- Email: [email protected]
Jr) rat strains
BEHAVIORAL AND N E U R A L BIOLOGY
57, 144-148 (1992)
Shared Maternal Influences in the Development of High Blood Pressure in the Spontaneously Hypertensive (SHR) and Dahl Salt-Sensitive (SS/Jr) Rat Strains CAROL A. MURPHY, CECILIA FIELDS-OKOTCHA, AND RICHARD MCCARTY 1
Department of Psychology, University of Virginia, Charlottesville, Virginia 22901
sistant (SR/Jr) strain were produced by selectively breeding S p r a g u e - D a w l e y rats for either sensitivity or resistance to the pressor effects of high dietary NaC1, respectively (Dahl, Heine, & Tassinari, 1963). Although the Dahl strains were originally developed specifically to further the study of salt-sensitive hypertension, the outbred animals (designated DS and DR) first studied by Dahl and co-workers have since been inbred such that the S S / J r strain exhibits elevated blood pressures when reared on either a low or high NaC1 diet (Rapp & Dene, 1985). Despite differences in the programs of genetic selection used to produce these two models of hypertension, a number of intriguing similarities have been observed between the SHR and S S / J r strains. For example, SHR blood pressures are also sensitive to the hypertensive effects of excess dietary NaC1 (Louis & Spector, 1975). Moreover, rats of both strains exhibit cardiovascular hyperreactivity to psychogenic stress (McCarty, 1983; Friedman & Iwai, 1976). Previous studies from this laboratory and others have used the technique of reciprocal cross-fostering to demonstrate the importance of early maternal environment in the normal expression of a hypertensive phenotype. SHR rats cross-fostered to either a WKY dam (Cierpial & McCarty, 1987) or a S p r a g u e - D a w l e y dam (DiNicolantonio, Marshall, Nicolaci, & Doyle, 1986) for the duration of the preweaning period exhibited significantly lower mean arterial pressures (MAP) compared to controlreared SHRs. Similarly, S S / J r rats cross-fostered at birth to S R / J r rats had reduced MAPs compared to control-reared S S / J r rats (Murphy & McCarty, 1989). In these same studies, normotensive WKYs fostered to SHR dams and S R / J r pups fostered to S S / J r dams did not exhibit elevated blood pressures in adulthood. These results suggest that the ma-
The influence of maternal environment on the development of high blood pressure in spontaneously hypertensive (SHR) and Dahl salt-sensitive (SS/Jr) rats was examined using the technique of reciprocal cross-fostering. Previous experiments from this laboratory demonstrated that adult blood pressures of the SHR and SS/Jr strains were significantly attenuated when hypertensivestrain pups were fostered to a dam of the respective normotensive control strain during the preweaning period. In this study, SHR and SS/Jr pups were assigned to either a cross-fostered (fostered to a dam of the opposite hypertensive strain) or control-reared condition within 24 h of birth. Adult resting blood pressures were similar in control and cross-fostered SHR rats and in control and crossfostered SS/Jr rats. Heart rates and heart, adrenal, and kidney weights were also similar in control and crossfostered rats of each strain. However, body weights of SHR rats reared by an SS/Jr dam were somewhat lower compared to control-reared SHR rats. These data indicate that the maternal environments provided by SHR and SS/Jr mothers are similar in some way such that they permit the development and full expression of the hypertensive phenotype in both same-strain and oppositestrain pups. ©1992 Academic Press, Inc. The spontaneously hypertensive (SHR) rat and the Dahl salt-sensitive (SS/Jr) rat are animal models of essential hypertension which have been widely used in investigations into the pathogenesis of high blood pressure. The SHR strain was developed through progressive genetic selection of Wistar rats for high systolic blood pressures (Okamoto & Aoki, 1963). Studies of hypertension in the SHR strain typically include the normotensive progenitor W i s t a r - K y o t o (WKY) rat as a control strain. The Dahl S S / J r strain and the normotensive salt-re1 Address correspondence and reprint requests to Carol A. Murphy at present address: 722 W. 168th St., Box 40, New York State Psychiatric Institute, New York, NY 10032. 144 0163-1047/92 $3.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERNALENVIRONMENTAND RAT BLOODPRESSURE ternal environment provided by both SHR and SS/Jr dams is in some way "hypertensinogenic," providing some form of necessary environmental stimulation for the normal expression of a genetic predisposition to high blood pressure. The fact that cross-fostering to a normotensive dam produces adult MAPs which are midway (approx 125-140) between traditional hypertensive and normotensive classifications suggests that maternal influences control the expression of only a portion of the total genetic loci responsible for elevating blood pressure in the SHR and SS/Jr strains. The present study extends these earlier findings by examining whether the maternal environment provided by dams of the SHR strain is sufficient to trigger full expression of the hypertensive phenotype in pups of the SS/Jr strain and, conversely, whether the SS/Jr maternal environment permits the normal development of high blood pressure in SHR pups. If the maternal environments provided by SS/Jr and SHR dams were sufficiently similar such that each permitted the development of hypertension in pups of the opposite hypertensive strain, this would indicate that the MAP-lowering effect of neonatal cross-fostering to a normotensive WKY or SR/Jr dam is mediated by altering an environmental senstivity common to both the SHR and SS/Jr strains. METHODS Breeding pairs of SS/Jr and SR/Jr rats (original stock supplied by Dr. J. Rapp, Medical College of Ohio, Toledo, OH) and SHR and WKY rats (Taconic Farms, Germantown, NY) were