Published online before print December 26, 2006, doi: 10.1161/01.HYP.0000254481.94570.74
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(Hypertension. 2007;49:653.)
© 2007 American Heart Association, Inc.
Original Articles, Part 2 |
Identification of Active Central Nervous System Sites in Renal Wrap Hypertensive Rats
From the Department of Pharmacology, University of Texas Health Science Center at San Antonio.
Correspondence to J. Thomas Cunningham, Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78229. E-mail cunninghamt{at}uthscsa.edu
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Abstract |
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To identify central neurons participating in cardiovascular regulation in hypertension, we studied Fos staining, a marker for synaptically activated neurons, in adult male normotensive and hypertensive (HT) rats. At 1 and 4 weeks after induction of unilateral nephrectomy, renal wrap hypertension mean arterial pressure was 138±4 mm Hg (n=6) in 1-week HT rats and 159±6 mm Hg (n=6) in 4-week HT rats. Mean arterial pressure was 103±2 mm Hg (n=6) in sham-operated, normotensive rats. Mean arterial pressure was greater in both HT groups compared with normotensive rats, and the mean arterial pressure in 4-week HT rats was greater than that in 1-week HT rats. Rats were anesthetized and perfused, brains sectioned and processed using a Fos antibody, and the number of Fos immunoreactive neurons counted in sections through various brain regions. Hypertension of 1 or 4 weeks did not alter the number of Fos immunoreactive neurons in the area postrema, the supraoptic nucleus, and the median preoptic nucleus. The number of Fos immunoreactive neurons was increased after 1 and 4 weeks in the nucleus of the solitary tract, both the caudal and ventral lateral medulla, and the organum vasculosum of the lamina terminalis. In addition, after 4 weeks of HT, the number of Fos immunoreactive neurons was increased in the parabrachial nucleus and the paraventricular nucleus of the hypothalamus. The results indicate central regions active in acute and chronic HT rats and suggest certain areas that may be differentially activated depending on the duration of the hypertension.
Key Words: baroreceptor • brain • immunohistochemistry
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Introduction |
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Immunocytochemistry for the protein Fos has been widely used to map the activation of specific regions of the central nervous system associated with acute homeostatic challenges.1–4 Fos is an immediate early gene transcription factor that has been shown to be present in synaptically activated cells.5 A number of studies in the rat have examined areas in the central nervous system where neurons express Fos after brief increases in arterial blood pressure6–13; however, few studies have sought to identify which of these areas might be active in chronically hypertensive (HT) rats.14–17
At present, our current understanding of central nervous system function in hypertension is limited because of the lack of quantitative answers to fundamental questions. Many of these questions deal with the overall operating characteristics of the peripheral and central sites that regulate blood pressure. For example, is the number of neurons active in a particular brain region different in an HT compared with a normotensive (NT) individual? Is the number of neurons active in a particular brain region graded as a function of the magnitude or the duration of the change in blood pressure?
Therefore, quantification of the number of neurons in key central sites active in renal wrap HT rats is important. Such information could provide insights into areas that are likely candidates to mediate the elevated sympathetic outflow18–20 and altered baroreflex function21 associated with this model of hypertension. For example, previous studies have demonstrated that the anteroventral region of the third ventricle,22 paraventricular nucleus (PVN) of the hypothalamus,23 and parabrachial nucleus (PBN)24 contribute to renal wrap hypertension. The anteroventral region of the third ventricle includes the organum vasculosum of the lamina terminalis (OVLT), circumventricular organ, and the median preoptic nucleus (MnPO). These regions are interconnected, and the PVN projects to the rostral ventrolateral medulla (RVLM), which contains sympathetic premotor neurons, and could provide a neural substrate for chronic sympathetic activation. In addition, chronic activation of the nucleus of the solitary tract (NTS) or caudal ventrolateral medulla (CVLM) could provide evidence of baroreflex resetting.
In the present study, we used Fos immunocytochemistry to quantify the number of neurons active in critical cardiovascular regulatory areas of the central nervous system in NT rats and during acute (1 week) and chronic (4 weeks) renal wrap hypertension. These 2 time points were chosen to determine whether there were any changes in the number of active neurons as a function of the duration of the hypertension.
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Methods |
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Animals
Experiments were performed on 18 adult, male Sprague–Dawley rats (375 to 500 g, Charles River Laboratories). Rats were housed 2 per cage in a fully accredited (Association for Assessment and Accreditation of Laboratory Animal Care and the US Department of Agriculture) laboratory animal room with free access to food and water. All of the rats were given
1 week to acclimate before being used for any procedures. The Institutional Animal Care and Use Committee approved all of the experimental protocols.
