow) and water were available. Rats had continuous access to the running wheel. S HR and W K Y rats assigned to the activity-stress control condition were individually housed in single cages. On Day 4, food was withdrawn from all rats at 9:30 a.m. On Day 5, and all subsequent days, rats were fed for 1 hr daily between 9:30 and 10:30 a.m. Food consumption and body weight were recorded daily for experimental and control rats. In addition, running-wheel activity was recorded daily for experimental rats. When an experimental animal was moribund it was killed with carbon dioxide. Our operational criteria to determine that an animal was seriously ill were based on previous research [32]; these included (a) daily food consumption below 2 g, (b) a dramatic drop in dally running activity from the previous day, and (c) a fall in body temperature as indicated by piloerection. Stomachs were removed and inspected in the same fashion as described for the coldrestraint procedure. Ulcers observed in rats as a result of exposure to the activity-stress procedure were referred to as activity-stress (A-S) ulcers. When two S H R experimental rats died, one S HR control rat was also sacrificed. The same procedure was followed for the W K Y rats. After 12 days of 1-hr feeding, all surviving rats were sacrificed and stomachs inspected.
TABLE 4 PEARSON CORRELATIONCOEFFICIENTS*BETWEEN OPEN-FIELD AMBULATIONSCORES FOR SHR AND WKY RATSAND THE MEAN DAILY ACTIVITYSCORES FOR THE HABITUATIONDAYSAND RESTRICTED FEEDING DAYSOF THE A-S PROCEDURE Activity-Stress Periods Open-Field Ambulation SHR Ambulation WKY Ambulation
Activity for Habituation Period
Activity for Restricted Feeding Period
0.09 -0.27
0.03 -0.31
*dr=8.
RESULTS
Open-field testing. Table 1 summarizes the behavioral data for the open-field. S H R rats were clearly more active as compared to WKY rats. Latency scores were significantly shorter for SHR, F(1,58)=6.85, p<0.05. In addition, S H R ' s displayed more rearing responses, F(1,58)=61.68, p<0.05, groomed more frequently, F(1,58)=4.87, p<0.05, and for longer time periods, F(1,58)=7.30, p <0.01. S H R ' s were also more active in exploring the open-field. Ambulation scores were significantly greater for S H R rats throughout the first 5-min and second 5-min observation periods, F(1,580)=332.37, p<0.01. These data are illustrated by Fig. 1. The mean boluse score was greater for WKY rats but the difference between groups was not significant, F(1,58)=0.64. Cold-restraint stress. Core body temperature losses during cold-restraint were approximately the same for both S H R and W K Y rats, F(1,18)=0.11. An analysis of the adrenal and thymus gland weight failed to reveal any significant differences between either experimental and control groups or between SHR and W K Y rats. These data are presented in Table 2. There were, however, group differences with the ulcer data. Significantly more W K Y rats developed stomach ulcers from the cold-restraint procedure as compared to SHR rats, X2=4.05, p<0.05. W K Y rats had more ulcers, F(1,18)=40.52, p<0.01, and larger ulcers, F(1,18)=31.33,
702
PARE ANDSCHIMMEI, TABLE 5 THE EFFECT OF ACTIVITY-STRESS ON ADRENAL AND THYMUS GLAND WEIGHT (MEANS _+.SE)
n,-
io,oool I ;=,.
/o o
8,000
n
Body Weight (g)*
Relativet Adrenal Weight
Relative i Thymus Weight
SHR Rats Experimental Control
10 5
177.8 +_ 2.68 249.0 _+ 2.86
0.37 _+_0.01 0.21 ± 0.02
0.21 ± 0.01 (I.52 ± (I.04
WKY Rats Experimental Control
10 5
188.3 + 3.57 256.0 ± 3.87
(I.32 ± 0.02 (J.19 + 0.01
0.22 ± 0.02 0.58 ± 0.02
Treatment Groups
~. 6,000
_z z z
4,000 _...._..._o,,~ °
n- 2,000
t Start
.J
2
3
4
5
~-------VS K y Rats
l-hr
feeding
6
7
8
i 9
DAYS
FIG. 2. Mean daily running activity for SHR rats (n= 10) and WKY rats (n= I0) in the activity-stress procedure. Data are illustrated for the first 4 habituation days and the next 5 days of 1-hr feeding.
TABLE 6 SUMMARY OF STOMACH CONDITIONS FOR RATS EXPOSED TO ACTIVITY-STRESS
n
Number of Rats with Ulcers
Mean (-+SE) Number Lesions/Rat
Mean (-+SE) Cumulative Length (ran)
SHR Rats Experimental Control
10 5
10 0
22.20 ± 2.61 0
87.70 ± 9.69 0
WKY Rats Experimental Control
10 5
10 0
46.30 +_ 5.62 0
104.70 + 8.49 0
Treatment Groups
p < 0 . 0 1 as c o m p a r e d to S H R ' s . These data are summarized in Table 3. Activity-stress. There were no significant differences between S H R and W K Y experimental rats with respect to daily food c o n s u m p t i o n during the 4-day habituation period in the activity wheels, F(1,18)=0.02, p > 0 . 0 5 , although S H R rats were more active in the running wheels during this period, F(1,18)=7.37, p <0.025. An analysis o f the first 5 days of l-hr feeding also r e v e a l e d that there were no differences in food intake b e t w e e n S H R and W K Y rats, F(1,18)=0.61, p > 0 . 0 5 , but S H R rats persisted in being m o r e active in the running wheels, F(1,18) = 5.40, p <0.05. The running-wheel activity data are illustrated by Fig. 2. Open-field ambulation scores, within strains, were not predictive o f activity in the running-wheel cages. Table 4 contains the correlations b e t w e e n open-field ambulation for both S H R and W K Y rats, and running-wheel activity for the 4-day A-S habituation period, as well as the subsequent first 5 days of 1-hr feeding during the activity-stress procedure. N o n e of these correlations w e r e significant for either S H R or W K Y rats. These non-significant correlations suggested that, w h e n a within strain c o m p a r i s o n was made, the activity in the open-field and activity in the running-wheel activity
*Body weight at the end of the activity-stress period. tExpressed as milligrams per gram of body weight.
