Influence of postnatal maternal stress on blood pressure and heart rate of juvenile and adult rat offspring
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Influence of Postnatal Maternal Stress on Blood Pressure and Heart Rate of Juvenile and Adult Rat Offspring ELLIOTT MILLS JOSEPH W. BRUCKERT PETER G. SMITH Department of Pharmacology Duke University Medical Center Durham, North Carolina
Autonomic control of blood pressure (BP) and heart rate (HR) was tested in offspring of rat dams that were unmanipulated (controls) or exposed repeatedly to either ( I ) Postnatal restraint, (2) Postnatal S.C. injections of alkaline saline, or (3) Prenatal S.C. alkaline saline. Under urethane anesthesia, BP was higher than control in 20-day-old offspring of restrained dams and lower at 80 days. The magnitude of the BP response to autonomic ganglionic blockade (chlorisondamine) changed in parallel; the control HR accelerator response was reversed at 20 days and enhanced at 80 days. Postnatal maternal injections increased BP in 20-day-old offspring and lowered it at 80 days. The BP response to blockade was unchanged; HR acceleration was attenuated at 20 days and increased at 80 days. N o influence of Prenarul maternal injections was seen in adult offspring. Conclusion: Post-not prenatal maternal stress disrupts BP and HR control in rat offspring; disruption is greater after restraint than injection. There is sympathetic hyperactivity in preweanlings and hypoactivity in adults.
Exposure of the rat to drugs or hormones in utero or via nursing can alter the postnatal development of the sympathetic nervous system (see for example Lau & Slotkin, 1980; Slotkin, Weigel, Barnes, Whitmore, & Seidler, 1981; Navarro, Seidler, Whitmore, & Slotkin, 1988). While testing for related cardiovascular effects, we found that a maternal sham treatment altered the offsprings blood pressure (Smith & Mills, 1983). The treatment was S.C. injection of alkaline saline, 2 pl/g (a vehicle for thyroid hormone, Lau & Slotkin, 1980) from gestational Day 19 to postnatal Day 9. The anesthetized offspring of injected dams had higher resting blood pressure (58% increase in MAP) at 19-23 days of age and lower
Reprint requests should be sent to Elliott Mills, Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, NC 27710, U.S.A.
Received for publication 28 March 1990 Revised for publication 12 June 1990 Accepted at Wiley 27 August 1990
Deuelopmenrul Psychobiology 23(8):839-847 ( 1990) 0 1990 by John Wiley & Sons, Inc. CCC 0012-1630/90/080839-W$04.00
840 MILLS, BRUCKERT, AND SMITH
pressure (35% decrease) at 82-86 days of age. In contrast, pups that were injected from birth to Day 9 remained normotensive. These observations suggested that manipulation of nursing dams might alter development of autonomic cardiovascu- lar control mechanisms in the offspring.
The influence of infantile experience on adult physiology is an area of long- standing interest in developmental psychobiology (Denenberg, 1971 ; Levine, 1971 ; Myers, Brunelli, Squire, Shindeldecker, & Hofer, 1989). Withal, there is scant evidence for an influence on cardiovascular function: adult rats handled as neo- nates are reported to have a reduced heart rate response to novel stimulation (Blizard, 1971). Suggestive of a maternal influence, correlation analysis indicates that differences in behavioral interactions between rat mothers and pups contribute to variance in adult blood pressure (Myers et al., 1989).
In the current experiments, nursing dams were exposed to two types of stress- ful intervention: restraint or S . C . injection (Brown & Martin, 1974; Pare & Glavin, 1986). Other, pregnant dams received S.C. injections. Offspring were studied at 17-20 or 80-85 days of age. To determine if tonic autonomic activity to the vasculature or heart was heightened or diminished, the blood pressure and heart rate response to pharmacologic blockade of ganglionic neurotransmission was measured.
Methods Sprague-Dawley rats (Charles River Breeding Laboratories, Raleigh, N.C.)
were used. Pregnant dams were caged individually, maintained in a 14 : 10 hr light/ dark cycle, and allowed free access to food and water.
Injecting or Restraining Dams Postnatally Pregnant dams were received during the second week of gestation. At birth,
the pups were randomized and redistributed to the nursing mothers; the litter size was set at 10. Six dams were controls, 6 received injections and 6 were restrained. Controls were handled only for cage maintenance. On postnatal days 1-23, dams were either injected S.C. with alkaline saline (0.4 ml adjusted to pH 9-10; Smith & Mills, 1983) or restrained for 45 min in the supine position beneath plastic mesh stapled to a padded board. Measurements were made on 18 of the offspring when they were 17-20 days of age: one pup was used from each of the litters nursed by the control, injected or restrained dams. The other pups were weaned at 24 days of age and housed with their littermates. At 80-85 days of age, experiments were performed on 18 of these offspring: one born of each of the dams in the control or treatments groups. Pups of both sexes were studied. Wherever possible, groups were comprised of equal numbers of males and females.