Chronic HT Model
Rats were anesthetized with medetomidine (0.5 mg/kg IP; Pfizer) and ketamine (75 mg/kg IP; Ft Dodge Laboratory), and, under aseptic conditions, hypertension was induced using a figure-8 Grollman renal wrap and contralateral nephrectomy (Grollman A 1944). NT rats were similarly anesthetized and received a unilateral nephrectomy but no wrap of the contralateral kidney (sham) or no surgical procedures before the day of the experiment. Because the neuronal responses of both groups of NT rats were identical, they were grouped for analysis. Anesthesia was terminated by atipamezole (1 mg/kg IP; Pfizer) at the conclusion of the surgical procedures. Postoperative analgesics (Children’s Tylenol, oral) were provided for the first 2 to 5 days after surgery.
Two days before the experiment, an arterial catheter was placed in the femoral artery while the animal was anesthetized with medetomidine/ketamine (0.5 mg/kg IP and 75 mg/kg IP, respectively). After a 2-day recovery period, blood pressure was measured in all of the rats while conscious and freely moving by connecting the arterial catheter to a pressure transducer (Kobe) and visualized using MacLab or Cambridge Electronics Design A/D converters. Blood pressure was measured for 3 hours, and measurements during the last hour were used as an index of mean arterial pressure (MAP).
Fos Immunocytochemistry
Three separate groups of rats were used for histology: NT (n=6), 1 week-HT (n=6), and 4-week HT (n=6). All of the rats were deeply anesthetized with pentobarbital (50 mg/kg IP) and perfused intracardially with 0.1 mol/L of PBS followed by 300 to 500 mL of 4% paraformaldehyde in PBS. After the perfusion, the brains were removed and placed in PBS with 30% sucrose for 3 to 4 days. The forebrain and hindbrain of each was separately sectioned at 40 µm in a cryostat. Three serial sets of coronal sections from each region were placed in cryoprotectant and stored at –20°C until they were processed for immunocytochemistry as described previously.25,26
One set of free-floating sections from each rat was stained for Fos using a commercially available antibody directed at the amino acid residues 4 to 17 in human Fos (rabbit anti-c-Fos Ab5, Calbiochem) as described previously. The sections were incubated in the primary antibody (1:30 000) for 72 hours at 4°C. Next, the sections were incubated in biotinylated horse anti-rabbit IgG (Vector Laboratories) diluted 1:200 in PBS for 2 hours at room temperature. After an additional 60-minute rinse in PBS, sections were reacted with an avidin-peroxidase conjugate (Vectastain ABC kit, Vector Laboratories) and PBS containing 0.04% 3,3'-diaminobenzidine hydrochloride and 0.04% nickel ammonium sulfate. After the staining was complete, sections were mounted on gelatin coated slides, air dried for 1 to 2 days, and coverslipped with Permount.
Sections were examined using light microscopy to identify Fos-positive cells in the OVLT, MnPO, PVN, and supraoptic nucleus from the forebrain and the area postrema (AP), NTS, CVLM, and RVLM from the hindbrain. Tissue sections containing regions of interest were recorded using an Olympus microscope (IX 50) equipped for epifluorescence. Digital images were acquired using a Spot camera (SPOT RT Slider, Diagnostic Instruments) connected to a Pentium computer running Spot imaging software (version 3.24). Regions of the forebrain were identified based on the rat brain stereotaxic atlas of Paxinos and Watson27 as described previously.28,29 The portion of the NTS used for analysis extends from 300 µm caudal to obex and 300 to 400 µm past the rostral edge of the AP. The CVLM was defined posteriorly by the pyramidal decussation and anteriorly by the appearance of the principle nucleus of the inferior olive.30,31 The anterior border of the RVLM was defined by the caudal pole of the facial nucleus, and the rostral hypoglossal nucleus was used for the posterior border.30,31 Analysis of the PBN included the ventral lateral, central medial, external lateral, and external medial regions, which included the pontine taste area. The numbers of Fos-positive cells in each region were tabulated for each section without knowledge of which treatment group the sections were from. The numbers of Fos-positive cells in each brain sections were averaged for the regions, and these averages were used in the statistical analysis. At least 5 sections containing the NTS were used from each rat. At least 3 sections containing the CVL, RVL, AP, supraoptic nucleus, PVN, and PBN from each rat were used for the cell counts. Because of the small anterior to posterior dimensions of the MnPO and OVLT, only 1 to 3 sections containing these regions were used. In bilateral structures, Fos-positive cells were present on both sides, but counts were made unilaterally. The number of Fos-positive cells was counted on each section, and these numbers were averaged for each region for each rat for statistical analysis.