mated. Females were placed in individual maternity cages when visibly pregnant and the date of birth was designated as the day on which pups were present by 1700 h. Litters of each strain were culled to eight pups on the day after birth and assigned to either a control or cross-fostered rearing condition. In this manner a total of six groups were included in this study: four control-reared groups (SHR, WKY, SS/Jr, and SR/Jr rats nursed by their natural dam) and two cross-fostered groups (SHR-fostered SS/Jr and SS/Jr-fostered SHR rats). Dam and pups were allowed ad libitum access to normal laboratory chow (Purina, 0.7% NaC1) and water and left undisturbed until the pups had reached the age of weaning. Thirty days is the recommended age for weaning the inbred Dahl strains (Rapp & Dene, 1985), while SHR and WKY pups are normally weaned at 21 days. To provide a uniform duration of preweaning exposure to the dam across all strains, all litters
145
were weaned at 30 days of age. Male pups were subsequently group-housed and maintained on chow containing 0.7% NaC1 into adulthood. To permit the direct measurement of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats/min), a polyethylene (PE50) catheter was inserted into the ventral tail artery using the method described by Chiueh and Kopin (1978). Surgeries were performed when subjects had reached 100 days of age. Rats were first anesthetized for surgery with the inhalation anesthetic, methoxyflurane (Metofane, Pittman-Moore, NY). Body weights were also measured at this time to the nearest 0.1 g. Following surgical insertion of the catheter into the tail artery, the exposed tubing was threaded dorsally under the tail sheath and skin to exit at the nape of the neck, where it was then covered with protective spring wire. The end of the tubing was led out of the topcenter of the cage and occluded with a tuberculin syringe, in such a way as to allow the animals to move freely about the cage. In order to maintain patency of the catheter line, heparinized saline (300 U, in a volume of 0.5 ml) was infused twice daily. Resting MAP and HR were measured 1 day following surgery. The end of each catheter, which extended outside the recovery cage, was connected to a Statham pressure transducer through which a pulse pressure signal was transmitted to a Grass Model 7 polygraph. Heart rates were obtained using a Grass Model 7p44b tachograph derived from the pulsations in the arterial pressure signal. Subjects remained undisturbed in their recovery cages while MAP and HR were being measured. When the animals were judged to be in an awake and quietly resting state, MAP and HR were recorded continuously over a period of at least 2 min for each subject during the light portion of the light-dark cycle (1000-1400 hr) and a median value for both signals was obtained through visual screening. Animals were subsequently sacrificed with an overdose of sodium pentobarbitol. The heart, kidneys, and adrenal glands of SS/Jr and SHR rats were removed, blotted dry, and weighed to the nearest 0.1 mg. Organ weight data were not obtained from WKY or SR/Jr rats due to the fact that these groups were being used in a separate experiment. Statistical analyses were performed using an SPSS-PC software package. The main effects of strain and rearing condition and strain x rearing condition interactions for the control and cross-fostered SHR and SS/Jr subject groups were determined using two-way analyses of variance for each variable measured. Additional analyses of variance were performed across six subject groups, including
146
MURPHY, FIELDS-OKOTCHA, AND MCCARTY
500.
600
CONTROL CROSS-FOSTERED TO HYPERTENSIVE STRAIN
500
._v.
400
0 gO
300
1 11
200 100.
SS/Jr
SR/Jr
SHR
the control WKY and SR/Jr groups. Pairwise comparisons were conducted with a two-tailed Student's t test. RESULTS
Body weight measurements at 100 days of age are shown in Fig. 1. A main effect of strain on body weight (F(3, 51) = 42.04, p < .001) was observed, reflecting the greater body weights of SS/Jr and WKY rats compared to SHR and SR/Jr rats. A strain × fostering condition interaction was detected with respect to the SHR and SS/Jr subject groups (F(1, 33) = 5.86, p < .05) whereby SHR rats reared by an SS/Jr dam had significantly lower body
I---I CONTROL Ir2pl CROSS-FOSTERED TO HYPERTENSIVE STRAIN
200
E w
16o -T.
,,m,
120
13-
~ im
'Y
SO 40
Z
~
v.a I.zJ
300-
~
20o-
0
1
SS/Jr
SR/Jr
SHR
-r
100
WKY
FIG. 1. Body weights (g) of Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) male rats of spontaneously hypertensive (SHR) and W i s t a r - K y o t o (WKY) normotensive male rats at 100 days of age. Rats of t h e hypertensive strains were reared from b i r t h to weaning by t h e i r n a t u r a l mothers or a foster mother of the opposite strain. Values are means for 7 - 1 0 rats per group and vertical bars denote 1 SEM. * p < .05 compared to SHR controls.
'•
400.
E
$
-rLU
.E
r-'-i CONTROL v'~ CROSS-FOSTERED TO HYPERTENSIVE STRAIN
WKY
FIG. 2. Mean arterial pressures (mmHg) of Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) male rats and of spontaneously hypertensive (SHR) and W i s t a r - K y o t o (WKY) normotensive male rats a t 100 days of age. Rats of the hypertensive strains were reared from b i r t h to weaning by t h e i r n a t u r a l mothers or a foster mother of the opposite strain. Values are means for 7 - 1 0 rats per group and vertical bars denote 1 SEM.
ss/.Jr
SR/Jr
111 SHR
WKY
FIG. 3. Baseline h e a r t rates (beats per minute) of Dahl saltsensitive (SS/Jr) and salt-resistant (SR/Jr) male rats and of spontaneously hypertensive (SHR) and W i s t a r - K y o t o (WKY) normotensive male rats a t 100 days of age. Rats of the hypertensive strains were reared from b i r t h to weaning by their natural mothers or a foster mother of the opposite strain. Values are m e a n s for 7 - 1 0 rats per group and vertical bars denote 1 SEM.