Injecting Dams Prenatally Dams used in this study were received on gestational Days 2-3. Daily S.C.
injections of alkaline saline were given to 6 of the rats on gestational Days 4 to 21. The 6 controls were handled only for cage maintenance. At birth, pups were randomized within group and redistributed to the nursing dams. Offspring of both
MATERNAL STRESS AND OFFSPRlNG BLOOD PRESSURE 841
sexes were studied when they were 80- to 85-days-old. As no significant effects were detected in adults, younger animals were not studied.
Measurements on Offspring of Injected or Restrained Nursing Dams Experiments on preweanlings were started at 0900 hr; the maternal treatments
were carried out starting at 1600 hr. The rats were weighed and anesthetized with urethane (ethyl carbamate, 1-1.5 mg/g, i.p.). A femoral artery and vein were cannulated. The rats breathed room air enriched by a stream of 40% 0, in N, directed toward the snout. Body temperature was measured with a rectal probe and maintained at 37"-38"C with a regulator and heat lamp. Blood pressure (Gould 23d transducer) and heart rate (Grass 7P4 Tachograph, triggered by pressure pulse) were recorded continuously on a polygraph. Thirty min were allowed for stabilization of blood pressure and heart rate. Control values were obtained during the next five min. Chlorisondarnine (Ecolid, CIBA Pharmaceuticals, Summitt NJ), a specific and long acting ganglionic blocking agent (Plummet-, Trapold, Schneider, Maxwell, & Earl, 1955), was then administered intravenously in a dose known to be supramaxirnum for blockade and hypotension in preweanling and adult rats (2.5 mg/kg, Mills & Smith, 1986). The post-ganglionic blockade values were obtained starting five minutes after drug administration when blood pressure and heart rate were restabilized.
Blood pressure and heart rate were read from the continuous polygraph record at 1 min intervals for 5 min during the control and post-ganglionic blockade periods. The variation among the five blood pressure or heart rate readings was 5 5%. The readings were averaged to obtain a single control value and single post-ganglionic blockade value for each experiment.
After measuring post-ganglionic blockade blood pressure in the offspring that were 80-85 days of age, we tested the capacity of the vasculature to respond to noradrenergic stimulation by measuring the maximum pressor response to the selective alpha- 1 agonist, methoxamine (Vasoxyl, Burroughs- Wellcome). The ago- nist was administered intravenously (injection volume of 2 pl/g body weight) in repeated cummulative doses (0.05-2.0 mg/kg) until the maximum increase in blood pressure was recorded (Mills et al., 1986).
Individuals reading the blood pressure records were unaware of the treatment group from which they were obtained.
Data from offspring of the postnatally restrained or injected dams was tested first by 2-way analyses of variance (ANOVA) for treatment effects and for age by treatment interactions. Factor 1 was treatment; the levels were control, restraint, and injection. Factor 2 was age; the levels were 20 days and 80 days. These ANOVAs were done on values for resting blood pressure, change in blood pressure after chlorisondamine, resting heart rate, and change in rate after chlorisondamine. The ANOVAs identified a significant treatment by age interaction for each of the variables but no treatment effect ( F 2,30 values respectively = 0.71, 1.54, 1.17 and 1.46, p > .05). Accordingly, the significance of differences between individual groups was assessed with Duncan's multiple range test. Note that the 6 replicates
842 MILLS, BRUCKERT, AND SMlTH
Table I Body Weights of Offspring on Which Experiments Were Performed. Each entry is the mean +- S.E . of 6 unimals.
Offspring Postnatal Prenatal Age (Days) Control Restraint Injection Injection
11-20 4 6 ? 2 4 3 t 2 5 4 t 2 80-85 318 ? 24 315 5 24 315 t 21 80-85 325 ? 20 301 ? 21
For offspring of postnatally restrained or injected dams. ANOVA (performed on log values) did not detect significant treat- ment ( F = 2.16) or age by treatment interaction ( F = 2.20). For offspring of prenatally injected dams, no significant treatment effect.
in the control or treatment groups consisted of one pup from each of the 6 litters in the group. One-way ANOVAs were performed on values for same variables from 80 to 85-day-old offspring of prenatally injected dams.
Results Maternal treatments did not influence the body weights of the offspring (Table
1). The control values of blood pressure and heart rate at the different age levels (Table 2) are typical of normal development in the rat (Mills & Smith, 1986).
Offspring of Restrained Dams Resting blood pressure was higher in offspring of restrained dams than in
offspring of control mothers at 17-20 days of age and lower at 80-85 days, ( F 2,30 = 12.20, p < .01, age by treatment interaction; p < .05 vs. control at 17-20 days, p < .01, at 80-85 days). In parallel, the hypotensive response to ganglionic blockade in offspring of treated dams was greater than control in the preweanlings and less in adults ( F 2,30 = 14.26, p < .01; p < .01 vs. the age level controls). In the adults, the maximum pressor response to the alpha-1 noradrenergic agonist, methoxamine, was comparable to control (maximum increase = 104 k 3 mm Hg, treated vs. 108 L 2 mm Hg, control).