TABLE 7 PEARSON CORRELATION COEFFICIENTS BETWEEN THE OPENFIELD AMBULATION SCORES FOR SHR AND WKY RATS AND ULCERATION MEASURES FOR BOTH COLD-RESTRAINT AND ACTIVITY-STRESS PROCEDURES Ulcerogenic Procedures Cold-Restraint
Activity-Stress
Open-Field Ambulation
No. of Ulcers
Size of Ulcers
No. of Ulcers
Size of Ulcers
SHR Ambulation+ WKY Ambulationt SHR & WKY Combined~
0.33 0.05 -0.71"
0.26 0.17 -0.66*
0.20 0.26 -0.51"
0.19 0.35 0.38*
*p <0.05. td#= 8. $(U= 18.
cages were unrelated and probably reflected different behaviors. S H R and W K Y experimental rats had larger adrenals as c o m p a r e d to controls, F(1,26)=61.21, p < 0 . 0 1 . These data are contained in Table 5. This table also reveals that S H R ' s had larger adrenals than W K Y rats, F(1,26)=5.90, p < 0 . 0 5 . Exposure to activity-stress also resulted in a significant involution of the thymus, F(1,26)=213.11, p < 0 . 0 1 , and this effect was not strain specific since that data analysis did not reveal differences b e t w e e n S H R and W K Y rats with respect to thymus involutions. Table 6 summarizes the ulcer data. All experimental rats d e v e l o p e d stomach ulcers. The control rats were ulcer-free. H o w e v e r , the ulcer scores for the activity-stress procedure were similar to that o b s e r v e d with the cold-restraint procedure, namely, W K Y rats had significantly more ulcers, F(1,18) = i 1.59, p <0.01, and larger ulcers, F(1,18)=5.02, p < 0 . 0 5 , as c o m p a r e d to S H R rats. The n u m b e r o f days survived in the activity-stress procedure differed slightly for S H R and W K Y rats, but a life table analysis of the survival rates for the two strains, using a generalized W i l c o x o n test was not significant, T = 1 . 6 5 , p >0.05. If the two ulcerogenic procedures are c o m p a r e d , we find
STRESS U L C E R IN H Y P E R T E N S I V E RATS that the activity-stress procedure, as compared to the coldrestraint procedure, resulted in more ulcers in both SHR, F(1,18)=87.17, p<0.01, and W K Y rats, F(1,18)=39.16, p<0.01. In order to investigate the possible relationship between open-field ambulation and ulcer susceptibility, correlations were computed separately between the ambulation scores of SHR and W K Y rats, and the ulceration measures for both ulcerogenic procedures. Those correlations are contained in Table 7. In none of the comparisons made was a significant correlation found, therefore suggesting that, within strains, open-field ambulation was not related to cold-restraint ulcer or A-S ulcer. However, when SHR and W K Y animals were combined, this procedure allowed a comparison between the low ambulation scores of W K Y rats and their high ulcer scores as well as the high ambulation and relatively low ulcer scores of SHR rats. This inverse relationship was reflected by the significant negative correlations for the comparisons, as illustrated in the last row of Table 7. DISCUSSION The adrenal and thymus gland data are in agreement with previous reports. Changes in adrenal and thymus gland weights represent relatively gross measures of endocrine reactivity to environmental stressors and may not be sufficiently sensitive to detect effects issuing from an acute stress situation. This may explain the lack of group differences for adrenal and thymus weights in the cold-restraint portion of this investigation. However, in the activity-stress procedure where the treatment is applied for several days, variations in gland function may be detected by differences in gland weight. In this instance the larger adrenal gland weights for SHR rats are suggestive of previous reports that the sympatho-adrenal system is more reactive in S H R ' s [7,19]. The significant thymus involutions for experimental rats exposed to activity-stress concurs with the reports by Hara and his colleagues [13-15] who have suggested that thymus involution reflects an immunodeficiency in activity-stress rats. The open-field data obtained in this investigation agree with previous reports [16, 17, 20, 25]. S H R ' s had shorter latencies and were generally more active in the open-field arena than W K Y rats. In contrast, W K Y rats took longer to initiate exploratory behavior in the field and were less active during the testing sessions. Previous studies have found that SHR rats are more reactive to stimulation and that stressrelated secretions of epinephrine and norepinephrine are greater in this strain [19, 22, 23]. These data would support a hypothesis that S H R ' s should be more vulnerable to stressinduced ulceration. However, the open-field test results of this investigation suggested that W K Y rats may be considered more inhibited and fearful in a novel situation, and on that basis it might be reasonable to hypothesize that W K Y rats would be more ulcer prone. The ulcer data obtained in this study supported the latter hypothesis. There are other reasons why one would expect more ulcers in SHR rats. F o r instance Ader [1,2] demonstrated that the incidence of restraint ulcer is highest in those rats restrained during their daily high activity hr as compared to rats restrained during those daily hr when they are least active. Thus, restraint applied during peak activity hr is more ulcerogenic than restraint applied during resting hr. This would suggest that the more active SHR rats should be more vulnerable to restraint ulcer. However, in this study, W K Y rats had more ulcers. It may be necessary to differentiate
703 between general motor activity and reactivity to stimulation. Different notions can be advanced to explain the greater prevalence of restraint ulcer in W K Y rats. One possible explanation is based on the fact that a decrease in core body temperature is related to restraint-induced gastric lesions [3, 4, 8, 27, 42] and it has been reported that cold represents a greater challenge to W K Y rats because these rats have greater difficulty in maintaining normal body temperature [45]. Therefore W K Y rats would be expected to be more susceptible to cold-restraint ulcer. In our study, W K Y rats did indeed have more restraint ulcers as compared to SHR's but there were no differences in loss of core body temperature between the two strains. Therefore, differences in maintaining body temperature cannot be used to explain these results. An alternative explanation is the fact that, while SHR rats are hyperadrenergic to some stressors, this hyperadrenergic response does not occur to all stressful stimulations. Chiueh and McCarty [7] have demonstrated that the sympathoadrenal medullary system is essentially the same in SHR and W K Y rats under basal conditions, and that SHR rats will respond to footshock with an increase in plasma norepinephrine, epinephrine and corticosterone, but this response does not occur when exposed to a cold environment. This would suggest that, on the basis of adrenergic reactivity, W K Y rats are just as vulnerable as SHR to cold stress. Another consideration pertains to the designation that W K Y rats are more fearful and that this trait predisposes to stress ulcer. The open-field data would