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Small for gestational age infants are known to develop hypertension in adulthood. This prenatal programming of hypertension (PPH) can result from several insults including maternal dietary protein deprivation, uteroplacental insufficiency and prenatal administration of glucocorticoids. The mechanisms underlying the development of hypertension remain unclear although the sympathetic nervous system has been indirectly implicated. This study was designed to directly measure renal sympathetic activity (RSNA) both at rest and during physical stress in an animal model of PPH. The adult male offspring of rats fed either a 6% (PPH) or 20% protein diet (control) were investigated. Conscious systolic blood pressure measured by tail cuff was significantly higher in PPH compared to control (140 ± 3 vs. 128 ± 3 mmHg, P < 0.05). Baseline mean arterial pressure (MAP), heart rate (HR) and RSNA were not different between groups during isoflurane anesthesia or after decerebration. Physical stress was induced in decerebrate animals by activating the exercise pressor reflex (EPR) during static muscle contraction. Stimulation of the EPR evoked significantly larger changes from baseline in MAP (40 ± 7 vs. 20 ± 4 mmHg, P < 0.05), HR (19 ± 3 vs. 5 ± 1 bpm, P < 0.05) and RSNA (198 ± 29 vs. 68 ± 14 %, P < 0.05) in PPH as compared to control. The data demonstrate that the sympathetic response to physical stress is markedly exaggerated in PPH and may play a significant role in the development of hypertension in adults born small for gestational age.
It has been demonstrated by Barker and colleagues 1–4 that there is an association between small for gestational age infants and the development of hypertension and cardiovascular mortality in later life. However, the cause for the predisposition of low birth weight infants to develop chronically elevated arterial blood pressure (ABP) when they become adults is unknown. To investigate the etiology for the increase in blood pressure in adults who were small for gestational age, studies have attempted to recapitulate common prenatal insults in animals such as prenatal administration of glucocorticoids, maternal dietary protein deprivation and uteroplacental insufficiency. In these models, exposure to such insults not only produces offspring that are small for gestational age but also leads to the prenatal programming of hypertension (PPH). As a result, the models can be used to investigate the mechanisms underlying the development of hypertension in adults born of low birth weight.
Investigations using such animal models as well as studies in humans have led to a number of hypotheses for the generation and maintenance of hypertension with prenatal programming. Brenner and colleagues 5–8 have proposed that intrauterine growth retardation results in a reduction in nephron number that predisposes the development of hypertension in later life. Certainly, human neonates with intrauterine growth retardation have a reduction in nephron number 9–11. Further, prenatal insults in animals also result in reductions in nephron number 12, 13. However, there is a poor correlation between nephron number after prenatal insult and hypertension in adult offspring 14–17. Recent studies have demonstrated that prenatal programming leads to increased renal tubular sodium transport. Indeed, there is an increase in apical sodium transporters and sodium transport in multiple nephron segments that could lead to salt sensitive hypertension 12, 18–23. An important clue to the etiology of the hypertension with prenatal programming has come from studies showing that renal sympathetic denervation results in the normalization of blood pressure as well as sodium transporter abundance 21, 24, 25. Clearly, these findings implicate a role for the sympathetic nervous system in the development of hypertension with prenatal programming. However, to date, direct assessment of renal sympathetic nerve activity (RSNA) at rest and during physical stress has not been examined with prenatal programming making it difficult to draw definitive conclusions.
The purpose of this study was to directly assess RSNA at rest and in response to physical stress in both control and PPH adult rats. The physical stressors chosen for this purpose were experimental activation of the exercise pressor reflex (EPR) and its individual functional components; the muscle mechanoreflex and metaboreflex. The EPR is an important source of neural input to the brainstem during exercise contributing to the control of the cardiovascular system 26. In this reflex, contraction-induced sensory signals are generated by stimulation of group III (predominantly mechanically-sensitive A-δ fibers associated with the mechanoreflex) and IV (primarily chemically-sensitive C fibers associated with the metaboreflex) skeletal muscle afferent neurons which reflexively increase ABP and heart rate (HR) 27. EPR induced alterations in cardiovascular hemodynamics are primarily mediated by increasing efferent sympathetic activity 28. Thus, activating the EPR (or its individual components) is a reliable means by which to assess RSNA responsiveness. It was hypothesized that prenatal programming i) induces enhanced levels of basal RSNA and ii) produces exaggerated increases in RSNA during selective activation of the EPR, mechanoreflex and metaboreflex. To test these hypotheses, RSNA was measured at rest and during activation of the EPR (via electrically-induced static muscle contraction), the muscle mechanoreflex (via passive muscle stretch), and the muscle metaboreflex (via intra-arterial capsaicin administration in the hindlimb) in control and PPH (produced by maternal dietary protein deprivation during the last half of pregnancy).