The resting heart rate was unchanged in the preweanling offspring of treated dams and was slightly less than control in the adults ( F 2,30 = 3.79, p < .05); p < .05 vs. control at 80-85 days). Ganglionic blockade produced a fall in heart rate at 17-20 days of age (a reversal of the control response) and an exaggerated increase in rate in the adults ( F 2,30 = 29.23, p < .01; p < .01 vs. age level controls).
Offspring of Postnatally Injected Dams Resting blood pressure of the offspring of treated dams was higher than in
offspring of control dams at 17-20 days of age and lower at 80-85 days. ( p < .01 vs. control at 17-20 days; p < .05 at 80-85 days). In contrast, the hypotensive
MATERNAL STRESS AND OFFSPRING BLOOD PRESSURE 843
Table 2 Cardiovascular Variables in Offspring of Postnatally Restrained or Injected Dams. Entries are the mean ? S.E. ofvaliws from 6 ruts. BP REST = mean arterial blood pressim before chlorisondamine. BP BLOCK = decrease after ganglionic blockade. HR REST = heart rate before chlorisondamine. HR BLOCK = chunge ujitrc>r ganglionic blockade.
Values in Offspring Daily Daily Offspring Age Control Restraint Injection
BP REST (mm Hg) 17-20 Days 4 7 t 3 60 t 3* 59 t 3* 80-85 Days 8 4 + 5 62 t 3* 68 t 5*
_ _ _ ~ ~ ~ ~
ABP BLOCK (mm Hg) 17-20 Days - 8 2 2 - 1 7 2 3* - 9 2 2 80-85 Days - 2 4 t 2 - 7 t 2* - 2 2 t 4
~ ~~~~~ ~~
HR REST (beatshin) 17-20 Days 428 ? 10 412 ? 15 440 t 10 80-85 Days 346 ? 12 290 t 14* 327 t 14
~~~~ ~~ ~~ ~~
AHR BLOCK (beatsimin) 17-20 Days + 4 5 2 8 -14 ? 7* + 2 3 t 3* 80-85 Days + 2 7 t 4 +76 t 13* +67 t 9*
* = different from control at same age level by Duncans multiple range test after ANOVA (see text for values).
response to ganglionic blockade remained comparable to control throughout. The pressor response to methoxamine was unaffected in the adults (maximum increase = 105 ? vs. 104 ? 5 mm Hg).
Resting heart rate was similar to control at both ages, but the cardioaccelerator response to ganglionic blockade was attenuated at 17-20 days and enhanced in the adults ( p < .01, age by treatment; p < .05 vs. control at 17-20 days; p < .01 vs. control at 80-85 days).
Adult Offspring of Prenatally Injected Dams There were no significant changes (l-way ANOVA) in the resting blood pres-
sure (84 ? 5 vs. 83 ? 2 mm Hg), hypotensive response to chlorisondamine (-22 2 3 vs. -28 ? 5 mm Hg), resting heart rate (347 ? 7 vs. 347 5 11 beats/min), heart rate response to chlorisondamine ( +44 5 12 vs. + 35 & 9 beatslmin) or the pressor response to methoxamine (99 ? vs. 96 * 3 mm Hg).
Discussion As the perturbations of autonomic control of blood pressure and heart rate
were detectable under general anesthesia, they are considered to originate at sub- cortical levels of the neuraxis. While peripheral target organ dysfunction cannot
844 MILLS, BRUCKERT, AND SMITH
be excluded completely, the unchanged vascular response to direct noradrenergic stimulation argues against the possibility. The disruptive influence of postnatal maternal interventions as opposed to prenatal interventions can be attributed to their coincidence with critical events in development of sympathetic nervous system and feedback control mechanisms in the offspring. These include: gangli- onic neurotransmission which matures at the end of postnatal week one (Smith et al., 1982; Smith, Evoniuk, Poston, & Mills, 1983; Slotkin, 1986); noradrenergic neurotransmission which matures through the 4th postnatal week (Behrens, La- celle, & Depocas, 1979; Lau & Slotkin, 1980; Seidler & Slotkin, 1981; Smith et al., 1982); a parallel development of presynaptic feedback inhibition of norepinephrine release and synthesis (Mills, Navarro, Slotkin, & Bruckert, 1988); baroreflex control of sympathetic and of cardiac vagal activity which appears in the 3rd postnatal week (Bartolome, Mills, Lau, & Slotkin, 1980; Mills, 1990).
A common response of the developing sympathetic nervous system to adverse environmental influences (drugs, hormones, toxic chemicals) is an acceleration of ganglionic synaptogenesis and precocious onset of neural control of target organs (Lau & Slotkin, 1980; Smith, Mills, & Slotkin, 1981; Seidler & Slotkin, 1985). Initially, in preweanlings there is enhancement of NE biosynthesis, release, synap- tosomal uptake, and postsynaptic receptor number; but later in life, deficiencies are evident that persist at least...