suggest that W K Y rats are more fearful, or "emotional," because these animals are relatively inactive and manifest very little exploratory behavior. Glavin [9] has already demonstrated that randomly selected Wistar rats with high emotionality scores, as determined by open-field testing, are also more susceptible to restraint-induced ulcers as compared to rats judged nonemotional. Another line of evidence also suggests that S H R ' s are less sensitive to pain, as induced by thermal stimulation in the hot-plate test [35,37], and that opiod paininhibiting mechanisms may be responsible for mediating this hypoalgesia [46]. The stress of thermal stimulation and coldrestraint may differ, but a common or similar opioid analgesic mechanism may be operating which could protect SHR rats from the stress of cold-restraint and thereby reduce ulcer incidence in these animals Previous research [31-35] had suggested that excessive running in the activity-wheel cages was related to the incidence of A-S ulcer. Since SHR rats were more active in the open-field arena, it was expected that this high open-field ambulation would transfer to greater running-wheel activity. When SHR rats were compared to W K Y rats, the greater open-field ambulation behavior of the SHR rats was associated with greater running-wheel activity; SHR rats were indeed more active than W K Y rats in the activity-stress situation. These higher activity scores were, however, contrary to expectations, not associated with more severe ulceration scores. Ulceration was less severe in SHR rats as compared to W K Y rats. It is also interesting to note that differences in open-field ambulation scores, within strains, were not related to corresponding differences in running-wheel activity. The non-significant correlations reported in Table 4 simply indicated that a high-active SHR rat in the open-field was not necessarily a high active rat in the running-wheel cages. Level of open-field ambulation was also unrelated to level of running-wheel activity for W K Y rats. These data may suggest that the exploratory ambulation behavior of the open-
704
PARE AND SCHIMMEL
field, and the running activity in the running wheels, may represent different behaviors and that high open-field ambulation scores is not a predictor of vulnerability to A-S ulcer. Knardahl and Sagvolden [17] have proposed that S H R ' s are not more hyperactive than W K Y rats under resting, baseline conditions, but that S H R rats are simply hyperactive to novel stimulation. Therefore, running-wheel and open-field activity may represent different behaviors and/or reactions in two unique test situations. In the past it was generally assumed that e x c e s s i v e running activity was a necessary element in the production of activity-stress ulcer [31-35], but the data from S H R and W K Y rats in this report imply that genetic factors may be more critical in the etiology of activity-stress ulceration. While running b e h a v i o r may be a contributing cause to ulcer d e v e l o p m e n t , it is not a sufficient cause. T h e r e are previous reports from this laboratory which are congruent with the present activity-stress data. Par6 and Vincent [35] reported a pilot study wherein Sprague-Dawley rats tested in the open-field and subsequently exposed to activity-stress produced some significant correlations between open-field b e h a v i o r and ulceration. Open-field rearing was inversely related to n u m b e r and size of ulcers. These
data would have predicted the reduced ulcer incidence of S H R rats in the present study. S H R rats may also more effectively tolerate chronic stress. Measures of cardiovascular and sympatho-adrenal medullary systems indicate a greater response by S H R ' s to initial stressors, but that repeated long term stress is accompanied by greater adaptive changes in S H R as c o m p a r e d to W K Y rats [19]. If activity-stress can be considered chronic stress, then S H R rats should be more resistant to A-S ulcer. The greater adaptability of S H R ' s to chronic stress, plus their hypoalgesia mentioned earlier [36,37] would help explain the low incidence of A-S ulcer in S H R as compared to W K Y rats. The present report supports the earlier report by Athey and lams [4]; cold-restraint ulcers are more prevalent in W K Y rats. H o w e v e r , the value of this report is that it emphasizes the importance of genetic and constitutional variables in the d e v e l o p m e n t of ulcer disease. S H R and W K Y rats exhibit different responses in various behavioral testing situations and they are also differentially susceptible to stress-ulcer. As such, S H R and W K Y rats represent a valuable tool for investigating how genetic factors, as well as behavioral traits, contribute to ulcer disease.
REFERENCES
1. Ader, R. Gastric erosions in the rat: Effects of immobilization at different points in the activity cycle. Science 145: 406-407, 1964. 2. Ader, R. Behavioral and physiological rhythms and the development of gastric erosions in the rat. Psychosom Med 29: 345353, 1967. 3. Anton, J. W. and R. V. Gregg. The influence of body temperature on the production of ulcers of restraint in the rat. Gastroenterology 70: 747-750, 1976. 4. Athey, G. R. and S. G. lams. Cold-restraint induced gastric lesions in normotensive and spontaneously hypertensive rats. Lift, Sci 28: 88%894, 1981. 5. Bartlett, R. G., Jr., V. C. Bohr and R. H. Helmendach. Ability of rat to adapt to stress of light restraint. Prae Soc l=~p Biol Med 86" 395-396, 1954. 6. Broadhurst, P. L. Determinants of emotionality in the rat. I. Situational factors. Br J Psychol 48: 1-12, 1957. 7. Chiueh, C. C. and R. McCarty. Sympatho-adrenal hyperreactivity to footshock stress but not to cold exposure in spontaneously hypertensive rats. Physiol Behav 26: 85-89, 1981. 8. Dalton, D. J. The effect of maintenance of normal body temperature during the alarm reaction. Anat Rec 78:110-111, 1940. 9. Glavin, B. P. Emotionality, coping behavior and ulcer production and healing in rats. Parlor .I Biol Sci 17: 101, 1982. 10. Goldenberg, M. M. Study of cold + restraint stress gastric lesions in spontaneously hypertensive, Wistar and SpragueDawley rats. Life Sei 12: 51%527, 1974. 11, Goldenberg, M. M. Stress-induced gastric lesions in spontaneously hypertensive rats. Am J Dig Dis 19: 353-360, 1974. 12. Hallback, M., G. Magnusson and L. Weiss. Stress-induced ulcer in spontaneously hypertensive rats. Acta Physiol Sound 19: 6-7, 1974. 13. Hara. C., K. Manabe and N. Ogawa. Influence of activity-stress on thymus, spleen and adrenal weights of rats: Possibility for an immunodeficiency model. Physiol Behav 27: 243-248, 1981. 14. Hara, C., N. Ogawa and Y. Imada. The activity-stress ulcer and antibody production in rats. Physiol Behav 27: 609-613, 1981. 15. Hara, C. and N. Ogawa. Influence of maturation on ulcerdevelopment and immunodeficiency induced by activity-stress in rats. Physiol Behav 27: 187-200, 1979. 16. Knardahl, S. and T. Sagvolden. Open-field behavior of spontaneously hypertensive rats. Behav Neural Biol 27: 187-200, 1979.