weights compared to control-reared SHRs (t(1, 18) = 2.75, p < .05). However, body weights of SS/Jr rats were not altered by cross-fostering to the SHR strain. As expected, there was also a main effect of strain on direct mean arterial pressure (F(3, 49) = 26.65, p < .001, see Fig. 2). MAPs of SHR rats were significantly higher than those of WKY rats (p < .001) and somewhat, although not significantly greater compared to SS/Jr rats (F(1, 31) = 3.73, p < .1). Blood pressures of SS/Jr rats were in turn elevated with respect to those of SR/Jr rats (p < .001). However, fostering environment did not alter MAP in rats of either the SHR or SR/Jr strain. Resting HRs in each of the six groups are depicted in Fig. 3. Despite the finding that HRs of SS/Jr and SR/Jr rats were higher than those of SHR and WKY rats (main effect of strain, (F(1, 33) = 7.67, p < .01), cross-fostering of hypertensive pups of either strain to a dam of the opposite hypertensive strain did not alter baseline heart rates in adulthood. No effect of cross-fostering was observed on heart, kidney, or adrenal weights in either SS/Jr or SHR rats (Table 1). However, mean values Of heart weight/100 g body weight were greater in SHR than in SS/Jr rats (F(1, 33) = 18.3, p < .001). Strain comparisons of kidney weight/100 g body weight revealed that SS/Jr rats had significantly heavier kidneys compared to SHR rats (F(1, 33) = 6.83, p <
.O2). DISCUSSION
The data presented here indicate that the maternal environments provided by SHR and SS/Jr moth-
147
MATERNAL ENVIRONMENT AND RAT BLOOD PRESSURE
TABLE 1 Mean Heart, Kidney, and Adrenal Weights (g) per 100 g Body Weight of SS/Jr and SR/Jr Rats Strain
Rearing condition
Heart
Kidney
Adrenal
SS/Jr
Control Cross-fostered to SHR dam
0.357 -+ 0.013 0.304 +- 0.005***
0.828 _+ 0.036 0.807 +- 0.035*
0.015 -+ 0.001 0.015 +- 0.001
SHR
Control Cross-fostered to S S / J r dam
0.441 -+ 0.048 0.416 _+ 0.008
0.708 - 0.021 0.749 +_ 0.027
0.013 +- 0.001 0.014 _+ 0.004
* p < .05 ~main effect of strain. *** p < .00 J
ers reflect some shared hypertensinogenic component, the nature of which has yet to be determined. Marked reductions in resting blood pressure have previously been observed in both WKY-fostered SHR rats (Cierpial & McCarty, 1987) and SR/Jr fostered SS/Jr rats (Murphy & McCarty, 1989), indicating that preweanling exposure to a normotensive dam has beneficial effects on the development of cardiovascular pathology in hypertensive-strain pups. Each of these earlier studies also included an in-fostered control group (hypertensive pup fostered to same-strain hypertensive foster dam) in which blood pressures were no different from those of control-reared hypertensive rats. In the present study, SHRs and SS/Jrs reciprocally cross-fostered to a dam of the opposite hypertensive strain also exhibited elevated blood pressures in adulthood similar to those of control-reared SHRs and SS/Jrs. Taken together, these findings indicate that not only is there some factor unique to the maternal environment provided by hypertensive dams which promotes full expression of the high blood pressure phenotype in genetically predisposed animals, but that the maternal environment provided by either an SHR or SS/Jr dam is sufficient to sustain the normal development of hypertension in pups of either hypertensive strain. A number of strain differences were observed between SHR and SS/Jr rats regardless of rearing condition. Measures of heart weight and mean arterial pressure were somewhat higher in SHR rats, although the strain difference in blood pressure approached but did not attain significance. In addition, values of kidney weight/100 g body weight were greater in SS/Jr than in SHR rats. This suggests that there are significant differences in the underlying pathology sustaining hypertension in the two strains, regardless of any shared developmental susceptibility to factors of the maternal environment. The extent of cardiac hypertrophy in 100-day-old SHR and SS/Jr rats was also similar in control and cross-fostered subjects. Data not presented here in-
dicate that SS/Jr pups fostered to an SR/Jr dam do not exhibit decreased heart weights compared to controls; thus, there was little reason to expect that exposure to an SHR maternal environment would affect cardiac hypertrophy in SS/Jr rats. In contrast, there is some evidence that the development of cardiac hypertrophy in preweanling SHR rats reared by WKY foster mothers is somewhat reduced compared to control-reared SHR rats (Cierpial & McCarty, unpublished findings). However, fostering of SHR pups to an SS/Jr dam was not attended by reduced heart weights in this study. Thus, while the hypertensive SS/Jr maternal environment is not essential for the expression of cardiac hypertrophy in SS/Jr pups, it does not inhibit the exaggerated myocardial development characteristic of SHR rats. These findings may reflect a peculiarity of the WKY dam's rearing influences in reducing cardiac hypertrophy, rather than an effect of a hypertensive maternal environment per se in promoting excess cardiac development. In contrast, cross-fostering to an SS/Jr dam did have a small but significant effect in lowering the body weights of SHR rats. In a previous study, body weights of SHR rats fostered to WKY dams were markedly increased compared to control-reared SHRs (Cierpial & McCarty, 1987). In contrast, SS/Jr rats exhibited no differences in body weight after cross-fostering to either SR/Jr (Murphy & McCarty, 1989) or SHR dams in the present study, indicating that strain differences in body weight between the SS/Jr and SR/Jr strains are not regulated by maternal factors. Hypertensive SS/Jr rats, like WKY rats, weigh significantly more than SHRs and yet SHR body weights were somewhat lower following exposure to the SS/Jr maternal environment. Despite the lack of an effect of the SS/Jr fostering environment on MAP or cardiac hypertrophy in SHRs, some aspect of a dysfunctional mother-pup interaction may be reflected in the decreased body weights of SHRs reared by SS/Jr foster dams.