For a complete description of the Materials and Methods see the online-only data supplement.
Pregnant Sprague Dawley rats were fed either a low 6% protein diet to produce PPH offspring (Teklad 6% Protein Diet) or an isocaloric control diet (Teklad 20% Protein Diet) from day 12 of gestation until birth 12, 29. All mothers were fed the control diet after delivery. Experiments were performed in male, age-matched (16–20 weeks) control (n = 13) and PPH (n = 14) rats. The procedures outlined were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas. All studies were conducted in accordance with the United States Department of Health and Human Services National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The EPR was stimulated in control and PPH animals by contracting the gastrocnemius and soleus muscles of the right hindlimb for 30 s via electrical stimulation of the isolated L4 and L5 ventral roots. Constant current stimulation was used at a 3 times motor threshold (i.e. the minimum current required to produce a muscle twitch) with a pulse duration of 0.1 ms at 40 Hz.
To selectively activate the mechanically-sensitive component of the EPR, hindlimb muscles were passively stretched in control and PPH animals using a calibrated 9.5 mm rack and pinion system (Harvard Apparatus).
Selective activation of chemically-sensitive afferent fibers innervating skeletal muscle was achieved by administering capsaicin into the arterial supply of the hindlimb (0.3 and 1.0 µg/100 µL). Capsaicin was injected into the right common iliac artery while the reversible ligature placed around the right common iliac vein was occluded for 2 min.
All values are expressed as means ± S.E.M. Data were analyzed using unpaired t-tests. The significance level was set at P < 0.05.
To quantitate the food and water intake of pregnant rats, some pregnant rats were placed in metabolic cages starting on day 12 of pregnancy. After a day of acclimatization, average daily food and water consumption and weight gain were recorded. The results are shown in Table 1. The PPH mothers ate more food (6% protein chow) and had a tendency to drink less water than the control mothers. The weight gain however was comparable. The 1-day-old offspring of the mothers fed a 20% protein diet weighed more than the offspring of mothers fed a 6% protein diet (6.3 ± 0.1 g vs 5.4 +0.2 g P<0.001).
Morphometric characteristics and baseline hemodynamics are summarized in Table 2. Morphometric characteristics were not different between groups. Systolic blood pressure in the conscious state was significantly higher in PPH than control animals whereas baseline cardiovascular and sympathetic parameters were not different between groups during administration of inhalant anesthesia or after decerebration.
Representative tracings from control and PPH animals during electrically induced static muscle contraction, a maneuver known to preferentially activate the EPR, are shown in Fig. 1. In response to the same amount of tension development, activation of the EPR elicited significantly larger elevations in MAP, HR and RSNA in PPH as compared to control (Fig. 2A). As depicted in Fig 1, these exaggerated changes in MAP in PPH resulted from enhanced increases in both systolic and diastolic pressure. Likewise, the integrated HR and RSNA responses were significantly greater in PPH than control (Fig. 2B). The integrated MAP response was similarly elevated in PPH although differences between groups did not reach statistical significance (P = 0.09).
The cardiovascular and sympathetic responses to activation of the mechanically-sensitive component of the EPR via passive muscle stretch were enhanced in PPH compared to control. This was true of both the peak as well as the integrated responses in MAP, HR and RSNA (Fig. 3A and 3B).
The cardiovascular and sympathetic responses to administration of capsaicin at the lower 0.3 µg/100 µL dose were not significantly different between control and PPH (Fig. 4A). At the higher concentration of capsaicin (1.0 µg/100 µL), however, HR and RNSA response were significantly greater in PPH as compared to control although the difference in the MAP response between the groups was not found to be statistically significant (P = 0.09, Fig.4B).