17. Knardahl, S. and T. Sagvolden. Regarding hyperactivity of the SHR in the open-field test. Behav Neural Bio132: 274--275, 1981. 18. Knardahl, S. and T. Sagvolden. Two-way active avoidance behavior of spontaneously hypertensive rats: Effects of intensity of discontinuous shock. Behav Neural Biol 35: 105-120, 1982. 19. Kvetnansky, R., R. McCarty, N. B. Thoa, C. R. Lake and I. J. Kopin. Sympatho-adrenal responses of spontaneously hypertensive rats to immobilization stress. Am ,I Physiol 236: 457462, 1979. 20. Martin, M. S., F. Martin, C. Andre and R. Lambert. Selection du rat Wistar en function de sa sensibilite a I'ulcere de constrainte. ('omp Soc Bial 162: 2126--2128, 1968. 21. McCarty, R., C. C. Chiueh and I. J. Kopin. Behavioral and cardiovascular responses of spontaneously hypertensive and normotensive rats to inescapable footshock. Behav Bial 22: 405-410, 1978. 22. McCarty, R. and I. J. Kopin. Alterations in plasma catecholamines and behavior during acute stress in spontaneously hypertensive and Wistar-Kyoto normotensive rats. Life Sei 22: 9971005, 1978. 23. McCarty, R. and I. J. Kopin. Sympatho-adrenal medullary activity and behavior during exposure to footshock stress: A comparison of seven rat strains. Physiol Behav 21: 567-572, 1978. 24. McCarty, R. and 1. J. Kopin. Patterns of behavioral development in spontaneously hypertensive rats and Wistar-Kyoto normotensive controls. Dev Psychabial 12: 23%243, 1979. 25. McCarty, R. and R. F. Kirby. Spontaneous hypertension and open-field behavior. Behav Neural Bio! 34: 450-452, 1982. 26. McCarty, R., R. Kvetnansky, C. R. Lake, N. B. Thoa and 1. J. Kopin. Sympatho-adrenal activity of SHR and WKY rats during recovery from forced immobilization. Physiol Behav 21: 951955, 1978. 27. Mersereau, W. A. and E. J. Hinchey. Hypothermia-induced gastric hypercontractility in the genesis of the restraint ulcer. Can J Surg 24: 622-625, 1981. 28. Myers, M. M., D. McCrorery, C. A. Bulman and E. D. Hendley. Open field behavior and brain norepinephrine uptake in spontaneously hypertensive rats and Wistar-Kyoto normotensive controls. Fed Proe 36: 1047, 1977. 29. Myers, M. M., R. E. Musty and E. D. Hendby. Attenuation of hyperactivity in the spontaneously hypertensive rat by amphetamine. Behav Neural Biol 34: 42-54, 1982.
S T R E S S U L C E R IN H Y P E R T E N S I V E R A T S 30. Okamoto, K. and K. Aoki. Development of a strain of spontaneously hypertensive rats. Jpn Circ J 27: 282-293, 1963. 31. Par6, W. P. The influence of food consumption and running activity on the activity-stress ulcer in the rat. Am J Dig Dis 20: 262-273, 1975. 32. Par6, W. P. The activity-stress ulcer: Frequency and chronicity. Physiol Behav 16: 699-704, 1976. 33. Par6, W. P. Psychological studies of stress ulcer in the rat. Brain Res Bull 5: Suppl l, 73-79, 1980. 34. Par6, W. P. and V. P. Houser. Activity food-restriction effects on gastric glandular lesions in the rat: The activity-stress ulcer. Bull Psychon Soc 2: 213-214, 1973. 35. Par6, W. P. and G. P. Vincent. The activity-stress ulcer model. In: Advanees in Experimental Ulcer, edited by S. Umehara and H. Ito. Tokyo: Tokyo Medical College Press, 1981, pp. 93-108. 36. Randich, A. and W. Maxiner. Acquisition of conditioned suppression and responsivity to thermal stimulation in spontaneously hypertensive, renal hypertensive and normotensive rats. Physiol Behav 27: 585-590, 1981. 37. Saavedra, J. M. Naloxone reversible decrease in pain sensitivity in young and adult spontaneously hypertensive rats. Brain Res 209: 245-249, 1981. 38. Sines, J. O. Selective breeding for development of stomach lesions following stress in the rat. J Comp Physiol Psychol 52: 615-617, 1959.
705 39. Sines, J. O. Strain differences in activity, emotionality, body weight and susceptibility to stress induced stomach lesions. J Genet Psychol 101: 209-217, 1962. 40. Sines, J. O. Physiological and behavioral characteristics of rats selectively bred for susceptibility to stomach lesion development. J Neuropsychiatr 4: 396-398, 1963. 41. Sines, J. O. and D. G. McDonald. Heritability of stressed-ulcer susceptibility in rats. Psychosom Med 30: 390-394, 1968. 42. Tran, T. A. and R. V. Gregg. Hypothermia in restraint-induced gastric ulcers in parabiotic rats. Gastroenterology 67: 271-275, 1974. 43. Vincent, G., G. Glavin, J. Rutkowski and W. Par& Body orientation, food deprivation and potentiation of restraint induced gastric lesions. Gastroenterol Clin Biol 1: 53%543, 1977. 44. Vincent, G. P. and W. P. Par6. Post-stress development and healing of supine-restraint induced stomach lesions in the rat. Physiol Behav 29: 721-725, 1982. 45. Wilson, L, M. Some developmental aspects of open-field behavior in the spontaneously hypertensive rat (SHR) and two normotensive strains. Paper presented to the International Society for Developmental Psychobiology, Toronto, Canada, 1976. 46. Zamir, N., R. Simantov and M. Segal. Pain sensitivity and opioid activity in genetically and experimentally hypertensive rats. Brain Res 184: 299-310, 1980.