148
MURPHY, FIELDS-OKOTCHA,AND MCCARTY
There is convincing evidence to suggest t h a t events which occur within the context of the "maternal" environment can be attributed to properties of both mother and pup. While strain differences in maternal behavior between SHR and WKY dams have been consistently observed (Cierpial, Shasby, & McCarty, 1987; Myers, Brunelli, Squire, Shindeldecker, & Hofer, 1989a), these differences reportedly disappear when dams of the two strains are presented with genetically identical (F1 cross-SHR × WKY) litters (Myers, Brunelli, Shair, Squire, & Hofer, 1989b). These studies have shown t h a t when SHR mothers rear their own litters, they are observed more frequently in an active nursing posture and in contact with their pups compared to WKY mothers nursing-WKY pups. This finding is suggestive of a direct effect of increased nursing on blood pressure, particularly given t h a t the cardiovascular response of SHR pups to milk letdown, regardless of the strain of the dam or even if milk is delivered via a tongue cannula, is significantly greater t h a n t h a t of WKY rats (Myers & Scalzo, 1988). Differing concentrations of milk electrolytes between hypertensive and normotensive rats (Azar & Hrushesky, 1985) have also been proposed to play a role in altering the development of hypertension. F u r t h e r study will be needed to elucidate which component(s) of the postnatal m o t h e r - p u p interaction constitute a mechanism by which cross-fostering of hypertensive pups to a normotensive dam reduces adult blood pressures. In conclusion, the results of this study suggest t h a t there is some quality of the hypertensive maternal environment required for the full development of hypertension in S S / J r and SHR pups which is shared between dams of the two strains. It can be inferred t h a t the same putative maternal factor(s) control the expression of the same set of environmentally susceptible genetic loci in rats of both strains. Thus, comprehensive study of physiological similarities between SHR and S S / J r rats should be instrumental in clarifying functional components of the total hypertensive phenotype which are sensitive to therapeutic manipulations occurring early in development, as well as pointing to aspects of the hypertensive phenotype which are expressed independently of early enviromental influences.
REFERENCES Azar, S., & Hrushesky,W. (1985). Environmentalfactor(s)during suckling exerts effects upon blood pressure in genetic hypertension. Clinical Research, 33, 882A. Chiueh, C. C., & Kopin, I. J. (1978). Hyperresponsivityof spontaneouslyhypertensiverats to indirect measurementofblood pressure. American Journal of Physiology, 234, H690-H695. Cierpial, M. A., & McCarty,R. (1987). Hypertensionin SHR rats: Contribution of maternal environment.American Journal of Physiology, 253, H980-H984. Cierpial, M. A., Shasby, D. E., & McCarty, R. (1987). Patterns of maternal behavior in the spontaneouslyhypertensiverat. Physiology and Behavior, 39, 633-637. Dahl, L. K., Heine, M., & Tassinari, L. (1963). Effectsof chronic salt ingestion: Evidence that genetic factors play an important role in susceptibility to experimental hypertension. Journal of Experimental Medicine, 115, 1173-1190. DiNicolantonio,R., Marshall, S. J., Nicolaci,J. A., & Doyle,A. E. (1986). Bloodpressure and saline preference of cross-suckled geneticallyhypertensiveand normotensiverats: Role ofmilk electrolytes. Journal of Hypertension, 4, $253-$254. Friedman, R., & Iwai, J. (1976). Genetic predisposition and stress-induced hypertension. Science, 193, 161-162. Louis, W. J., & Spector, S. (1975). The effect of salt intake and adrenal steroids on blood pressure in a genetic strain of hypertensive rats. Clinical and Experimental Pharmacological Physiology, (Suppl.) 2, 131-163. McCarty, R. (1983). Stres, behavior and experimentalhypertension. Neuroscience and Biobehavioral Reviews, 7, 493-502. Murphy, C. A., & McCarty,R. (1989). Maternal environmentand development of high blood pressure in Dahl hypertensive rats. American Journal of Physiology, 257, H1396-H1401. Myers, M. M., Brunelli, S. A., Squire,J. M., Shindeldecker,R. D., & Hofer, M. A. (1989a). Maternal behavior of SHR rats and its relationship to offspringblood pressures. Developmental Psychobiology, 22, 29-53. Myers, M. M., Brunelli, S. A., Shair, H. N., Squire, J. M., & Hofer, M. A. (1989b). Relationships between maternal behavior of SHR and WKY dams and adult blood pressures of cross-fosteredF1 pups. Developmental Psychobiology, 22, 5567. Myers, M. M., & Scalzo, F. M. (1988). Bloodpressure and heart rate responses of SHR and WKY rat pups during feeding. Physiology and Behavior, 44, 75-83. Okamoto, K., & Aoki, K. (1963). Development of a strain of spontaneouslyhypertensiverats. Japanese Circulation Journal, 27, 282-293. Rapp, J. R., & Dene, H. (1985). Developmentand characteristics of inbred strains of Dahl salt-sensitive and salt-resistant rats. Hypertension, 7, 340-349.
