Sympatho-adrenal responses of spontaneously hypertensive rats to immobilization stress RICHARD KVETNANSKY, RICHARD MCCARTY, NGUYEN B. THOA, C. RAYMOND LAKE, AND IRWIN J. KOPIN Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014
B. Sympathoadrenal responses of spontaneously hypertensive rats to immobilization stress. Am. J. Physiol. 236(3): H457-H462, 1979 or Am. J. PhysioZ.: Heart Circ. PhysioZ. 5(3): H457-H462, levels of 1979.-Blood pressure, heart rate, and circulating norepinephrine, epinephrine, and corticosterone were measured before and during the first or seventh period of immobilization stress (150 min per day) in spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive male rats. A catheter was inserted into the tail artery of each rat to permit direct measurement of blood pressure and heart rate and serial sampling of blood in conscious, unhandled animals. During the first immobilization, SHR rats had significantly higher circulating levels of norepinephrine, epinephrine, and corticosterone than did WKY rats. One day after the sixth immobilization, basal levels of norepinephrine and epinephrine were significantly higher and mean blood pressure was significantly lower in repeatedly stressed SHRs compared to unstressed SHRs. In addition, adaptation to the repeated stress in SHRs was attended by reduced adrenomedullary secretion and an increased blood pressure response. These results demonstrate that adaptive changes in the cardiovascular and sympatho-adrenal medullary systems of repeatedly immobilized rats are greater in SHR than in WKY rats. KVETNANSKY, RICHARD, THOA, C. RAYMOND LAKE,
RICHARD MCCARTY, AND IRWIN J. KOPIN.
NGUYEN
velopment of blood pressure elevation in spontaneously hypertensive (SHR) rats (8, 12, 27, 28). SHR animals have greater cardiovascular responses provoked by a variety of alerting stimuli (9, 10). Furthermore, previous studies in our laboratory have shown that SHR rats differ from genetically related normotensive WKY rats in their behavioral responses to foot shock (22)) anticipation of foot shock (23), and handling procedures (24). These behavioral differences are attended by greater elevations of plasma catecholamines (epinephrine from the adrenal medulla and norepinephrine from sympathetic neurons) as well as by higher levels of corticosterone. On the basis of these observations it might be expected that repeated stress would accelerate the hypertension of SHR rats. In the present study, the changes in plasma levels of catecholamines and corticosterone as well as heart rate and blood pressure evoked by forced immobilization are compared in SHR and WKY rats during a first interval of stress and after a week of repeated daily intervals of forced immobilization. MATERIALS
AND METHODS
SHR and WKY male rats (250-325 g) were purchased from Taconic Farms, Germantown, NY and were housed four to five per cage for l-2 wk prior to use in experiments. Rats were provided with food and water ad lib, and a diurnal light cycle was maintained (lights on from IT IS WELL ESTABLISHED that a variety of emotional and 0600- 1800 h) . As a model of severe stress, animals were immobilized physical stressesprovoke release of catecholamines from the sympatho-adrenal medullary system (1, 5, 21). Par- in a prone position up to 2.5 h daily, as previously ticipation of the adrenal cortical hormones in the re- described (19). Briefly, the head of each rat was inserted sponse and adaptation to stress is also an integral part of through two parallel concentric metal loops fixed on a stainless steel plate, and the limbs were secured with the response (23). Forced immobilization of rats has been a useful model adhesive tape to four specially designed metal clips. To obtain blood samples and for direct measurement of stress, which produces activation of the sympathoof blood pressure, cannulas (PE-50) were inserted into adrenal medullary system (15, 17) and the hypothalamopituitary-adrenocortical axis (16,29). With repeated daily the tail arteries (2). The tubing (75 cm in length and immobilization, there is a decrease in the sympatho- filled with 0.9% saline containing 500 U heparin per ml) adrenal response with reductions in the elevations of was passed under the skin to emerge at the back of the plasma levels of norepinephrine (NE), epinephrine (Epi) , neck; a 30-cm length of spring wire was placed over the and corticosterone (17, 26). Normotensive rats immobi- tubing for protection and was secured to the rat with an lized for 2 h daily over a period of 4 wk develop significant adhesive tape collar. The end of the tubing was occluded elevations in arterial blood pressure (20). with a no. 23 needle attached to a l-ml plastic syringe Several studies have suggested that enhanced sympa- filled with the heparinized saline solution. Rats were thetic nerve activity plays an important role in the de- placed in individual cages (25 x 25 x 16 cm), and the Wistar-Kyoto rats; plasma sure; corticosterone
catecholamines;
mean blood pres-
H457
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 18, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
H458 tubing extended from a hole in the top of the cage. To measure mean blood pressure (MBP, mmHg) and heart rate (HR, beats/min) the end of the catheter was attached to a Statham pressure transducer and the pulsations were recorded on a Grass polygraph (Quincy, MA). Blood was removed via the tail arterial cannula. In control experiments removal of up to 3.0 ml (five 0.6-ml samples at 5-min intervals) did not significantly alter plasma catecholamine levels. The blood was collected in iced heparinized tubes and centrifuged at 3,000 g for 15 min, and an aliquot (50 ,ul) of plasma was removed from each sample and frozen at -20°C until assayed for corticosterone as described by Glick et al. (6). A ZOO+1 portion of plasma was deproteinated by the addition of an equal volume of 0.6 N perchloric acid containing 2% EGTA and 0.2% MgC12. ZHZO. Samples were vortexed, centrifuged at 3,000 g for 15 min, and 200 ~1 portions of the clear supernatant were frozen until assayed for content of catecholamines (3, 31). In separate experiments, catheters were inserted into the tails of unstressed SHR and WKY and into rats previously subjected to five daily intervals of forced immobilization. The next day, MBP and HR were measured while rats were at rest in their home cages (OSOO0900 h) and after 5, 20, 60, and 120 min during an initial period of immobilization. On the day after insertion of the arterial cannulas between 0800-0930 h, MBP and HR were measured while the animals were undisturbed in their home cages and at 5, 20, 60, and 120 min after beginning of forced immobilization. Unstressed control rats remained in their home cages without being disturbed. Blood samples (0.6 ml) were obtained via the catheter from rats between 08000900 h while at rest in their home cages and during immobilization (for the first or seventh time) at 5, 20, 60, and 120 min immediately after measurement of MBP and HR. Results are expressed as mean values t SE for the indicated nurnbers of animals. Statistical significance was determined according to Student’s t test (for unpaired or paired data as appropriate).
KVETNANSKY
q
AL.
CONTROL
Ezl 6 x IMMOBILIZED 2 0.2
cn 4 h
-J 0.1 2
SHR
WKY
SHR
WKY
FIG. 1. Effects of repeated daily immobilization on plasma levels of norepinephrine (NE), epinephrine (Epi), and corticosterone in SHR and WKY rats. Blood samples were obtained via an arterial catheter while rats were conscious and undisturbed in the home cage. Results are means t, SE for groups of 6-8 animals. *P < 0.01; **p < 0.001 compared to nonimmobilized SHR or either group of WKY rats.