0031-9384/86$3.00 + .00
Stress Ulcer in Normotensive and Spontaneously Hypertensive Rats W I L L I A M P. PARI~ A N D G R E G G T. S C H I M M E L
V e t e r a n s A d m i n i s t r a t i o n M e d i c a l C e n t e r (15R), P e r r y P o i n t , M D 21902 R e c e i v e d 11 M a r c h 1985 PARE, W. P, AND G, T. SCH|MMEL. Stress ulcer in normotensive and spontaneously hypertensive rats. PHYSIOL BEHAV 36(4) 699-705, 1986.--Spontaneously hypertensive rats (SHR) and their normotensive progenitors, the WistarKyoto (WKY) rats, were tested in the open-field arena and subsequently exposed to either cold-restraint stress or activity-stress. SHR rats were more active and judged less fearful in the open-field test. Changes in core body temperature, and adrenal and thymus weights did not differentiate between SHR and WKY rats in the cold-restraint procedure. A significant adrenal hypertrophy was observed for SHR rats in the activity-stress procedure. WKY rats were more susceptible to stress ulcer in both the cold-restraint and the activity-stress procedures. While running-wheel activity had been considered an important etiological variable for activity-stress ulcer, the lower activity demonstrated by the ulcer-prone WKY rats suggested that genetic variables might be more relevant to stress ulcer disease. Activity-stress Stress ulcer Cold restraint Core body temperature Adrenal Thymus
Spontaneously hypertensive
SOME investigators have emphasized the importance of genetic factors in the etiology of stress ulcer. For instance, Martin, Martin, Andre and Lambert [20] described differential susceptibility to restraint-induced stomach ulcers in two distinct strains of Wistar rats. In a series of studies, Sines [38-41] reported his procedure of selectively breeding Sprague-Dawley rats which were susceptible to restraint ulcer. This procedure yielded a Sprague-Dawley strain with a high incidence of stress ulcer. Another approach to this problem would involve studying animal strains with characteristics which predispose to ulcer development. Such a strain is the spontaneously hypertensive rat (SHR) and its normotensive progenitor, the Wistar-Kyoto (WKY) control which were developed by Okamoto and his co-workers [30]. The SHR rats are characterized as being more reactive since these animals demonstrate higher levels of epinephrine (EP) and norepinephrine (NE) in response to footshock [21-24], and when subjected to restraint stress [19,26]. SHR rats are also more active behaviorally when tested in the open-field arena [16, 17, 28, 45] or exposed to a novel environment [24]. Since the SHR rat is considered hyperresponsive to stress [26], the hypothesis might be advanced that the SHR rat is more susceptible to stress ulcer. Unfortunately, investigators who have studied this problem have produced conflicting results. Goldenberg [10,11] exposed male Sprague-Dawley, Wistar, and SHR rats to restraint plus cold for 3 hr and reported that a greater percentage of SHR rats had gastric lesions as compared to Wistars. Ulcer incidence, however, was highest in the SpragueDawley rats. Hallback, Magnusson and Weiss [12] failed to observe any differences in ulcer incidence between SHR and normotensive control Wistar Kyoto (WKY) rats. In their study, SHR and WKY rats were restrained for 12 hr but not subjected to the additional cold stressor. More recently
SHR
Open-field
Athey and Iams [4] reported a higher incidence of stress ulcer in WKY rats exposed to restraint plus cold as compared to SHR rats. Thus, none of the investigations which have studied ulcer susceptibility of SHR and WKY rats have produced comparable results. The aim of the present work was to further study stress ulcer susceptibility in SHR and WKY rats, but in this instance the investigation was more rigorous since rats were subjected to two ulcerogenic procedures: restraint plus cold [44] and activity-stress [31-35]. Since SHR rats are considered more reactive, it was hypothesized that SHR rats would manifest more ulcers as a result of exposure to restraint plus cold. SHR's were also considered more vulnerable to the activity-stress procedure since SHR rats are behaviorally more active than WKY rats in the open-field test and excessive running activity apparently contributes to the ulcerogenic process in the activity-stress procedure [31-35]. In order to confirm that SHR's were more reactive to a novel environment, all rats were tested in an open-field arena before being subjected to one of the ulcerogenic procedures. In addition since control of core body temperature is considered to be a factor in the development of restraint ulcer [3, 5, 8, 27, 42], and since WKY rats are reported to have difficulty in maintaining body temperature [45], core body temperature was recorded from all rats exposed to restraint plus cold. METHOD
Animals Thirty SHR and 30 WKY male rats were secured from Taconic Farms, Germantown, NY. These animals were 50-60 days old. Animals were housed in group cages with no more than 4 rats per cage. Group housing continued for two weeks, after which all animals were housed in single cages. Food and water were always available.
699
700
P*\RI:~ AN I) S{ 'HI M M t':l TABLE l
G R O U P M E A N S (-+SE) O F O P E N F I E L D M E A S U R E S F O R SPONTANEOUSLY HYPERTENSIVE (SHR) AND NORMOTENSIVE (WKY) RATS
Ill
Trial
AMBULATION k~ Trial
1
Z
W
Rat Strains Open Field Measures
SHR Rats (n=30)
WKY Rats (n=30)
p*
¢a~ 10 I-Z iii
2
SHR R a t s
'O~,J J "\ "o - -o
• ~/-
WKY
o
Rats
"0
W
Latency-see Rearings-number Grooming-number Grooming, duration-see Boluses-number
2.10 21.70 0.66 11.13 1.93
+_ 0.39 + 2.57 +_ 0.15 +_ 3.01 _+_0.30
3.73 1.36 0.26 2.33 2.06
+ 0.48 +_ 0.26 _+ 0.09 + 1.22 + 0.34
0.05 0.01 0.05 0.01 NS
*Statistical significance of t-test comparing SHR and WKY groups.
Apparatus The open-field arena was designed after the unit described by Broadhurst [6]. The arena was round with a diameter of 82 cm. The round wall was 30 cm high and was constructed of aluminum sheeting. The arena was situated on a p l y w o o d floor. The floor and wall were painted with black enamel paint. The arena was divided by three concentric circles. The smallest inner circle had a diameter of 20 cm; the second circle had a d i a m e t e r of 50 cm and the outside circle was defined by the arena wall. Each circle was divided into essentially equal areas. The number o f areas in the inner, middle and outer circles were one, six, and twelve, respectively. A ceiling was situated 132 cm a b o v e the arena floor. Five 100-W bulbs were mounted in the ceiling and served as the illumination for the arena. Cheese cloth was draped from the ceiling and dropped outside the arena wall. The cloth served to diffuse the light and functioned as a one-way vision screen. The immobilization device consisted of a 23 x 31 cm plastic board with cleats at each corner. The cleats were used to secure the braided umbilical tape which was used to tie the rat's four limbs. The cages for the activity-stress ulcer procedure consisted of standard running-wheel activity cages (Wahmann Manufacturing Co., Baltimore, MD). Each activity wheel was equipped with an adjoining cage measuring 25 x 15 x13 cm. Wheel revolutions were recorded on digital counters. Daylight conditions were artificially maintained in all housing and testing areas b e t w e e n 6 a.m. and 6 p.m.
Procedure The open-field testing phase was initiated after all rats were placed in single cages. The single cage, with the rat inside, was transported to the open-field testing room. Rats were placed in the inner circle. Five behaviors were measured: latency (in sec) to leave the inner circle, n u m b e r of field segments entered, n u m b e r of rearings, n u m b e r of groomings, and number of fecal boluses. Open-field testing consisted of two trials. The first trial lasted 5 min after which the rat was placed in its transport cage for l0 rain. Data for the four behaviors was recorded and the arena was cleaned with a damp sponge. Finally, the rat was returned to the arena for a second 5-min trial. After the second trial, the data for the same five behav-
1
2
3
4
.b
16
17
18
19
20
MINUTES
FIG. I. Mean open-field activity scores, in terms of segments entered for SHR rats (n=30) and WKY rats (n=30). Data are presented for the first 5 min during Trial 1 and for Trial 2 when rats were returned 10 rain later to the open-field for another 5 rain.