57, 144-148 (1992)
Shared Maternal Influences in the Development of High Blood Pressure in the Spontaneously Hypertensive (SHR) and Dahl Salt-Sensitive (SS/Jr) Rat Strains CAROL A. MURPHY, CECILIA FIELDS-OKOTCHA, AND RICHARD MCCARTY 1
Department of Psychology, University of Virginia, Charlottesville, Virginia 22901
sistant (SR/Jr) strain were produced by selectively breeding S p r a g u e - D a w l e y rats for either sensitivity or resistance to the pressor effects of high dietary NaC1, respectively (Dahl, Heine, & Tassinari, 1963). Although the Dahl strains were originally developed specifically to further the study of salt-sensitive hypertension, the outbred animals (designated DS and DR) first studied by Dahl and co-workers have since been inbred such that the S S / J r strain exhibits elevated blood pressures when reared on either a low or high NaC1 diet (Rapp & Dene, 1985). Despite differences in the programs of genetic selection used to produce these two models of hypertension, a number of intriguing similarities have been observed between the SHR and S S / J r strains. For example, SHR blood pressures are also sensitive to the hypertensive effects of excess dietary NaC1 (Louis & Spector, 1975). Moreover, rats of both strains exhibit cardiovascular hyperreactivity to psychogenic stress (McCarty, 1983; Friedman & Iwai, 1976). Previous studies from this laboratory and others have used the technique of reciprocal cross-fostering to demonstrate the importance of early maternal environment in the normal expression of a hypertensive phenotype. SHR rats cross-fostered to either a WKY dam (Cierpial & McCarty, 1987) or a S p r a g u e - D a w l e y dam (DiNicolantonio, Marshall, Nicolaci, & Doyle, 1986) for the duration of the preweaning period exhibited significantly lower mean arterial pressures (MAP) compared to controlreared SHRs. Similarly, S S / J r rats cross-fostered at birth to S R / J r rats had reduced MAPs compared to control-reared S S / J r rats (Murphy & McCarty, 1989). In these same studies, normotensive WKYs fostered to SHR dams and S R / J r pups fostered to S S / J r dams did not exhibit elevated blood pressures in adulthood. These results suggest that the ma-
The influence of maternal environment on the development of high blood pressure in spontaneously hypertensive (SHR) and Dahl salt-sensitive (SS/Jr) rats was examined using the technique of reciprocal cross-fostering. Previous experiments from this laboratory demonstrated that adult blood pressures of the SHR and SS/Jr strains were significantly attenuated when hypertensivestrain pups were fostered to a dam of the respective normotensive control strain during the preweaning period. In this study, SHR and SS/Jr pups were assigned to either a cross-fostered (fostered to a dam of the opposite hypertensive strain) or control-reared condition within 24 h of birth. Adult resting blood pressures were similar in control and cross-fostered SHR rats and in control and crossfostered SS/Jr rats. Heart rates and heart, adrenal, and kidney weights were also similar in control and crossfostered rats of each strain. However, body weights of SHR rats reared by an SS/Jr dam were somewhat lower compared to control-reared SHR rats. These data indicate that the maternal environments provided by SHR and SS/Jr mothers are similar in some way such that they permit the development and full expression of the hypertensive phenotype in both same-strain and oppositestrain pups. ©1992 Academic Press, Inc. The spontaneously hypertensive (SHR) rat and the Dahl salt-sensitive (SS/Jr) rat are animal models of essential hypertension which have been widely used in investigations into the pathogenesis of high blood pressure. The SHR strain was developed through progressive genetic selection of Wistar rats for high systolic blood pressures (Okamoto & Aoki, 1963). Studies of hypertension in the SHR strain typically include the normotensive progenitor W i s t a r - K y o t o (WKY) rat as a control strain. The Dahl S S / J r strain and the normotensive salt-re1 Address correspondence and reprint requests to Carol A. Murphy at present address: 722 W. 168th St., Box 40, New York State Psychiatric Institute, New York, NY 10032. 144 0163-1047/92 $3.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERNALENVIRONMENTAND RAT BLOODPRESSURE ternal environment provided by both SHR and SS/Jr dams is in some way "hypertensinogenic," providing some form of necessary environmental stimulation for the normal expression of a genetic predisposition to high blood pressure. The fact that cross-fostering to a normotensive dam produces adult MAPs which are midway (approx 125-140) between traditional hypertensive and normotensive classifications suggests that maternal influences control the expression of only a portion of the total genetic loci responsible for elevating blood pressure in the SHR and SS/Jr strains. The present study extends these earlier findings by examining whether the maternal environment provided by dams of the SHR strain is sufficient to trigger full expression of the hypertensive phenotype in pups of the SS/Jr strain and, conversely, whether the SS/Jr maternal environment permits the normal development of high blood pressure in SHR pups. If the maternal environments provided by SS/Jr and SHR dams were sufficiently similar such that each permitted the development of hypertension in pups of the opposite hypertensive strain, this would indicate that the MAP-lowering effect of neonatal cross-fostering to a normotensive WKY or SR/Jr dam is mediated by altering an environmental senstivity common to both the SHR and SS/Jr strains. METHODS Breeding pairs of SS/Jr and SR/Jr rats (original stock supplied by Dr. J. Rapp, Medical College of Ohio, Toledo, OH) and SHR and WKY rats (Taconic Farms, Germantown, NY) were mated. Females were placed in individual