Statistics
Data were analyzed by 1-way ANOVA with Student—Newman–Keuls t test for posthoc analysis of significant main effects (SigmaStat, version 2.03, Systat Software Inc). Alternatively, Kruskal–Wallis ANOVA on ranks and Dunn’s multiple comparison procedure were used when data were not normally distributed.32 Significance was set at P<0.05. All of the values are presented as mean±SEM.
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Results |
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MAP in NT rats was 103±2 mm Hg (n=6); in 1-week HT rats, MAP was 138±4 mm Hg (n=6); and in 4-week HT rats, MAP was 159±6 mm Hg (n=6). MAP was greater in both HT groups compared with NT rats, and the MAP in 4-week HT rats was greater than that in 1-week HT rats.
Fos immunoreactivity was fairly low in most brain regions in NT rats (Table), although there was a tendency for a larger number of immunoreactive neurons in forebrain regions. One week of hypertension was associated with significant increases in the number of Fos immunoreactive neurons in the NTS, CVLM, RVLM, and the OVLT as compared with the NT controls (Table). In contrast, the number of Fos-positive cells in the AP, PBN, PVN, supraoptic nucleus, and MnPO of 1-week HT rats was not different from NT rats (Table). In the NTS, Fos-positive cells were located in the caudal and subpostremal portions of the nucleus (Figure 1). CVLM Fos-positive cells were located mostly at the level of the AP (Figure 1). In the RVLM, Fos-positive cells were evenly distributed throughout the region (Figure 1). In the OVLT, significant Fos staining was observed in both the dorsal cap and the lateral margins of the nucleus (Figure 2). After 4 weeks of hypertension, the number of Fos immunoreactive neurons was greater compared with NT rats in the NTS, CVLM, RVLM, PBN, PVN, and OVLT. After 4 weeks of hypertension, the number of Fos immunoreactive neurons was greater compared with 1-week HT rats in the NTS, RVLM, PBN, and PVN, with there being no difference in any of the other regions analyzed between 1 week and 4 weeks of hypertension (Table). For the NTS, this represented an increase in Fos-positive cells in the same regions that were activated after 1 week of hypertension (Figure 1). In the PBN, the Fos-positive cells were located almost exclusively in the lateral portion of the nucleus. The increase in Fos staining in the PVN was found mostly in the parvocellular subnuclei, especially in the dorsal cap and lateral parvocellular cell groups (Figure 2).
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Discussion |
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Fos is the protein product of the immediate early gene transcription factor c-fos that has been shown to be present in synaptically activated cells. Increasing neuronal discharge via antidromic activation does not induce expression of Fos.5 This is the first study of Fos immunoreactive neurons in the renal wrap HT rat. The data provide important, quantitative information on the number of neurons active at any given point in time, which is of importance to attempts to model baroreflex regulation of blood pressure in NT and HT states. In addition, the results extend observations in other HT models by incorporating duration and amplitude of change in blood pressure as additional variables. Considering these aspects of the data provides possible insights into the organization of the central nervous system pathways that modulate cardiovascular function in hypertension.
