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Blunted responsiveness of the neuronal activation marker Fos in brainstem cardiovascular nuclei of cirrhotic rats
Original Articles Blunted Responsiveness of the Neuronal Activation Marker Fos in Brainstem Cardiovascular Nuclei of Cirrhotic Rats DEANNE R. BREITMAN
Cardiovascular function in cirrhosis is deranged, with indirect evidence of abnormal central cardiovascular regulation. We aimed to elucidate the role of brainstem cardiovascular nuclei in hemodynamic regulation by examining the protein product, Fos, of the immediate-early gene c-fos, in cirrhotic rats. Cirrhosis was induced by chronic bile duct ligation (BDL) of 25-days duration, while controls underwent a sham operation. To examine the effects of jaundice per se in the absence of cirrhosis, a third group of 5-day BDL rats was also studied. All rats were anesthetized with pentobarbital, and catheters were inserted to measure baseline blood pressure and heart rate. Separate groups were then subjected to volume manipulation by a hypotensive hemorrhage or isotonic saline infusion, or no challenge. Ninety minutes after the volume manipulation, the animals were killed and the medulla sectioned and stained for Fos by immunohistochemisty. The nucleus tractus solitarius (NTS) of the sham-operated unchallenged rats showed scant Fos immunoreactivity (27.8 { 3.3 cells), but both hemorrhage and volume infusion significantly increased Fos staining (86.0 { 3.7 and 95.2 { 8.5, respectively). In contrast, the unchallenged cirrhotic rats showed markedly increased Fos in the NTS (154.6 { 27.0), but neither hemorrhage nor volume infusion significantly changed the amount of Fos staining. Fos staining in the ventrolateral medulla (VLM) followed a similar pattern with low staining in the unchallenged sham rats and increased staining in the other groups, but no differences between the unchallenged and the volume-manipulated cirrhotic groups. The 5-day BDL jaundiced rats showed no baseline increase in Fos staining, nor any significant increase after hemorrhage. These results showing baseline activation of central neuronal regions responsible for blood pressure homeostasis, but completely blunted responsiveness in cirrhotic rats, confirm a central origin of disordered cardiovascular regulation. The presence of jaundice may also contribute to the central cardiovascular hyporesponsiveness. (HEPATOLOGY 1997;26:1380-1385.)
Abbreviations: BDL, bile duct–ligated; PBS, phosphate-buffered saline; NTS, nucleus tractus solitarius; VLM, ventrolateral medulla. From the Liver Unit, Gastroenterology Research Group, University of Calgary, Calgary, Alberta, Canada. Received October 23, 1996; accepted May 12, 1997. Funded by an operating research grant from the Medical Research Council of Canada. Ms. Breitman was supported by an Alberta Heritage Foundation for Medical Research (AHFMR) Summer Studentship award. Dr. Lee was supported by an AHFMR Scholarship award. Address reprint requests to: S. S. Lee, M.D., 3330 Hospital Dr NW, Calgary, AB, T2N 4N1, Canada. Fax: (403) 270-0995. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2606-0002$3.00/0
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SAMUEL S. LEE
Cirrhosis is associated with abnormal cardiovascular phenomena. These include the development of hyperdynamic circulation and impairment of cardiac contractility.1-4 Hyperdynamic circulation is manifested by elevated cardiac output, increased blood flow, and lowered peripheral vascular resistance and arterial blood pressure.1-3 However the precise causes and mechanisms of this hyperdynamic circulation remain unclear. Humoral factors, such as nitric oxide, glucagon, bile salts, and prostaglandins have been proposed, but the results to date have proved inconclusive.5-9 Neural factors have also been implicated. Sympathetic nervous activity has been shown to be enhanced in cirrhosis,10-14 although the mechanism by which sympathetic activation leads to hyperdynamic circulation, and the level at which these alterations arise, remains to be elucidated. Although indirect evidence has suggested that central sympathetic tone is increased,10-14 to date, this hypothesis has not been directly tested. C-fos is an immediate-early gene that is rapidly and transiently expressed within cells in the central nervous system as a result of membrane depolarization and voltage-gated calcium influx.15,16 Its protein product, Fos, can be detected by immunohistochemistry. Basal levels of Fos immunoreactivity are known to be low,15 and the presence of Fos immunoreactivity within neurons is therefore indicative of activation of these cells. Fos has been extensively used and validated as a sensitive marker for identifying and characterizing neuronal activation as a response to stimulation by external factors.17 More particularly, Fos staining has been used to identify and characterize neuronal activation and pathways in the brainstem in response to cardiovascular challenge.17-21 We therefore hypothesized that central regulatory mechanisms are indeed disordered, and that this would be manifested by neuronal activation in the brainstem cardiovascular control centers. Accordingly, we aimed to test this notion by examining Fos expression in these neurons in 4-week bile duct-ligated (BDL) cirrhotic and control sham-operated rats, in the basal state and following a cardiovascular stimulation of either volume depletion or infusion. We also studied a group of 5-day BDL rats with acute cholestasis but no cirrhosis, to delineate the effect of jaundice per se. MATERIALS AND METHODS Animals and Treatment Protocols. The protocol was approved by