iors was recorded and a total score was derived for the two trials. The ambulation measure (i,e., segments entered) was r e c o r d e d for each min of both trials. After the animal was returned to the colony room, the arena was washed with soap and water. Each rat was exposed to only one open-field test session. S H R rats were rank ordered on the basis of their ambulation scores and subsequently assigned to one of four treatment conditions. These consisted of cold-restraint stress (n= 10), cold-restraint control (n=5), activity-stress experimental (n---10) and activity-stress control (n=5). These four groups were equated on the basis of the ambulation scores. The W K Y rats were also rank ordered and assigned to treatment groups in the same fashion. Coht-restraint. Mean body weights for S H R and W K Y rats, assigned to cold-restraint, were 279 and 307 g respectively. T h e s e rats had continuous access to food and water until 24 hr prior to the start of the stress session, at which time food was r e m o v e d . W a t e r was always available during the prerestraint period. Cold-restraint was accomplished by restraining the rat in a supine position on the plastic boards [43]. Each leg was e x t e n d e d at a 45 ° angle from the body midline and secured with a loop of braided nylon tape. Rats were then placed in a ventilated unilluminated refrigerator with an ambient temperature of 5°C. The restraint treatment lasted 3 hr. Core body temperature was measured at the beginning and end of the 3-hr restraint period with a Model 43TA Yellow Spring t h e r m o m e t e r and a No. 423 flexible nylon rectal temperature probe. S H R and W K Y rats in the cold-restraint control groups were food-deprived for 24 hr but were n e v e r restrained. Following the 3-hr restraint period, all experimental and control rats were sacrificed. The stomach was r e m o v e d immediately and cut along the greater curvature. It was rinsed with water, spread and pinned on a flat surface and c o v e r e d with 10% formalin to fix the stomach in a flat attitude. The stomach was e x a m i n e d with a binocular microscope. One e y e p i e c e of the scope was fitted with a reticle permitting ulcers to be m e a s u r e d in terms of millimeters of abnormal tissue. The n u m b e r and size of lesions were recorded. All stomach inspections w e r e c o n d u c t e d by a technician w h o was unaware of the rats' treatment condition. In addition, adrenal and thymus glands were r e m o v e d and wet weights were obtained. Weights w e r e expressed as milligrams per gram of body weight. Activity-stress. S H R and W K Y rats assigned to the exper-
STRESS U L C E R IN H Y P E R T E N S I V E RATS
701
TABLE 2 THE EFFECT OF COLD-STRESSON BODYTEMPERATURE,ADRENALGLAND AND THYMUS GLAND WEIGHT (MEANS --- SE)
n
Body Weight
Change in Body Temperature, %
Relative* Adrenal Weight
Relative* Thymus Weight
SHR Rats Experimental Control
10 5
279 ± 4.04 298 _+ 3.11
-12.9 ± 0.81 0
0.18 ± 0.01 0.19 ± 0.01
0.74 ± 0.03 0.73 ± 0.05
WKY Rats Experimental Control
10 5
307 ± 5.29 306 ± 4.91
-12.4 ± 1.44 0
0.19 ± 0.01 0.17 ± 0.01
0.73 ± 0.02 0.72 ± 0.04
Treatment Groups
*Expressed as milligrams per gram of body weight.
TABLE 3 SUMMARYOF STOMACHCONDITIONSFOR RATS EXPOSED TO COLD-RESTRAINT Treatment Groups SHR Rats Experimental Control WKY Rats Experimental Control
n
10 5 10 5
Number of Rats with Ulcers
5 0 9 0
Mean (-SE) Number Lesions/Rat
0.70 ± 0.26 0 10.00 ± 1.43 0
Mean (-SE) Cumulative Length (mn)
1.80 + 0.71 0 19.62± 3.09 0
imental activity-stress group were individually housed in running-wheel activity cages for a 4-day habituation period during which food (granular Purina Rat Chow) and water were available. Rats had continuous access to the running wheel. S HR and W K Y rats assigned to the activity-stress control condition were individually housed in single cages. On Day 4, food was withdrawn from all rats at 9:30 a.m. On Day 5, and all subsequent days, rats were fed for 1 hr daily between 9:30 and 10:30 a.m. Food consumption and body weight were recorded daily for experimental and control rats. In addition, running-wheel activity was recorded daily for experimental rats. When an experimental animal was moribund it was killed with carbon dioxide. Our operational criteria to determine that an animal was seriously ill were based on previous research [32]; these included (a) daily food consumption below 2 g, (b) a dramatic drop in dally running activity from the previous day, and (c) a fall in body temperature as indicated by piloerection. Stomachs were removed and inspected in the same fashion as described for the coldrestraint procedure. Ulcers observed in rats as a result of exposure to the activity-stress procedure were referred to as activity-stress (A-S) ulcers. When two S H R experimental rats died, one S HR control rat was also sacrificed. The same procedure was followed for the W K Y rats. After 12 days of 1-hr feeding, all surviving rats were sacrificed and stomachs inspected.
TABLE 4 PEARSON CORRELATIONCOEFFICIENTS*BETWEEN OPEN-FIELD AMBULATIONSCORES FOR SHR AND WKY RATSAND THE MEAN DAILY ACTIVITYSCORES FOR THE HABITUATIONDAYSAND RESTRICTED FEEDING DAYSOF THE A-S PROCEDURE Activity-Stress Periods Open-Field Ambulation SHR Ambulation WKY Ambulation
Activity for Habituation Period
Activity for Restricted Feeding Period
0.09 -0.27
0.03 -0.31
*dr=8.
RESULTS
Open-field testing. Table 1 summarizes the behavioral data for the open-field. S H R rats were clearly more active as compared to WKY rats. Latency scores were significantly shorter for SHR, F(1,58)=6.85, p<0.05. In addition, S H R ' s displayed more rearing responses, F(1,58)=61.68, p<0.05, groomed more frequently, F(1,58)=4.87, p<0.05, and for longer time periods, F(1,58)=7.30, p <0.01. S H R ' s were also more active in exploring the open-field. Ambulation scores were significantly greater for S H R rats throughout the first 5-min and second 5-min observation periods, F(1,580)=332.37, p<0.01. These data are illustrated by Fig. 1. The mean boluse score was greater for WKY rats but the difference between groups was not significant, F(1,58)=0.64. Cold-restraint stress. Core body temperature losses during cold-restraint were approximately the same for both S H R and W K Y rats, F(1,18)=0.11. An analysis of the adrenal and thymus gland weight failed to reveal any significant differences between either experimental and control groups or between SHR and W K Y rats. These data are presented in Table 2. There were, however, group differences with the ulcer data. Significantly more W K Y rats developed stomach ulcers from the cold-restraint procedure as compared to SHR rats, X2=4.05, p<0.05. W K Y rats had more ulcers, F(1,18)=40.52, p<0.01, and larger ulcers, F(1,18)=31.33,
702
PARE ANDSCHIMMEI, TABLE 5 THE EFFECT OF ACTIVITY-STRESS ON ADRENAL AND THYMUS GLAND WEIGHT (MEANS _+.SE)
n,-
io,oool I ;=,.