maternity cages when visibly pregnant and the date of birth was designated as the day on which pups were present by 1700 h. Litters of each strain were culled to eight pups on the day after birth and assigned to either a control or cross-fostered rearing condition. In this manner a total of six groups were included in this study: four control-reared groups (SHR, WKY, SS/Jr, and SR/Jr rats nursed by their natural dam) and two cross-fostered groups (SHR-fostered SS/Jr and SS/Jr-fostered SHR rats). Dam and pups were allowed ad libitum access to normal laboratory chow (Purina, 0.7% NaC1) and water and left undisturbed until the pups had reached the age of weaning. Thirty days is the recommended age for weaning the inbred Dahl strains (Rapp & Dene, 1985), while SHR and WKY pups are normally weaned at 21 days. To provide a uniform duration of preweaning exposure to the dam across all strains, all litters
145
were weaned at 30 days of age. Male pups were subsequently group-housed and maintained on chow containing 0.7% NaC1 into adulthood. To permit the direct measurement of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats/min), a polyethylene (PE50) catheter was inserted into the ventral tail artery using the method described by Chiueh and Kopin (1978). Surgeries were performed when subjects had reached 100 days of age. Rats were first anesthetized for surgery with the inhalation anesthetic, methoxyflurane (Metofane, Pittman-Moore, NY). Body weights were also measured at this time to the nearest 0.1 g. Following surgical insertion of the catheter into the tail artery, the exposed tubing was threaded dorsally under the tail sheath and skin to exit at the nape of the neck, where it was then covered with protective spring wire. The end of the tubing was led out of the topcenter of the cage and occluded with a tuberculin syringe, in such a way as to allow the animals to move freely about the cage. In order to maintain patency of the catheter line, heparinized saline (300 U, in a volume of 0.5 ml) was infused twice daily. Resting MAP and HR were measured 1 day following surgery. The end of each catheter, which extended outside the recovery cage, was connected to a Statham pressure transducer through which a pulse pressure signal was transmitted to a Grass Model 7 polygraph. Heart rates were obtained using a Grass Model 7p44b tachograph derived from the pulsations in the arterial pressure signal. Subjects remained undisturbed in their recovery cages while MAP and HR were being measured. When the animals were judged to be in an awake and quietly resting state, MAP and HR were recorded continuously over a period of at least 2 min for each subject during the light portion of the light-dark cycle (1000-1400 hr) and a median value for both signals was obtained through visual screening. Animals were subsequently sacrificed with an overdose of sodium pentobarbitol. The heart, kidneys, and adrenal glands of SS/Jr and SHR rats were removed, blotted dry, and weighed to the nearest 0.1 mg. Organ weight data were not obtained from WKY or SR/Jr rats due to the fact that these groups were being used in a separate experiment. Statistical analyses were performed using an SPSS-PC software package. The main effects of strain and rearing condition and strain x rearing condition interactions for the control and cross-fostered SHR and SS/Jr subject groups were determined using two-way analyses of variance for each variable measured. Additional analyses of variance were performed across six subject groups, including
146
MURPHY, FIELDS-OKOTCHA, AND MCCARTY
500.
600
CONTROL CROSS-FOSTERED TO HYPERTENSIVE STRAIN
500
._v.
400
0 gO
300
1 11
200 100.
SS/Jr
SR/Jr
SHR
the control WKY and SR/Jr groups. Pairwise comparisons were conducted with a two-tailed Student's t test. RESULTS
Body weight measurements at 100 days of age are shown in Fig. 1. A main effect of strain on body weight (F(3, 51) = 42.04, p < .001) was observed, reflecting the greater body weights of SS/Jr and WKY rats compared to SHR and SR/Jr rats. A strain × fostering condition interaction was detected with respect to the SHR and SS/Jr subject groups (F(1, 33) = 5.86, p < .05) whereby SHR rats reared by an SS/Jr dam had significantly lower body
I---I CONTROL Ir2pl CROSS-FOSTERED TO HYPERTENSIVE STRAIN
200
E w
16o -T.
,,m,
120
13-
~ im
'Y
SO 40
Z
~
v.a I.zJ
300-
~
20o-
0
1
SS/Jr
SR/Jr
SHR
-r
100
WKY
FIG. 1. Body weights (g) of Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) male rats of spontaneously hypertensive (SHR) and W i s t a r - K y o t o (WKY) normotensive male rats at 100 days of age. Rats of t h e hypertensive strains were reared from b i r t h to weaning by t h e i r n a t u r a l mothers or a foster mother of the opposite strain. Values are means for 7 - 1 0 rats per group and vertical bars denote 1 SEM. * p < .05 compared to SHR controls.
'•
400.
E
$
-rLU
.E
r-'-i CONTROL v'~ CROSS-FOSTERED TO HYPERTENSIVE STRAIN
WKY
FIG. 2. Mean arterial pressures (mmHg) of Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) male rats and of spontaneously hypertensive (SHR) and W i s t a r - K y o t o (WKY) normotensive male rats a t 100 days of age. Rats of the hypertensive strains were reared from b i r t h to weaning by t h e i r n a t u r a l mothers or a foster mother of the opposite strain. Values are means for 7 - 1 0 rats per group and vertical bars denote 1 SEM.
ss/.Jr
SR/Jr
111 SHR
WKY
FIG. 3. Baseline h e a r t rates (beats per minute) of Dahl saltsensitive (SS/Jr) and salt-resistant (SR/Jr) male rats and of spontaneously hypertensive (SHR) and W i s t a r - K y o t o (WKY) normotensive male rats a t 100 days of age. Rats of the hypertensive strains were reared from b i r t h to weaning by their natural mothers or a foster mother of the opposite strain. Values are m e a n s for 7 - 1 0 rats per group and vertical bars denote 1 SEM.