The significant findings of the current investigation were that i) PPH did not exhibit alterations in baseline RSNA (contrary to our original hypothesis) and ii) PPH did display markedly exaggerated increases in RSNA, ABP and HR in response to physical stress (in concurrence with our original hypothesis). The latter finding supports a role for the sympathetic nervous system in the development of hypertension with prenatal programming and is in agreement with a growing body of evidence, albeit predominately indirect, that there is an alteration in sympathetic nerve activity in human adults born small for gestational age. For example, while an imperfect assessment of sympathetic tone, resting HR is higher in adults that were of low birth weight compared to controls 30. Furthermore, the increases in blood pressure in response to psychological stresses such as public speaking were greater in offspring of mothers exposed to the Dutch famine during their first trimester of pregnancy than unexposed control subjects 31. Direct measurement of sympathetic activity has been reported in adults who were small for gestational age and compared to those who were of normal weight at birth. While one study showed an increase in resting sympathetic nerve activity in adults who were small for gestational age 32 another did not 33. However, the latter study found a significant increase over basal sympathetic nerve activity in response to the stress of breath holding in those who were born of low birth weight compared to controls 33. These latter findings are comparable to our study where we demonstrated similar sympathetic nerve activity at baseline but an augmented RSNA response to physical stress.
The current study demonstrating that there is an enhanced RSNA response to physical stress in rats which were the offspring of mothers fed a low protein diet is consistent with previous studies examining the effect of renal denervation on blood pressure in this animal model 21, 24, 25. In the previous studies, blood pressure was elevated in adult rats exposed to uteroplacental insufficiency 24, 25 or prenatal exposure of glucocorticoids 21. The offspring of mothers exposed to uteroplacental insufficiency had greater renal norepinephrine content than controls at 6 weeks of age 25. Renal norepinephrine content was also greater in offspring whose mothers were administered prenatal dexamethasone than control when studied at 3 weeks of age but not at 8 weeks of age 21. While denervation did not affect blood pressure of control rats, blood pressure normalized to control levels in offspring of mothers that had a prenatal insult 21, 24, 25. However, there are still some inconsistencies between these studies and our investigation that need to be resolved. In the current study, baseline RSNA was comparable in both groups of animals under anesthesia as well as after decerebration. An augmented increase in sympathetic activity in PPH was only noted in response to activation of the EPR, mechanoreflex and metaboreflex. It remains unknown if the conscious PPH rat would exhibit enhanced basal sympathetic activity and/or an exaggerated increase in RSNA in response to physical stress. In addition, baseline blood pressures significantly declined as compared to the conscious state in both control and PPH when the animals were placed on isoflurane anesthesia and/or decerebrated. One possibility for these reductions in pressure was the performance of the extensive surgery requisite for EPR testing. Another was the use of the pre-collicular decerebrate technique. For example, it has been suggested that portions of the hypothalamus (e.g. paraventricular nucleus) contribute to generating the elevated baseline SNA characteristic of hypertension34, 35. Removal of such areas within the hypothalamus by pre-collicular decerebration may have abrogated baseline elevations in RSNA in PPH animals and/or produced the reductions in baseline ABP as compared to the conscious state. It has been proposed that utilizing a mid-collicular decerebrate procedure may be preferable when investigating EPR function 36, 37 although use of this technique would likewise remove the hypothalamus. These technical limitations must be taken into account when interpreting the results of the current study. Resolving these inconsistencies will only be answered by directly measuring blood pressure and sympathetic nerve activity in conscious unanesthetized rats, the latter of which is currently a technically difficult experimental endeavor.