1. .Mean blood pressures and heart rates
TABLE RESULTS
Unstressed
Response of rats to the first immobilization. Before the first immobilization, while animals were at rest and undisturbed in their home cages, basal plasma levels of NE, Epi, and corticosterone were not significantly different between SHR and WKY rats (Fig. 1). While the mean blood pressure of unstressed SHR rats was significantly higher than that of unstressed WKY rats, basal heart rates did not differ between the two strains (Table 1) During the first immobilization, in both SHR and WKY rats, there were marked elevations (lo- to 20-fold above basal) in circulating levels of plasma NE and Epi after 5 min of the stress. After 120 min of immobilization, in rats of both strains, plasma levels of NE and Epi remained 8-12 times higher than basal values (Figs. 2 and 3). Plasma corticosterone levels increased to twice base line after 5 min and in SHR reached about fivefold basal levels after 60 min, whereas in WKY rats the levels
ET
MBP, mmHg SHR WKY HR, beats/min SHR WKY
Controls
Repeated
Immobilization
165 t, 4 120 Z!I 5
139 t 3” 115 t 4
229 t 6 277 t 7
309 -+ 7 285 t 9
Rats
were forcibly immobilized daily for 5 days as described in and mean blood pressure (MBP) and heart rate (HR) were measured via an arterial catheter in these rats on the 6th day and in unstressed animals while they were awake and undisturbed in their home cages. Results are ‘mean values t, SE for groups of 8-10 animals. * P < 0.001 compared to unstressed controls of the same strain. METHODS,
were only about three-fold basal (Fig. 4). When compared to WKY rats, SHR rats had significantly higher circulating levels of NE after 5 and 20 min (Fig. 2), Epi after 5, 20, 60, and 120 min (Fig. 3), and corticosterone after 20, 60, and 120 min (Fig. 4) of the first immobilization.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 18, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
STRESS
IN
SHR
H459
RATS SHR
az
WKY
4c
O FIRST IMMOBILIZATION SEVENTH
l
IMMOBILIZATION
-
FIG. 2. Plasma levels of norepinephrine (NE) during the first or seventh daily immobilization of SHR and WKY rats. Results are means t SE for groups of 6-8 animals. *P < 0.05; **p < 0.01; ***P < 0.001 compared to rats of the same strain that were immobilized for the first time.
m MINUTES
OF IMMOBILIZATION
WKY
SHR 0 FIRST 2.5
2.5
l
SEVENTH
IMMOBILIZATION IMMOBILIZATION
2 2.0 cn 4 L 4 1.5 2 3 5
FIG. 3. Plasma levels of epinephrine (Epi) during the first or seventh daily immobilization of SHR and WKY rats. Results are means t SE for groups of 6-8 animals. *P < 0.05; **p < 0.01 compared to rats of the same strain that were immobilized for the first time.
1.0
0.5
0
05
m
I
1
60
120 MINUTES
-05
20
60
120
OF IMMOBILIZATION
The net decrease in the mean blood pressure of SHR rats during the first immobilization was not statistically significant (P > 0.05), but in WKY rats there were significant increases (P < 0.05) above home cage levels in mean blood pressures after 5 and 120 min but not at 20 or 60 min of immobilization stress. Although the changes in blood pressure were slight (
B. Sympathoadrenal responses of spontaneously hypertensive rats to immobilization stress. Am. J. Physiol. 236(3): H457-H462, 1979 or Am. J. PhysioZ.: Heart Circ. PhysioZ. 5(3): H457-H462, levels of 1979.-Blood pressure, heart rate, and circulating norepinephrine, epinephrine, and corticosterone were measured before and during the first or seventh period of immobilization stress (150 min per day) in spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive male rats. A catheter was inserted into the tail artery of each rat to permit direct measurement of blood pressure and heart rate and serial sampling of blood in conscious, unhandled animals. During the first immobilization, SHR rats had significantly higher circulating levels of norepinephrine, epinephrine, and corticosterone than did WKY rats. One day after the sixth immobilization, basal levels of norepinephrine and epinephrine were significantly higher and mean blood pressure was significantly lower in repeatedly stressed SHRs compared to unstressed SHRs. In addition, adaptation to the repeated stress in SHRs was attended by reduced adrenomedullary secretion and an increased blood pressure response. These results demonstrate that adaptive changes in the cardiovascular and sympatho-adrenal medullary systems of repeatedly immobilized rats are greater in SHR than in WKY rats. KVETNANSKY, RICHARD, THOA, C. RAYMOND LAKE,
RICHARD MCCARTY, AND IRWIN J. KOPIN.