the University of Calgary Faculty of Medicine Animal Care Committee, and all animals received humane care in accordance with guidelines established by the Canadian Council on Animal Care. Male Sprague-Dawley rats were anesthetized with inhalational halothane, and, through a midline abdominal incision, the bile ducts were
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ligated, as previously described.8 Controls were subjected to sham operation, which included only the abdominal incision and gentle manipulation, but not ligation of the bile duct. Following the operation, the rats were injected intramuscularly with 30,000 units of benzathine penicillin G. The animals also received 0.25 mg vitamin K1 subcutaneously immediately following the operation, and once weekly thereafter. The cirrhotic and its sham-operated control groups were studied 24 to 27 days after their operations, while the acutely jaundiced group was studied 5 days after BDL, and its separate control group 5 days after sham operation. For the protocol, the rats were anesthetized intraperitoneally with 50 mg/kg sodium pentobarbital (MTC Pharmaceuticals, Mississauga, ON). To avoid Fos expression caused by stress, rats that struggled excessively or squealed during the injection were removed from further study. The right femoral artery and vein were cannulated with PE-50 tubing (Becton Dickinson, Parsippany, NJ). Blood pressure and heart rate were recorded using a polygraph recorder (Gould 2400, Oxnard, CA), calibrated at each use with a pressure manometer. A total of nine separate groups of rats were used in this study (n Å 5 in each group). Four groups of rats, a cirrhotic (25-day BDL), cholestatic (5-day BDL), and their respective sham-operated controls were not subjected to any volume challenge; their femoral veins and arteries were cannulated, but they were neither hemorrhaged nor volume-infused. Three groups were subjected to a graded hemorrhage (a cirrhotic, a cholestatic, and a 25-day sham-operated control group). After a baseline blood pressure recording was obtained, a graded hemorrhage was induced by manually withdrawing 12 mL blood per kilogram of body weight (estimated 15% blood volume), at 2 mL/min through the femoral vein catheter. Two groups of rats, cirrhotic and 26-day sham-operated, received an infusion of isotonic saline (12 mL/kg body weight at 2 mL/min) into the femoral vein. For the hemorrhage and volume-infusion protocols, the rats were observed for 90 minutes after the volume manipulation, during which their blood pressure and heart rate were recorded immediately after treatment (3 minutes), and every 30 minutes thereafter. After 90 minutes, the rats were injected with an excess intravenous dose of 60 mg/kg sodium pentobarbital through the femoral catheter. This injection induced cessation of respiration, but the heart continued beating. The rats were then perfused transcardially with 1 L/kg body weight cold phosphate-buffered saline (PBS) at pH 7.2, followed by 1 L/kg cold, filtered, 4% paraformaldehyde in 0.1 mol/L phosphate buffer at pH 7.4. Preparation of the brainstem sections and Fos immunohistochemistry were performed as previously described.22 The brainstems were removed and postfixed in 4% paraformaldehyde overnight at 47C. The brainstems were then cryoprotected in 30% sucrose in 0.1 mol/L phosphate buffer overnight to 3 days at 47C. Serial 30-mm sections of brainstem were cut with a cryostat at 0197C and collected in PBS. Every fourth section was used for Fos immunohistochemical staining, and every other fourth section was used for preabsorption staining to control for antibody specificity. Fos Immunohistochemistry. Sections were processed for Fos immunohistochemical detection using rabbit anti-Fos polyclonal antisera, directed against residues 7 to 14 of human Fos (Oncogene Science, Manhasset, NY). Sections were incubated in a blocking serum, consisting of 1.5% normal goat serum (Vector Labs, Burlingame, CA), diluted in PBS containing 0.4% Triton X-100, for 1 hour at room temperature. The blocking serum was removed and the sections were incubated with the primary antibody, rabbit anti-Fos polyclonal antisera diluted 1:20,000 with the blocking serum, for 48 to 72 hours at 47C. For the preabsorption staining, 1:20,000 primary antibody was preabsorbed overnight (47C) with a 10-fold concentration (100 mg/mL Ab) of the Fos peptide, residues 7 to 14 (Oncogene Science), before being incubated with the brainstem sections. Sections were then washed with PBS for 10 minutes at room tempera-