It has been well documented that, in hypertension, the cardiovascular response to exercise is abnormally heightened and characterized by exaggerated increases in ABP, HR and vascular resistance 38–42. To this end, our laboratory has previously demonstrated that selective activation of the EPR elicits greater increases in MAP, HR and RSNA in spontaneously hypertensive rats, a model of essential hypertension, compared to normotensive rats 43–45. These findings provide evidence that the exaggerated cardiovascular response to exercise in hypertension is mediated, in part, by a dysfunctional EPR. There are differences between our findings in the spontaneously hypertensive rat and PPH rats compared to their respective controls. While the blood pressure was not different under anesthesia in the PPH compared to the control group, it was over 50 mm Hg higher in the SHR compared to their respective controls. In addition, the normalized heart weight was not different between PPH and control groups unlike previous studies in spontaneously hypertensive rats which exhibit significant cardiac hypertrophy 43–45. Despite the possibility of PPH being a milder form of hypertension than that in the SHR model, the magnitude of the EPR overactivity was comparable 43–45. Collectively, these studies suggest the EPR contributes significantly to the abnormally exaggerated sympathetically-mediated cardiovascular response to exercise in multiple forms of hypertension. Given that the accentuated cardiovascular response to physical activity is associated with elevated risks for myocardial ischemia, myocardial infarction, cardiac arrest and/or stroke during or immediately after a bout of exercise 46–48 it is clinically important to determine the mechanisms underlying EPR dysfunction in each etiology of the disease.
There are several viable possibilities for the enhanced cardiovascular and sympathetic responses demonstrated during activation of the EPR in this study. Since prenatal programming by maternal dietary protein deprivation results in a reduction in nephron number 13 it is possible that renal dysfunction or injury contributes to EPR overactivity in PPH. Renal sympathetic afferents can be activated by minor injury resulting in an increase in central sympathetic nerve activity and hypertension, which is prevented by renal denervation 49. In patients with chronic kidney disease, there is an increase in muscle sympathetic nerve activity 50, 51, which normalizes after bilateral nephrectomy 51. Thus it is possible that the sympathetic overactivity may be initiated by renal afferents. However, prenatal programming can also have a primary effect on the brain 52, 53, which may result in increased sympathetic nerve activity when animals are put under stress. The relative roles of the kidney and central nervous system in mediating EPR overactivity with prenatal programming will have to be resolved in future studies.
The mechanisms underlying the development of hypertension in adults born small for gestational age is now being elucidated using animal models. This study has implicated a role for the sympathetic nervous system in the generation of hypertension in adult rats born small for gestational age. The applicability of these findings to humans born small for gestational age is unclear and requires translational investigation. Although these data do not support the contention that a chronic basal elevation in sympathetic activity contributes to the development of hypertension under resting conditions, it shows that there are markedly exaggerated elevations in sympathetic nerve activity in response to physical stress, which may have a significant impact in mediating the hypertension due to prenatal insults. Future studies will focus on telemetric measurements of sympathetic nerve activity and blood pressure at rest, during physical activity and with environmental stress to determine if these environmental perturbations cause parallel changes in sympathetic nerve activity and blood pressure in rats that had a prenatal insult. Telemetric measurements of blood pressure and sympathetic nerve activity may also provide insight into the best therapeutic regimen for the treatment of hypertension seen with prenatal programming.
The mechanisms underlying the development of hypertension in adults born small for gestational age due to maternal dietary protein deprivation remain largely undetermined. For the first time, the present study directly measures sympathetic nerve activity at rest and during physical stress in an animal model of prenatally programmed hypertension (induced by maternal dietary protein restriction) to assess the role of the sympathetic nervous system in the generation of chronic high blood pressure in adulthood.
Baseline renal sympathetic activity in adult hypertensive rats subjected to prenatal programming is not different compared to normal healthy adult rats. In contrast, the sympathetic response to physical stress is markedly exaggerated in prenatally programmed hypertensive rats.
This investigation has implicated a role for the sympathetic nervous system in the generation of hypertension in adults born of small birth weight due to dietary restriction during gestation. Although this study does not support the contention that chronic basal elevations in sympathetic activity contribute to the development of hypertension under these conditions, it does suggest that acute, markedly augmented elevations in sympathetic activity in response to physical stress may have a significant impact.
SOURCES OF FUNDING
This research was supported by grants from the National Institutes of Health Heart, Lung and Blood Institute (HL-088422 to SAS), the National Institutes of Diabetes and Digestive and Kidney
DK41612 (MB) and DK078596 (MB), and the O’Brien Center P30DK 079328 (Peter Igarashi).
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