weights compared to control-reared SHRs (t(1, 18) = 2.75, p < .05). However, body weights of SS/Jr rats were not altered by cross-fostering to the SHR strain. As expected, there was also a main effect of strain on direct mean arterial pressure (F(3, 49) = 26.65, p < .001, see Fig. 2). MAPs of SHR rats were significantly higher than those of WKY rats (p < .001) and somewhat, although not significantly greater compared to SS/Jr rats (F(1, 31) = 3.73, p < .1). Blood pressures of SS/Jr rats were in turn elevated with respect to those of SR/Jr rats (p < .001). However, fostering environment did not alter MAP in rats of either the SHR or SR/Jr strain. Resting HRs in each of the six groups are depicted in Fig. 3. Despite the finding that HRs of SS/Jr and SR/Jr rats were higher than those of SHR and WKY rats (main effect of strain, (F(1, 33) = 7.67, p < .01), cross-fostering of hypertensive pups of either strain to a dam of the opposite hypertensive strain did not alter baseline heart rates in adulthood. No effect of cross-fostering was observed on heart, kidney, or adrenal weights in either SS/Jr or SHR rats (Table 1). However, mean values Of heart weight/100 g body weight were greater in SHR than in SS/Jr rats (F(1, 33) = 18.3, p < .001). Strain comparisons of kidney weight/100 g body weight revealed that SS/Jr rats had significantly heavier kidneys compared to SHR rats (F(1, 33) = 6.83, p <
.O2). DISCUSSION
The data presented here indicate that the maternal environments provided by SHR and SS/Jr moth-
147
MATERNAL ENVIRONMENT AND RAT BLOOD PRESSURE
TABLE 1 Mean Heart, Kidney, and Adrenal Weights (g) per 100 g Body Weight of SS/Jr and SR/Jr Rats Strain
Rearing condition
Heart
Kidney
Adrenal
SS/Jr
Control Cross-fostered to SHR dam
0.357 -+ 0.013 0.304 +- 0.005***
0.828 _+ 0.036 0.807 +- 0.035*
0.015 -+ 0.001 0.015 +- 0.001
SHR
Control Cross-fostered to S S / J r dam
0.441 -+ 0.048 0.416 _+ 0.008
0.708 - 0.021 0.749 +_ 0.027
0.013 +- 0.001 0.014 _+ 0.004
* p < .05 ~main effect of strain. *** p < .00 J
ers reflect some shared hypertensinogenic component, the nature of which has yet to be determined. Marked reductions in resting blood pressure have previously been observed in both WKY-fostered SHR rats (Cierpial & McCarty, 1987) and SR/Jr fostered SS/Jr rats (Murphy & McCarty, 1989), indicating that preweanling exposure to a normotensive dam has beneficial effects on the development of cardiovascular pathology in hypertensive-strain pups. Each of these earlier studies also included an in-fostered control group (hypertensive pup fostered to same-strain hypertensive foster dam) in which blood pressures were no different from those of control-reared hypertensive rats. In the present study, SHRs and SS/Jrs reciprocally cross-fostered to a dam of the opposite hypertensive strain also exhibited elevated blood pressures in adulthood similar to those of control-reared SHRs and SS/Jrs. Taken together, these findings indicate that not only is there some factor unique to the maternal environment provided by hypertensive dams which promotes full expression of the high blood pressure phenotype in genetically predisposed animals, but that the maternal environment provided by either an SHR or SS/Jr dam is sufficient to sustain the normal development of hypertension in pups of either hypertensive strain. A number of strain differences were observed between SHR and SS/Jr rats regardless of rearing condition. Measures of heart weight and mean arterial pressure were somewhat higher in SHR rats, although the strain difference in blood pressure approached but did not attain significance. In addition, values of kidney weight/100 g body weight were greater in SS/Jr than in SHR rats. This suggests that there are significant differences in the underlying pathology sustaining hypertension in the two strains, regardless of any shared developmental susceptibility to factors of the maternal environment. The extent of cardiac hypertrophy in 100-day-old SHR and SS/Jr rats was also similar in control and cross-fostered subjects. Data not presented here in-
dicate that SS/Jr pups fostered to an SR/Jr dam do not exhibit decreased heart weights compared to controls; thus, there was little reason to expect that exposure to an SHR maternal environment would affect cardiac hypertrophy in SS/Jr rats. In contrast, there is some evidence that the development of cardiac hypertrophy in preweanling SHR rats reared by WKY foster mothers is somewhat reduced compared to control-reared SHR rats (Cierpial & McCarty, unpublished findings). However, fostering of SHR pups to an SS/Jr dam was not attended by reduced heart weights in this study. Thus, while the hypertensive SS/Jr maternal environment is not essential for the expression of cardiac hypertrophy in SS/Jr pups, it does not inhibit the exaggerated myocardial development characteristic of SHR rats. These findings may reflect a peculiarity of the WKY dam's rearing influences in reducing cardiac hypertrophy, rather than an effect of a hypertensive maternal environment per se in promoting excess cardiac development. In contrast, cross-fostering to an SS/Jr dam did have a small but significant effect in lowering the body weights of SHR rats. In a previous study, body weights of SHR rats fostered to WKY dams were markedly increased compared to control-reared SHRs (Cierpial & McCarty, 1987). In contrast, SS/Jr rats exhibited no differences in body weight after cross-fostering to either SR/Jr (Murphy & McCarty, 1989) or SHR dams in the present study, indicating that strain differences in body weight between the SS/Jr and SR/Jr strains are not regulated by maternal factors. Hypertensive SS/Jr rats, like WKY rats, weigh significantly more than SHRs and yet SHR body weights were somewhat lower following exposure to the SS/Jr maternal environment. Despite the lack of an effect of the SS/Jr fostering environment on MAP or cardiac hypertrophy in SHRs, some aspect of a dysfunctional mother-pup interaction may be reflected in the decreased body weights of SHRs reared by SS/Jr foster dams.