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ture, then incubated with secondary antibody, biotinylated goat anti-rabbit immunoglobulin G (1:200, Vector) for 30 minutes at room temperature. After the sections were washed in PBS for 10 minutes, they were incubated in Vecastain Elite ABC Reagent (Vector) for 30 minutes at room temperature. The sections were then washed in PBS for 10 minutes and incubated in diaminobenzidinenickel peroxidase substrate (Vector) until the reaction was complete. The sections were then washed three times with PBS and mounted on chrome-alum (Sigma Chemicals, St. Louis, MO)– coated slides. The slides were left to air-dry overnight, and were then dehydrated and cover-slipped. The diaminobenzidine-nickel–stained Fos cells were identified by their size, shape, and black color, discernible from background stain. The brainstem regions were defined by the atlas of stereotaxic coordinates by Paxinos and Watson.23 Preliminary analysis of the sections with the largest degree of Fos staining was performed, after which, to standardize the counting procedures, Fos-immunostaining cells were counted at specific sections according to the stereotaxic coordinates: nucleus tractus solitarius (NTS) at interaural 05.30 mm, bregma 014.30 mm; ventrolateral medulla (VLM) and lateral reticular nucleus at interaural 05.08 mm, bregma 014.08 mm; inferior olivary nucleus at interaural 04.80 mm, bregma 013.80 mm. Counting was performed visually with a 1400 magnification light microscope. All slides were analyzed together, and at two separate times, to decrease the variability in counting error. The final value used was the mean of the two counts. There was always less than 10% discordance between the two counts. Statistical Analysis. All data are presented as means { SEM. Repeat-measures ANOVA was used to analyze blood pressures and heart rates within a group of animals. Nonparametric Kruskal-Wallis ANOVA with a post hoc Dunn’s test was used to compare differences between groups. Significance was set at P õ .05. RESULTS Hemodynamic Measurements. None of the unchallenged groups of rats displayed any significant change in mean arterial blood pressure or heart rate throughout the 90-minute experiment (data not shown). In the saline-infused groups, the arterial pressure initially dropped slightly in both sham and cirrhotic animals, but this did not reach statistical significance (Fig. 1A). Similarly, the heart rates in both the sham and cirrhotic rats dropped slightly, but showed no significant change throughout the 90 minutes (Fig. 1C). The mean arterial pressures in all three groups of hemorrhaged rats initially declined by a similar amount, and then gradually returned toward the baseline values (Fig. 1B). However, whereas the cholestatic and sham rats’ pressures increased by 30 minutes to a value not significantly different from the baseline, the cirrhotic rats’ pressures increased more slowly, and after 90 minutes, were still slightly but significantly decreased compared with the basal value. There was no statistically significant change in heart rate in any group of hemorrhaged rats (Fig. 1D). As previously documented in other studies, baseline heart rates and blood pressures were significantly lower in the cirrhotic compared with the sham groups (data not shown). Fos Expression. Fos immunoreactivity was found in various areas of the medulla, including, most prevalently, the NTS, the VLM, the inferior olivary nucleus, and the lateral reticular nucleus. The greatest staining was located, however, in the NTS and the VLM. Preabsorption controls were performed to control for primary antibody specificity to residues 7-14 of the Fos peptide. No Fos immunoreactivity was detected in these sections. Although Fos immunoreactivity was variably detected in the inferior olivary and the lateral reticular nuclei,
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), cirrhotic FIG. 1. Hemodynamic measurements in 25-day sham ( (- - -), and cholestatic (rrrr) rats. Data are represented as mean { SEM, n Å 5 for each group. Cardiovascular challenge begins at 0 minutes and is complete at 3 minutes. (A) Mean arterial pressure before and after saline infusion. (B) Mean arterial pressure before and after hemorrhage. Mean arterial pressures after hemorrhage are significantly different from baseline in all three groups, and then return to baseline except in the cirrhotic group. *Significantly different from cirrhotic, t Å 0 minutes (P õ .05). (C) Heart rate before and after saline infusion. (D) Heart rate before and after hemorrhage. There were no significant differences in heart rates before and after saline or hemorrhage.