The overall pattern of activation of central neurons is similar to that reported in previous studies in spontaneously hypertensive rats16 and in obesity33 and angiotensin17,34 infusion models of hypertension. Fos immunoreactivity was increased in the RVLM in the obese dogs and in the present study but not in dogs chronically infused with angiotensin. Increased Fos immunoreactivity expression in the RVLM, as well as the PBN and PVN, supports the observations that sympathetic activity to the kidneys and other vascular beds is increased in renal wrap of hypertension.18–20 In contrast, the absence of increased Fos immunoreactivity in the RVLM in angiotensin infusion hypertension is consistent with recent findings indicating that renal sympathetic nerve activity is suppressed during chronic angiotensin infusion.35
A previous study found no significant difference in the basal discharge rate of presumed sympatho-excitatory neurons in the RVLM in the spontaneously hypertensive rats.36 This suggests that, if the RVLM mediates increased sympathetic outflow in hypertension, this may be mediated by increasing the number of RVLM neurons providing excitatory drive to sympathetic preganglionic neurons rather than grading the discharge frequency of individual RVLM neurons. Our data are consistent with this interpretation. The study of Sun and Guyenet36 also found no difference in the ability of the arterial baroreflex to inhibit the discharge of RVLM neurons comparing NT and HT rats. Our data suggest that an increased number of synaptically activated NTS neurons comparing 1- and 4-week HT rats is not associated with an increased number of synaptically activated CVLM neurons. This suggests that the CVLM input to the RVLM could diverge to multiple RVLM neurons to produce an equivalent baroreflex modulation of RVLM discharge in HT rats.36
The number of neurons in the CVLM with Fos immunoreactivity did not increase between 1 week and 4 weeks of hypertension in spite of a significantly greater MAP after 4 weeks compared with 1 week of hypertension. The graded increase in the number of synaptically activated NTS neurons comparing 1 week and 4 weeks of hypertension suggests that baroreflex activation of the CVLM is saturated after 1 week of hypertension, which could play a permissive role in tonically elevated sympathetic outflow. Of course, Fos provides no insights into whether or not cells within the CVLM that are already active at 1 week of hypertension increase their discharge in response to increased excitatory inputs from the NTS after 4 weeks of hypertension.
Both the PVN23 and the PBN24 have been shown to contribute to the elevated blood pressure observed after 4 weeks of renal wrap hypertension. There was no change in the number of Fos immunoreactive neurons in the PBN and PVN after 1 week of hypertension; however, an increase was observed after 4 weeks of hypertension. This suggests that the PBN and PVN might contribute to chronic, but not acute, renal wrap hypertension. However, it is important to keep in mind that Fos is only 1 indicator of synaptically activated cells. The data could also be explained if, in acute hypertension, sympathetic outflow is elevated by increasing the discharge of already active PVN neurons, whereas in chronic hypertension, the number of discharging neurons is increased. Information on the action potential discharge of single, sympatho-excitatory neurons in the PVN in NT and HT rats is required to answer this question.
The stimuli that induce Fos expression in central neurons remain conjectural at this point. Increased baroreceptor afferent input in hypertension37 could increase Fos expression in the NTS. In addition, descending inputs from other central areas known to be important in this model of hypertension23,24 could induce Fos expression in NTS.38,39 Descending inputs from these areas could also provide an excitatory drive and induce Fos expression in sympathoexcitatory neurons within the RVLM.
The renal wrap model of hypertension is angiotensin dependent,40 so one might anticipate that angiotensin could induce expression of Fos in angiotensin-sensitive forebrain regions. Therefore, the absence of a significant change in Fos immunoreactivity in the AP, supraoptic nucleus, and MnPO may be surprising. However, a significant increase in Fos immunoreactivity was observed in the OVLT, and this area has been proposed to be one site whereby circulating angiotensin mediates its central sympatho-excitatory effects.41 Unfortunately, the fragile subfornical organ, another such site, was not present in enough tissue to permit quantification of Fos immunoreactivity.
In conclusion, the present study indicates that synaptically activated neurons are present in most major central nuclei implicated in cardiovascular and sympathetic regulation in HT. Within the hindbrain, only within the NTS and the RVLM, and not within the CVLM, is the number of synaptically activated neurons graded as a function of the duration and/or the degree of hypertension. Within the forebrain circumventricular organ the OVLT, the number of synaptically activated neurons is increased in HT, but the number is not graded as a function of the duration or degree of hypertension. Within the midbrain PBN and the forebrain PVN, the number of synaptically activated neurons is only increased after 4 weeks of hypertension. The factors that could initiate increased Fos expression are numerous (eg, increased baroreceptor afferent input to the central nervous system, increased circulating or tissue-generated angiotensin, and metabolic factors) and provide avenues for future study. Alterations in the activation patterns of central neurons are considered adaptive responses to any or all of these factors and not causal to the HT state.
Perspectives
These data indicate a differential pattern of neuronal activation in hypertension. Hindbrain, midbrain, and forebrain areas show different temporal patterns of activation and differing sensitivities to the degree of hypertension. These data emphasize the need for a characterization of the basal discharge and response characteristics of single neurons in these areas in this and in other models of hypertension.
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Acknowledgments |
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We gratefully acknowledge the technical assistance of Maurice Penny and Joel Little.
Sources of Funding
This research was supported by the National Institutes of Health (NIH R01 HL63579 and R01 DK57822 to J.T.C. and HL56637 to S.W.M).
Disclosures
None.
Received October 15, 2006; first decision November 5, 2006; accepted November 28, 2006.
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