NGUYEN
velopment of blood pressure elevation in spontaneously hypertensive (SHR) rats (8, 12, 27, 28). SHR animals have greater cardiovascular responses provoked by a variety of alerting stimuli (9, 10). Furthermore, previous studies in our laboratory have shown that SHR rats differ from genetically related normotensive WKY rats in their behavioral responses to foot shock (22)) anticipation of foot shock (23), and handling procedures (24). These behavioral differences are attended by greater elevations of plasma catecholamines (epinephrine from the adrenal medulla and norepinephrine from sympathetic neurons) as well as by higher levels of corticosterone. On the basis of these observations it might be expected that repeated stress would accelerate the hypertension of SHR rats. In the present study, the changes in plasma levels of catecholamines and corticosterone as well as heart rate and blood pressure evoked by forced immobilization are compared in SHR and WKY rats during a first interval of stress and after a week of repeated daily intervals of forced immobilization. MATERIALS
AND METHODS
SHR and WKY male rats (250-325 g) were purchased from Taconic Farms, Germantown, NY and were housed four to five per cage for l-2 wk prior to use in experiments. Rats were provided with food and water ad lib, and a diurnal light cycle was maintained (lights on from IT IS WELL ESTABLISHED that a variety of emotional and 0600- 1800 h) . As a model of severe stress, animals were immobilized physical stressesprovoke release of catecholamines from the sympatho-adrenal medullary system (1, 5, 21). Par- in a prone position up to 2.5 h daily, as previously ticipation of the adrenal cortical hormones in the re- described (19). Briefly, the head of each rat was inserted sponse and adaptation to stress is also an integral part of through two parallel concentric metal loops fixed on a stainless steel plate, and the limbs were secured with the response (23). Forced immobilization of rats has been a useful model adhesive tape to four specially designed metal clips. To obtain blood samples and for direct measurement of stress, which produces activation of the sympathoof blood pressure, cannulas (PE-50) were inserted into adrenal medullary system (15, 17) and the hypothalamopituitary-adrenocortical axis (16,29). With repeated daily the tail arteries (2). The tubing (75 cm in length and immobilization, there is a decrease in the sympatho- filled with 0.9% saline containing 500 U heparin per ml) adrenal response with reductions in the elevations of was passed under the skin to emerge at the back of the plasma levels of norepinephrine (NE), epinephrine (Epi) , neck; a 30-cm length of spring wire was placed over the and corticosterone (17, 26). Normotensive rats immobi- tubing for protection and was secured to the rat with an lized for 2 h daily over a period of 4 wk develop significant adhesive tape collar. The end of the tubing was occluded elevations in arterial blood pressure (20). with a no. 23 needle attached to a l-ml plastic syringe Several studies have suggested that enhanced sympa- filled with the heparinized saline solution. Rats were thetic nerve activity plays an important role in the de- placed in individual cages (25 x 25 x 16 cm), and the Wistar-Kyoto rats; plasma sure; corticosterone
catecholamines;
mean blood pres-
H457
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 18, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
H458 tubing extended from a hole in the top of the cage. To measure mean blood pressure (MBP, mmHg) and heart rate (HR, beats/min) the end of the catheter was attached to a Statham pressure transducer and the pulsations were recorded on a Grass polygraph (Quincy, MA). Blood was removed via the tail arterial cannula. In control experiments removal of up to 3.0 ml (five 0.6-ml samples at 5-min intervals) did not significantly alter plasma catecholamine levels. The blood was collected in iced heparinized tubes and centrifuged at 3,000 g for 15 min, and an aliquot (50 ,ul) of plasma was removed from each sample and frozen at -20°C until assayed for corticosterone as described by Glick et al. (6). A ZOO+1 portion of plasma was deproteinated by the addition of an equal volume of 0.6 N perchloric acid containing 2% EGTA and 0.2% MgC12. ZHZO. Samples were vortexed, centrifuged at 3,000 g for 15 min, and 200 ~1 portions of the clear supernatant were frozen until assayed for content of catecholamines (3, 31). In separate experiments, catheters were inserted into the tails of unstressed SHR and WKY and into rats previously subjected to five daily intervals of forced immobilization. The next day, MBP and HR were measured while rats were at rest in their home cages (OSOO0900 h) and after 5, 20, 60, and 120 min during an initial period of immobilization. On the day after insertion of the arterial cannulas between 0800-0930 h, MBP and HR were measured while the animals were undisturbed in their home cages and at 5, 20, 60, and 120 min after beginning of forced immobilization. Unstressed control rats remained in their home cages without being disturbed. Blood samples (0.6 ml) were obtained via the catheter from rats between 08000900 h while at rest in their home cages and during immobilization (for the first or seventh time) at 5, 20, 60, and 120 min immediately after measurement of MBP and HR. Results are expressed as mean values t SE for the indicated nurnbers of animals. Statistical significance was determined according to Student’s t test (for unpaired or paired data as appropriate).