there was no consistent trend in the counts in these areas for the different groups of animals. A representative photomicrograph of NTS staining in unchallenged 25-day sham and cirrhotic rats is shown in Fig. 2, and Fig. 3 depicts a schematic diagram of the NTS region of the brainstem. As noted in previous studies, Fos counts were low in the NTS of unchallenged 25-day sham rats (27.8 { 3.3), but increased significantly with saline infusion (95.2 { 8.5) or hemorrhage (86.0 { 3.7) (Fig. 4). Five-day sham and 5-day BDL (cholestatic) rats showed similar low NTS Fos staining, and this did not change in the cholestatic rats after hemorrhage. In the cirrhotic rats, however, there was a high basal level of Fos-immunoreactive cells in the NTS of unchallenged animals (154.6 { 27.0), significantly greater than the unchallenged sham animals (P õ .01). In the NTS, the number of Fos-immunoreactive cells did not significantly increase from this high basal expression following cardiovascular challenges of hemorrhage (198.4 { 17.9) or saline infusion (158.4 { 32.2). The VLM showed a similar pattern of Fos expression (Fig. 5). The unchallenged sham rats displayed the least Fos immunoreactivity in the VLM (30.6 { 2.7), and this compares with a value of 48.6 { 6.5 in the saline-infused shams (P Å .08), and 61.4 { 6.0 in the hemorrhaged shams (P õ .05).
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FIG. 2. Fos expression in the NTS region of (A) an unchallenged 25day sham-operated rat and (B) an unchallenged cirrhotic rat. (Original magnification 1125.) Darkly-stained oval or round cells indicate the Fos-immunoreactive neurons.
All three of the 5-day animal groups (the shams, cholestatic, and cholestatic-hemorrhaged) displayed low levels of VLM Fos staining. The unchallenged cirrhotic rats again showed a higher Fos activity (54.8 { 12.3 cells; P õ .05) than their unchallenged sham counterparts, and again it was clear that there was also absolutely no effect of volume manipulation in cirrhotic animals; hemorrhaged and saline-infused rats showed remarkably similar numbers of Fos-immunoreactive cells in the VLM. Because blood pressure homeostasis is thought to involve both the NTS and VLM, we thought it reasonable to calculate the total NTS / VLM fos activity to obtain an idea of the overall medullary cardiovascular neural activation (Fig. 6). Fos immunoreactivity was lowest in the sham control rats
FIG. 3. Schematic diagram of Fig. 2B, showing the NTS region (dashed line). CC Å central canal.
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FIG. 4. Number of Fos-like immunoreactive (FLI) cells in the NTS. Data are represented as mean { SEM, n Å 5 for each group. *Significantly different from its corresponding sham control (P õ .05).
(58.4 { 5.4), and significantly different from shams receiving a saline infusion (143.8 { 11.3) and hemorrhage challenge (147.4 { 7.5). The combined NTS / VLM Fos staining patterns for the cholestatic and cirrhotic rats again showed the same profile noted for the NTS and VLM individually; cholestatic rats had no baseline increased expression and no response to hemorrhage, whereas the cirrhotic rats had baseline increased counts, and also no changes with saline infusion or hemorrhage. DISCUSSION
Cardiovascular perturbations in cirrhosis have been extensively documented, and, in particular, evidence of abnormal regulation of parameters such as blood pressure, volume, and flow has been well reported. Because neural input, predominantly the sympathetic nervous system, is involved in this regulation, attention has been focused on this area. It has been assumed, based on indirect evidence, that the sympathetic system is activated in cirrhosis. For example, plasma catecholamine levels have been found to be increased in both patients and animal models with cirrhosis,10-12 and direct measurement of skeletal muscle sympathetic nerve activity in patients with cirrhosis has shown increased spike discharge
FIG. 5. Number of Fos-like immunoreactive (FLI) cells in the ventrolateral medulla. Data are represented as mean { SEM, n Å 5 for each group. *Significantly different from its corresponding sham control (P õ .05).