148
MURPHY, FIELDS-OKOTCHA,AND MCCARTY
There is convincing evidence to suggest t h a t events which occur within the context of the "maternal" environment can be attributed to properties of both mother and pup. While strain differences in maternal behavior between SHR and WKY dams have been consistently observed (Cierpial, Shasby, & McCarty, 1987; Myers, Brunelli, Squire, Shindeldecker, & Hofer, 1989a), these differences reportedly disappear when dams of the two strains are presented with genetically identical (F1 cross-SHR × WKY) litters (Myers, Brunelli, Shair, Squire, & Hofer, 1989b). These studies have shown t h a t when SHR mothers rear their own litters, they are observed more frequently in an active nursing posture and in contact with their pups compared to WKY mothers nursing-WKY pups. This finding is suggestive of a direct effect of increased nursing on blood pressure, particularly given t h a t the cardiovascular response of SHR pups to milk letdown, regardless of the strain of the dam or even if milk is delivered via a tongue cannula, is significantly greater t h a n t h a t of WKY rats (Myers & Scalzo, 1988). Differing concentrations of milk electrolytes between hypertensive and normotensive rats (Azar & Hrushesky, 1985) have also been proposed to play a role in altering the development of hypertension. F u r t h e r study will be needed to elucidate which component(s) of the postnatal m o t h e r - p u p interaction constitute a mechanism by which cross-fostering of hypertensive pups to a normotensive dam reduces adult blood pressures. In conclusion, the results of this study suggest t h a t there is some quality of the hypertensive maternal environment required for the full development of hypertension in S S / J r and SHR pups which is shared between dams of the two strains. It can be inferred t h a t the same putative maternal factor(s) control the expression of the same set of environmentally susceptible genetic loci in rats of both strains. Thus, comprehensive study of physiological similarities between SHR and S S / J r rats should be instrumental in clarifying functional components of the total hypertensive phenotype which are sensitive to therapeutic manipulations occurring early in development, as well as pointing to aspects of the hypertensive phenotype which are expressed independently of early enviromental influences.
REFERENCES Azar, S., & Hrushesky,W. (1985). Environmentalfactor(s)during suckling exerts effects upon blood pressure in genetic hypertension. Clinical Research, 33, 882A. Chiueh, C. C., & Kopin, I. J. (1978). Hyperresponsivityof spontaneouslyhypertensiverats to indirect measurementofblood pressure. American Journal of Physiology, 234, H690-H695. Cierpial, M. A., & McCarty,R. (1987). Hypertensionin SHR rats: Contribution of maternal environment.American Journal of Physiology, 253, H980-H984. Cierpial, M. A., Shasby, D. E., & McCarty, R. (1987). Patterns of maternal behavior in the spontaneouslyhypertensiverat. Physiology and Behavior, 39, 633-637. Dahl, L. K., Heine, M., & Tassinari, L. (1963). Effectsof chronic salt ingestion: Evidence that genetic factors play an important role in susceptibility to experimental hypertension. Journal of Experimental Medicine, 115, 1173-1190. DiNicolantonio,R., Marshall, S. J., Nicolaci,J. A., & Doyle,A. E. (1986). Bloodpressure and saline preference of cross-suckled geneticallyhypertensiveand normotensiverats: Role ofmilk electrolytes. Journal of Hypertension, 4, $253-$254. Friedman, R., & Iwai, J. (1976). Genetic predisposition and stress-induced hypertension. Science, 193, 161-162. Louis, W. J., & Spector, S. (1975). The effect of salt intake and adrenal steroids on blood pressure in a genetic strain of hypertensive rats. Clinical and Experimental Pharmacological Physiology, (Suppl.) 2, 131-163. McCarty, R. (1983). Stres, behavior and experimentalhypertension. Neuroscience and Biobehavioral Reviews, 7, 493-502. Murphy, C. A., & McCarty,R. (1989). Maternal environmentand development of high blood pressure in Dahl hypertensive rats. American Journal of Physiology, 257, H1396-H1401. Myers, M. M., Brunelli, S. A., Squire,J. M., Shindeldecker,R. D., & Hofer, M. A. (1989a). Maternal behavior of SHR rats and its relationship to offspringblood pressures. Developmental Psychobiology, 22, 29-53. Myers, M. M., Brunelli, S. A., Shair, H. N., Squire, J. M., & Hofer, M. A. (1989b). Relationships between maternal behavior of SHR and WKY dams and adult blood pressures of cross-fosteredF1 pups. Developmental Psychobiology, 22, 5567. Myers, M. M., & Scalzo, F. M. (1988). Bloodpressure and heart rate responses of SHR and WKY rat pups during feeding. Physiology and Behavior, 44, 75-83. Okamoto, K., & Aoki, K. (1963). Development of a strain of spontaneouslyhypertensiverats. Japanese Circulation Journal, 27, 282-293. Rapp, J. R., & Dene, H. (1985). Developmentand characteristics of inbred strains of Dahl salt-sensitive and salt-resistant rats. Hypertension, 7, 340-349.