KVETNANSKY
q
AL.
CONTROL
Ezl 6 x IMMOBILIZED 2 0.2
cn 4 h
-J 0.1 2
SHR
WKY
SHR
WKY
FIG. 1. Effects of repeated daily immobilization on plasma levels of norepinephrine (NE), epinephrine (Epi), and corticosterone in SHR and WKY rats. Blood samples were obtained via an arterial catheter while rats were conscious and undisturbed in the home cage. Results are means t, SE for groups of 6-8 animals. *P < 0.01; **p < 0.001 compared to nonimmobilized SHR or either group of WKY rats.
1. .Mean blood pressures and heart rates
TABLE RESULTS
Unstressed
Response of rats to the first immobilization. Before the first immobilization, while animals were at rest and undisturbed in their home cages, basal plasma levels of NE, Epi, and corticosterone were not significantly different between SHR and WKY rats (Fig. 1). While the mean blood pressure of unstressed SHR rats was significantly higher than that of unstressed WKY rats, basal heart rates did not differ between the two strains (Table 1) During the first immobilization, in both SHR and WKY rats, there were marked elevations (lo- to 20-fold above basal) in circulating levels of plasma NE and Epi after 5 min of the stress. After 120 min of immobilization, in rats of both strains, plasma levels of NE and Epi remained 8-12 times higher than basal values (Figs. 2 and 3). Plasma corticosterone levels increased to twice base line after 5 min and in SHR reached about fivefold basal levels after 60 min, whereas in WKY rats the levels
ET
MBP, mmHg SHR WKY HR, beats/min SHR WKY
Controls
Repeated
Immobilization
165 t, 4 120 Z!I 5
139 t 3” 115 t 4
229 t 6 277 t 7
309 -+ 7 285 t 9
Rats
were forcibly immobilized daily for 5 days as described in and mean blood pressure (MBP) and heart rate (HR) were measured via an arterial catheter in these rats on the 6th day and in unstressed animals while they were awake and undisturbed in their home cages. Results are ‘mean values t, SE for groups of 8-10 animals. * P < 0.001 compared to unstressed controls of the same strain. METHODS,
were only about three-fold basal (Fig. 4). When compared to WKY rats, SHR rats had significantly higher circulating levels of NE after 5 and 20 min (Fig. 2), Epi after 5, 20, 60, and 120 min (Fig. 3), and corticosterone after 20, 60, and 120 min (Fig. 4) of the first immobilization.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 18, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
STRESS
IN
SHR
H459
RATS SHR
az
WKY
4c
O FIRST IMMOBILIZATION SEVENTH
l
IMMOBILIZATION
-
FIG. 2. Plasma levels of norepinephrine (NE) during the first or seventh daily immobilization of SHR and WKY rats. Results are means t SE for groups of 6-8 animals. *P < 0.05; **p < 0.01; ***P < 0.001 compared to rats of the same strain that were immobilized for the first time.
m MINUTES
OF IMMOBILIZATION
WKY
SHR 0 FIRST 2.5
2.5
l
SEVENTH
IMMOBILIZATION IMMOBILIZATION
2 2.0 cn 4 L 4 1.5 2 3 5
FIG. 3. Plasma levels of epinephrine (Epi) during the first or seventh daily immobilization of SHR and WKY rats. Results are means t SE for groups of 6-8 animals. *P < 0.05; **p < 0.01 compared to rats of the same strain that were immobilized for the first time.
1.0
0.5
0
05
m
I
1
60
120 MINUTES
-05
20
60
120
OF IMMOBILIZATION
The net decrease in the mean blood pressure of SHR rats during the first immobilization was not statistically significant (P > 0.05), but in WKY rats there were significant increases (P < 0.05) above home cage levels in mean blood pressures after 5 and 120 min but not at 20 or 60 min of immobilization stress. Although the changes in blood pressure were slight (