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FIG. 6. Number of Fos-like immunoreactive (FLI) cells in the medullary cardiovascular centers (NTS / VLM). Data are represented as mean { SEM, n Å 5 for each group. BDL Å bile duct ligated. *Significantly different from its corresponding sham control (P õ .05).
activity, suggestive of increased central sympathetic trafficking.14 In the present study, we directly document for the first time clearcut evidence of central neuronal activation, using Fos expression in the medullary cardiovascular control centers. The NTS receives direct vagal sensory innervation, and bidirectional pathways exist between the NTS and the rostral and caudal VLM.24,25 Accordingly, it is thought that these two areas of the medulla are dominantly involved in regulation of blood pressure homeostasis.25,26 The present results therefore suggest that the manifestations of increased peripheral sympathetic activity are directly caused by increased central neuronal activity, rather than other proposed factors such as decreased extraction of norepinephrine from the neurovascular junction27 and other peripheral nervous abnormalities. The increases in both the NTS and VLM Fos staining with cardiovascular challenge in the sham-operated rats agrees with values previously reported in the literature.18-20,28,32 As another ‘‘internal control,’’ the lack of significant change in the inferior olivary or lateral reticular nuclei in the controls of cirrhotic rats is reassuring, because neither of these areas is thought to be important in blood pressure/volume homeostasis.25,26 Moreover, several previous studies have also found that acute hemorrhage activates Fos immunoreactivity in the NTS and VLM, and generally not other medullary nuclei.18-20,28 We were surprised by the marked elevation in Fos activation in the unchallenged cirrhotic rats. Because c-fos is an early-response gene, one might have expected that chronic long-standing sympathetic activation would lead to downregulation of baseline Fos expression, with a ‘‘resetting’’ of the central cardiovascular control centers. This is clearly not the case, and the reasons for this remain obscure. Interestingly, a similar lack of down-regulation was noted with c-fos staining in the brainstem of chronically salt-loaded (and thus, presumably chronically volume-overloaded) rats.28 Whatever the mechanism, the Fos overexpression in our unchallenged cirrhotic rats is very much in keeping with the chronically activated sympathetic system, with high plasma catecholamines, and evidence of end-organ ‘‘overdrive’’ with peripheral vasodilatation, increased heart rate, and cardiac output. The peculiar pattern of the Fos expression with baseline increased staining, and complete failure to show further acti-
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vation with severe cardiovascular stimulation, generally agrees with the pattern of cirrhotic cardiovascular physiological phenomena previously reported. For example, cirrhotic cardiomyopathy is also manifested by increased baseline cardiac output and blunted contractile responsiveness to stimuli.4 A similar pattern has been extensively documented, with attenuation of cardiovascular reflexes in patient and animal models with liver disease (reviewed in Thuluvath29 and Lee30). Indeed, the present results of completely blunted neuronal responsiveness are strikingly similar to the results of peroneal nerve sympathetic activity measured by microneurography, in patients with decompensated cirhosis.14 In that study, baseline tonic nervous electrical activity was significantly increased in cirrhotic patients, but, whereas a Valsalva maneuver induced a significant increase in the nervous electrical activity in normal subjects, in cirrhotic patients, a completely blunted response was observed.14 Up to now, it had been assumed that such hyporesponsiveness was mediated by peripheral humoral factors or peripheral end-organ failure, i.e., defects in the blood vessel per se, but the present results are the first clear demonstration of what had long been suspected: central cardiovascular regulatory mechanisms are also disordered. Why the cirrhotic rats failed to exhibit any neuronal activation to either volume depletion or infusion remains unclear. One might question whether the magnitude of the cardiovascular challenges were equivalent in the cirrhotic and its sham-operated control groups, because plasma volume is known to be augmented in cirrhosis. Accordingly, it may have been possible that normalizing the volume hemorrhaged or infused by means of body weight produced an alteration of a proportionately smaller ratio of total blood volume in the cirrhotic rats, and thus not enough to activate central neuronal cardiovascular pathways. However, we think this possibility is extremely unlikely because changes in blood pressure, especially with hemorrhage, were dramatic and of generally similar magnitudes in the cirrhotic and control rats. Indeed, if anything, the