/o o
8,000
n
Body Weight (g)*
Relativet Adrenal Weight
Relative i Thymus Weight
SHR Rats Experimental Control
10 5
177.8 +_ 2.68 249.0 _+ 2.86
0.37 _+_0.01 0.21 ± 0.02
0.21 ± 0.01 (I.52 ± (I.04
WKY Rats Experimental Control
10 5
188.3 + 3.57 256.0 ± 3.87
(I.32 ± 0.02 (J.19 + 0.01
0.22 ± 0.02 0.58 ± 0.02
Treatment Groups
~. 6,000
_z z z
4,000 _...._..._o,,~ °
n- 2,000
t Start
.J
2
3
4
5
~-------VS K y Rats
l-hr
feeding
6
7
8
i 9
DAYS
FIG. 2. Mean daily running activity for SHR rats (n= 10) and WKY rats (n= I0) in the activity-stress procedure. Data are illustrated for the first 4 habituation days and the next 5 days of 1-hr feeding.
TABLE 6 SUMMARY OF STOMACH CONDITIONS FOR RATS EXPOSED TO ACTIVITY-STRESS
n
Number of Rats with Ulcers
Mean (-+SE) Number Lesions/Rat
Mean (-+SE) Cumulative Length (ran)
SHR Rats Experimental Control
10 5
10 0
22.20 ± 2.61 0
87.70 ± 9.69 0
WKY Rats Experimental Control
10 5
10 0
46.30 +_ 5.62 0
104.70 + 8.49 0
Treatment Groups
p < 0 . 0 1 as c o m p a r e d to S H R ' s . These data are summarized in Table 3. Activity-stress. There were no significant differences between S H R and W K Y experimental rats with respect to daily food c o n s u m p t i o n during the 4-day habituation period in the activity wheels, F(1,18)=0.02, p > 0 . 0 5 , although S H R rats were more active in the running wheels during this period, F(1,18)=7.37, p <0.025. An analysis o f the first 5 days of l-hr feeding also r e v e a l e d that there were no differences in food intake b e t w e e n S H R and W K Y rats, F(1,18)=0.61, p > 0 . 0 5 , but S H R rats persisted in being m o r e active in the running wheels, F(1,18) = 5.40, p <0.05. The running-wheel activity data are illustrated by Fig. 2. Open-field ambulation scores, within strains, were not predictive o f activity in the running-wheel cages. Table 4 contains the correlations b e t w e e n open-field ambulation for both S H R and W K Y rats, and running-wheel activity for the 4-day A-S habituation period, as well as the subsequent first 5 days of 1-hr feeding during the activity-stress procedure. N o n e of these correlations w e r e significant for either S H R or W K Y rats. These non-significant correlations suggested that, w h e n a within strain c o m p a r i s o n was made, the activity in the open-field and activity in the running-wheel activity
*Body weight at the end of the activity-stress period. tExpressed as milligrams per gram of body weight.
TABLE 7 PEARSON CORRELATION COEFFICIENTS BETWEEN THE OPENFIELD AMBULATION SCORES FOR SHR AND WKY RATS AND ULCERATION MEASURES FOR BOTH COLD-RESTRAINT AND ACTIVITY-STRESS PROCEDURES Ulcerogenic Procedures Cold-Restraint
Activity-Stress
Open-Field Ambulation
No. of Ulcers
Size of Ulcers
No. of Ulcers
Size of Ulcers
SHR Ambulation+ WKY Ambulationt SHR & WKY Combined~
0.33 0.05 -0.71"
0.26 0.17 -0.66*
0.20 0.26 -0.51"
0.19 0.35 0.38*
*p <0.05. td#= 8. $(U= 18.
cages were unrelated and probably reflected different behaviors. S H R and W K Y experimental rats had larger adrenals as c o m p a r e d to controls, F(1,26)=61.21, p < 0 . 0 1 . These data are contained in Table 5. This table also reveals that S H R ' s had larger adrenals than W K Y rats, F(1,26)=5.90, p < 0 . 0 5 . Exposure to activity-stress also resulted in a significant involution of the thymus, F(1,26)=213.11, p < 0 . 0 1 , and this effect was not strain specific since that data analysis did not reveal differences b e t w e e n S H R and W K Y rats with respect to thymus involutions. Table 6 summarizes the ulcer data. All experimental rats d e v e l o p e d stomach ulcers. The control rats were ulcer-free. H o w e v e r , the ulcer scores for the activity-stress procedure were similar to that o b s e r v e d with the cold-restraint procedure, namely, W K Y rats had significantly more ulcers, F(1,18) = i 1.59, p <0.01, and larger ulcers, F(1,18)=5.02, p < 0 . 0 5 , as c o m p a r e d to S H R rats. The n u m b e r o f days survived in the activity-stress procedure differed slightly for S H R and W K Y rats, but a life table analysis of the survival rates for the two strains, using a generalized W i l c o x o n test was not significant, T = 1 . 6 5 , p >0.05. If the two ulcerogenic procedures are c o m p a r e d , we find
STRESS U L C E R IN H Y P E R T E N S I V E RATS that the activity-stress procedure, as compared to the coldrestraint procedure, resulted in more ulcers in both SHR, F(1,18)=87.17, p<0.01, and W K Y rats, F(1,18)=39.16, p<0.01. In order to investigate the possible relationship between open-field ambulation and ulcer susceptibility, correlations were computed separately between the ambulation scores of SHR and W K Y rats, and the ulceration measures for both ulcerogenic procedures. Those correlations are contained in Table 7. In none of the comparisons made was a significant correlation found, therefore suggesting that, within strains, open-field ambulation was not related to cold-restraint ulcer or A-S ulcer. However, when SHR and W K Y animals were combined, this procedure allowed a comparison between the low ambulation scores of W K Y rats and their high ulcer scores as well as the high ambulation and relatively low ulcer scores of SHR rats. This inverse relationship was reflected by the significant negative correlations for the comparisons, as illustrated in the last row of Table 7. DISCUSSION The adrenal and thymus gland data are in agreement with previous reports. Changes in adrenal and thymus gland weights represent relatively gross measures of endocrine reactivity to environmental stressors and may not be sufficiently sensitive to detect effects issuing from an acute stress situation. This may explain the lack of group differences for adrenal and thymus weights in the cold-restraint portion of this investigation. However, in the activity-stress procedure where the treatment is applied for several days, variations in gland function may be detected by differences in gland weight. In this instance the larger adrenal gland weights for SHR rats are suggestive of previous reports that the sympatho-adrenal system is more reactive in S H R ' s [7,19]. The significant thymus involutions for experimental rats exposed to activity-stress concurs with the reports by Hara and his colleagues [13-15] who have suggested that thymus involution reflects an immunodeficiency in activity-stress rats. The open-field data obtained in this investigation agree with previous reports [16, 17, 20, 25]. S H R ' s had shorter latencies and were generally more active in the open-field arena than W K Y rats. In contrast, W K Y rats took longer to initiate exploratory behavior in the field and were less active during the testing sessions. Previous studies have found that SHR rats are more reactive to stimulation and that stressrelated secretions of epinephrine and norepinephrine are greater in this strain [19, 22, 23]. These data would support a hypothesis that S H R ' s should be more vulnerable to stressinduced ulceration. However, the open-field test results of this investigation suggested that W K Y rats may be considered more inhibited and fearful in a novel situation, and on that basis it might be reasonable to hypothesize that W K Y rats would be more ulcer prone. The ulcer data obtained in this study supported the latter hypothesis. There are other reasons why one would expect more ulcers in SHR rats. F o r instance Ader [1,2] demonstrated that the incidence of restraint ulcer is highest in those rats restrained during their daily high activity hr as compared to rats restrained during those daily hr when they are least active. Thus, restraint applied during peak activity hr is more ulcerogenic than restraint applied during resting hr. This would suggest that the more active SHR rats should be more vulnerable to restraint ulcer. However, in this study, W K Y rats had more ulcers. It may be necessary to differentiate