hemorrhagic stimulus may have been greater in the cirrhotic rats, because, even at 90 minutes, the blood pressure had not returned to baseline values in this group, whereas, in the sham-operated and cholestatic rats, the pressures had been restored. A blunting of neuronal activation in the hypothalamic region has recently been reported by Swain and Maric in response to restraint stress in the 5-day BDL cholestatic rat.31 Our results showing that this acutely cholestatic model has no baseline increase in NTS and VLM Fos staining, and no discernible neuronal response to a hemorrhagic stimulus, agrees remarkably well with their data. Therefore, these results suggest that at least part of the blunted neuronal responsiveness may be caused by the presence of jaundice per se. We also cannot definitively rule out a confounding effect of pentobarbital anesthesia on the results. The anesthetic induced some baseline depression of heart rate and arterial pressure, as reported previously.32 Moreover, the lack of significant heart rate changes with volume manipulation indicates blunting of baroreflex activity, even in the control sham-operated rats, and the pentobarbital may be responsible for this. However, the protocol involved a large volume depletion or infusion over a short period, and we felt that attempting this degree of volume manipulation in a conscious animal would result in severe stress, which is known
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to dramatically activate brainstem Fos staining.28,31,33 Accordingly, the use of a general anesthetic, while making the interpretation of the results more complicated, was felt to be unavoidable. Significant stress just at the time of pentobarbital injection might also have affected the results. We tried to avoid this by eliminating from further study any rats that had struggled or squealed during the injection. The general lack of Fos activation in the control rats not subjected to any volume manipulation would compellingly argue that we were indeed successful in avoiding major stress. In conclusion, central medullary nuclei involved in cardiovascular regulation of cirrhotic rats showed neuronal activation, but volume manipulation failed to induce any further activation. Rats with acute cholestasis but not cirrhosis also had blunted responses, suggesting that at least part of the central hyporesponsiveness may be related to jaundice. These results for the first time confirm a prominent central role of disordered cardiovascular regulation in chronic liver disease. Acknowledgment: We are grateful to Lorraine Oland for helpful advice on the Fos assay; Yikun Zhang for technical assistance; and Dr. Stefan Urbanski for taking the photomicrograph of the brainstem section. REFERENCES 1. Abelmann WH, Kowalski HJ, McNeely WF. The hemodynamic response to exercise in patients with Laennec’s cirrhosis. J Clin Invest 1955;34: 690-695. 2. Kontos HA, Shapiro W, Mauck HP, Patterson JP. General and regional circulatory alterations in cirrhosis of the liver. Am J Med 1964;37:526535. 3. Abelmann WH. Hyperdynamic circulation in cirrhosis: a historical perspective. HEPATOLOGY 1994;20:1356-1358. 4. Ma Z, Lee SS. Cirrhotic cardiomyopathy: getting to the heart of the matter. HEPATOLOGY 1996;24:451-459. 5. Bomzon A, Blendis LM. The nitric oxide hypothesis and the hyperdynamic circulation in cirrhosis. HEPATOLOGY 1994;20:1343-1350. 6. Benoit JN, Barrowman JA, Harper SL, Kvietys PR, Granger DN. Role of humoral factors in the intestinal hyperemia associated with chronic portal hypertension. Am J Physiol 1984;247:G486-G493. 7. Pak JM, Lee SS. Glucagon in portal hypertension. J Hepatol 1994;20: 825-832. 8. Pak JM, Lee SS. Vasoactive effects of bile salts in cirrhotic rats: in vivo and in vitro studies. HEPATOLOGY 1993;18:1175-1181. 9. Sitzmann JV, Li SS, Adkinson NF. Evidence for a role of prostacyclin as a systemic hormone in portal hypertension. Surgery 1991;109:149153. 10. Henriksen JH, Ring-Larsen H, Kanstrup IL, Christensen NJ. Splanchnic and renal elimination and release of catecholamines in cirrhosis. Evidence of enhanced sympathetic nervous activity in patients with decompensated cirrhosis. Gut 1984;25:1034-1043. 11. Willet I, Esler M, Burke F, Leonard P, Dudley F. Total and renal sympathetic nervous system activity in alcoholic cirrhosis. J Hepatol 1985;1: 639-648. 12. Gaudin C, Ruget G, Braillon A, Selz F, Cuche JL, Lebrec D. Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats. Life Sci 1989;45:1333-1339. 13. Moreau R, Lee SS, Hadengue A, Braillon A, Lebrec D. Hemodynamic effects of a clonidine-induced decrease in sympathetic tone in patients with cirrhosis. HEPATOLOGY 1987;7:149-154. 14. Floras JS, Legault L, Morali GA, Hara K, Blendis LM. Increased sympathetic outflow in cirrhosis and ascites: direct evidence from intraneural recordings. Ann Intern Med 1991;114:373-380. 15. Morgan JL, Curran T. Role of ion flux in the control of c-fos expression. Nature 1986;22:552-555. 16. Sheng M, Greenberg ME. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 1990;4:477-485. 17. Dragunow M, Faull R. The use of c-fos as a metabolic marker in neuronal pathway tracing. J Neurosci Methods 1989;29:261-265. 18. Erickson JT, Millhorn DE. Fos-like protein is induced in neurons of
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the medulla oblongata after stimulation of the carotid sinus nerve in awake anesthetized rats. Brain Res 1991;567:11-24. Dun NJ, Dun SL, Chiaia NL. Hemorrhage induces Fos immunoreactivity in rat medullary catecholaminergic neurons. Brain Res 1993;608:223232. Morgan JL, Curran T. Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci 1989;12:459-462. Murphy AZ, Ennis M, Shipley MT, Behbehani MM. Directionally specific changes in arterial pressure induce differential patterns of fos expression in discrete areas of the rat brainstem: a double-labeling study for fos and catecholamines. J Comp Neurol 1994;349:36-50. Fraser KA, Davison JS. Meal-induced c-fos expression in brainstem is not dependent on cholecystokinin release. Am J Physiol 1993;265: R235-R239. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd ed. San Diego: Academic Press, 1986. Sumal KK, Blessing WW, Joh TH, Reis DJ, Pickel VM. Synaptic interactions of vagal afferents and catecholaminergic neurons in the rat nucleus solitarius. Brain Res 1983;277:31-40. Spyer KM. The central nervous organization of reflex circulatory control. In: Loewy AD, Spyer KM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press, 1990:168-188. Guyenet PG. The role of the ventral medulla oblongata in blood pressure
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regulation. In: Loewy AD, Spyer KM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press, 1990:129-149. Westfall TC, Meldrum MJ. Alterations in release of norepinephrine at the vascular neuroeffector junction in hypertension. Annu Rev Pharmacol Toxicol 1985;25:621-641. Chan RKW, Brown ER, Ericsson A, Kovacs KJ, Sawchenko PE. A comparison of two immediate-early genes, c-fos and NGFI-B, as markers for functional activation in stress-related neuroendocrine circuitry. J Neurosci 1993;13:5126-5138. Thuluvath PJ, Triger DR. Autonomic neuropathy and chronic liver disease. Q J Med 1989;72:737-747. Lee SS. Cardiac abnormalities in liver cirrhosis. West J Med 1989;151: 530-535. Swain MG, Maric M. Impaired stress and interleukin-1b induced hypothalamic expression of the neuronal activation marker FOS in cholestatic rats. HEPATOLOGY 1996;24:914-918. Lee SS, Hadengue A, Girod C, Braillon A, Lebrec D. Hemodynamic characterization of bile duct ligated cirrhotic rats: effects of pentobarbital sodium. Am J Physiol 1986;251:G176-G180. Umemoto S, Noguchi K, Kawai Y, Senba E. Repeated stress reduces the subsequent stress-induced expression of Fos in rat brain. Neurosci Lett 1994;167:101-104.
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Cardiovascular function in cirrhosis is deranged, with indirect evidence of abnormal central cardiovascular regulation. We aimed to elucidate the role of brainstem cardiovascular nuclei in hemodynamic regulation by examining the protein product, Fos, of the immediate-early gene c-fos, in cirrhotic rats. Cirrhosis was induced by chronic bile duct ligation (BDL) of 25-days duration, while controls underwent a sham operation. To examine the effects of jaundice per se in the absence of cirrhosis, a third group of 5-day BDL rats was also studied. All rats were anesthetized with pentobarbital, and catheters were inserted to measure baseline blood pressure and heart rate. Separate groups were then subjected to volume manipulation by a hypotensive hemorrhage or isotonic saline infusion, or no challenge. Ninety minutes after the volume manipulation, the animals were killed and the medulla sectioned and stained for Fos by immunohistochemisty. The nucleus tractus solitarius (NTS) of the sham-operated unchallenged rats showed scant Fos immunoreactivity (27.8 { 3.3 cells), but both hemorrhage and volume infusion significantly increased Fos staining (86.0 { 3.7 and 95.2 { 8.5, respectively). In contrast, the unchallenged cirrhotic rats showed markedly increased Fos in the NTS (154.6 { 27.0), but neither hemorrhage nor volume infusion significantly changed the amount of Fos staining. Fos staining in the ventrolateral medulla (VLM) followed a similar pattern with low staining in the unchallenged sham rats and increased staining in the other groups, but no differences between the unchallenged and the volume-manipulated cirrhotic groups. The 5-day BDL jaundiced rats showed no baseline increase in Fos staining, nor any significant increase after hemorrhage. These results showing baseline activation of central neuronal regions responsible for blood pressure homeostasis, but completely blunted responsiveness in cirrhotic rats, confirm a central origin of disordered cardiovascular regulation. The presence of jaundice may also contribute to the central cardiovascular hyporesponsiveness. (HEPATOLOGY 1997;26:1380-1385.)