703 between general motor activity and reactivity to stimulation. Different notions can be advanced to explain the greater prevalence of restraint ulcer in W K Y rats. One possible explanation is based on the fact that a decrease in core body temperature is related to restraint-induced gastric lesions [3, 4, 8, 27, 42] and it has been reported that cold represents a greater challenge to W K Y rats because these rats have greater difficulty in maintaining normal body temperature [45]. Therefore W K Y rats would be expected to be more susceptible to cold-restraint ulcer. In our study, W K Y rats did indeed have more restraint ulcers as compared to SHR's but there were no differences in loss of core body temperature between the two strains. Therefore, differences in maintaining body temperature cannot be used to explain these results. An alternative explanation is the fact that, while SHR rats are hyperadrenergic to some stressors, this hyperadrenergic response does not occur to all stressful stimulations. Chiueh and McCarty [7] have demonstrated that the sympathoadrenal medullary system is essentially the same in SHR and W K Y rats under basal conditions, and that SHR rats will respond to footshock with an increase in plasma norepinephrine, epinephrine and corticosterone, but this response does not occur when exposed to a cold environment. This would suggest that, on the basis of adrenergic reactivity, W K Y rats are just as vulnerable as SHR to cold stress. Another consideration pertains to the designation that W K Y rats are more fearful and that this trait predisposes to stress ulcer. The open-field data would suggest that W K Y rats are more fearful, or "emotional," because these animals are relatively inactive and manifest very little exploratory behavior. Glavin [9] has already demonstrated that randomly selected Wistar rats with high emotionality scores, as determined by open-field testing, are also more susceptible to restraint-induced ulcers as compared to rats judged nonemotional. Another line of evidence also suggests that S H R ' s are less sensitive to pain, as induced by thermal stimulation in the hot-plate test [35,37], and that opiod paininhibiting mechanisms may be responsible for mediating this hypoalgesia [46]. The stress of thermal stimulation and coldrestraint may differ, but a common or similar opioid analgesic mechanism may be operating which could protect SHR rats from the stress of cold-restraint and thereby reduce ulcer incidence in these animals Previous research [31-35] had suggested that excessive running in the activity-wheel cages was related to the incidence of A-S ulcer. Since SHR rats were more active in the open-field arena, it was expected that this high open-field ambulation would transfer to greater running-wheel activity. When SHR rats were compared to W K Y rats, the greater open-field ambulation behavior of the SHR rats was associated with greater running-wheel activity; SHR rats were indeed more active than W K Y rats in the activity-stress situation. These higher activity scores were, however, contrary to expectations, not associated with more severe ulceration scores. Ulceration was less severe in SHR rats as compared to W K Y rats. It is also interesting to note that differences in open-field ambulation scores, within strains, were not related to corresponding differences in running-wheel activity. The non-significant correlations reported in Table 4 simply indicated that a high-active SHR rat in the open-field was not necessarily a high active rat in the running-wheel cages. Level of open-field ambulation was also unrelated to level of running-wheel activity for W K Y rats. These data may suggest that the exploratory ambulation behavior of the open-
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field, and the running activity in the running wheels, may represent different behaviors and that high open-field ambulation scores is not a predictor of vulnerability to A-S ulcer. Knardahl and Sagvolden [17] have proposed that S H R ' s are not more hyperactive than W K Y rats under resting, baseline conditions, but that S H R rats are simply hyperactive to novel stimulation. Therefore, running-wheel and open-field activity may represent different behaviors and/or reactions in two unique test situations. In the past it was generally assumed that e x c e s s i v e running activity was a necessary element in the production of activity-stress ulcer [31-35], but the data from S H R and W K Y rats in this report imply that genetic factors may be more critical in the etiology of activity-stress ulceration. While running b e h a v i o r may be a contributing cause to ulcer d e v e l o p m e n t , it is not a sufficient cause. T h e r e are previous reports from this laboratory which are congruent with the present activity-stress data. Par6 and Vincent [35] reported a pilot study wherein Sprague-Dawley rats tested in the open-field and subsequently exposed to activity-stress produced some significant correlations between open-field b e h a v i o r and ulceration. Open-field rearing was inversely related to n u m b e r and size of ulcers. These
data would have predicted the reduced ulcer incidence of S H R rats in the present study. S H R rats may also more effectively tolerate chronic stress. Measures of cardiovascular and sympatho-adrenal medullary systems indicate a greater response by S H R ' s to initial stressors, but that repeated long term stress is accompanied by greater adaptive changes in S H R as c o m p a r e d to W K Y rats [19]. If activity-stress can be considered chronic stress, then S H R rats should be more resistant to A-S ulcer. The greater adaptability of S H R ' s to chronic stress, plus their hypoalgesia mentioned earlier [36,37] would help explain the low incidence of A-S ulcer in S H R as compared to W K Y rats. The present report supports the earlier report by Athey and lams [4]; cold-restraint ulcers are more prevalent in W K Y rats. H o w e v e r , the value of this report is that it emphasizes the importance of genetic and constitutional variables in the d e v e l o p m e n t of ulcer disease. S H R and W K Y rats exhibit different responses in various behavioral testing situations and they are also differentially susceptible to stress-ulcer. As such, S H R and W K Y rats represent a valuable tool for investigating how genetic factors, as well as behavioral traits, contribute to ulcer disease.
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