Abbreviations: BDL, bile duct–ligated; PBS, phosphate-buffered saline; NTS, nucleus tractus solitarius; VLM, ventrolateral medulla. From the Liver Unit, Gastroenterology Research Group, University of Calgary, Calgary, Alberta, Canada. Received October 23, 1996; accepted May 12, 1997. Funded by an operating research grant from the Medical Research Council of Canada. Ms. Breitman was supported by an Alberta Heritage Foundation for Medical Research (AHFMR) Summer Studentship award. Dr. Lee was supported by an AHFMR Scholarship award. Address reprint requests to: S. S. Lee, M.D., 3330 Hospital Dr NW, Calgary, AB, T2N 4N1, Canada. Fax: (403) 270-0995. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2606-0002$3.00/0
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SAMUEL S. LEE
Cirrhosis is associated with abnormal cardiovascular phenomena. These include the development of hyperdynamic circulation and impairment of cardiac contractility.1-4 Hyperdynamic circulation is manifested by elevated cardiac output, increased blood flow, and lowered peripheral vascular resistance and arterial blood pressure.1-3 However the precise causes and mechanisms of this hyperdynamic circulation remain unclear. Humoral factors, such as nitric oxide, glucagon, bile salts, and prostaglandins have been proposed, but the results to date have proved inconclusive.5-9 Neural factors have also been implicated. Sympathetic nervous activity has been shown to be enhanced in cirrhosis,10-14 although the mechanism by which sympathetic activation leads to hyperdynamic circulation, and the level at which these alterations arise, remains to be elucidated. Although indirect evidence has suggested that central sympathetic tone is increased,10-14 to date, this hypothesis has not been directly tested. C-fos is an immediate-early gene that is rapidly and transiently expressed within cells in the central nervous system as a result of membrane depolarization and voltage-gated calcium influx.15,16 Its protein product, Fos, can be detected by immunohistochemistry. Basal levels of Fos immunoreactivity are known to be low,15 and the presence of Fos immunoreactivity within neurons is therefore indicative of activation of these cells. Fos has been extensively used and validated as a sensitive marker for identifying and characterizing neuronal activation as a response to stimulation by external factors.17 More particularly, Fos staining has been used to identify and characterize neuronal activation and pathways in the brainstem in response to cardiovascular challenge.17-21 We therefore hypothesized that central regulatory mechanisms are indeed disordered, and that this would be manifested by neuronal activation in the brainstem cardiovascular control centers. Accordingly, we aimed to test this notion by examining Fos expression in these neurons in 4-week bile duct-ligated (BDL) cirrhotic and control sham-operated rats, in the basal state and following a cardiovascular stimulation of either volume depletion or infusion. We also studied a group of 5-day BDL rats with acute cholestasis but no cirrhosis, to delineate the effect of jaundice per se. MATERIALS AND METHODS Animals and Treatment Protocols. The protocol was approved by
the University of Calgary Faculty of Medicine Animal Care Committee, and all animals received humane care in accordance with guidelines established by the Canadian Council on Animal Care. Male Sprague-Dawley rats were anesthetized with inhalational halothane, and, through a midline abdominal incision, the bile ducts were
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ligated, as previously described.8 Controls were subjected to sham operation, which included only the abdominal incision and gentle manipulation, but not ligation of the bile duct. Following the operation, the rats were injected intramuscularly with 30,000 units of benzathine penicillin G. The animals also received 0.25 mg vitamin K1 subcutaneously immediately following the operation, and once weekly thereafter. The cirrhotic and its sham-operated control groups were studied 24 to 27 days after their operations, while the acutely jaundiced group was studied 5 days after BDL, and its separate control group 5 days after sham operation. For the protocol, the rats were anesthetized intraperitoneally with 50 mg/kg sodium pentobarbital (MTC Pharmaceuticals, Mississauga, ON). To avoid Fos expression caused by stress, rats that struggled excessively or squealed during the injection were removed from further study. The right femoral artery and vein were cannulated with PE-50 tubing (Becton Dickinson, Parsippany, NJ). Blood pressure and heart rate were recorded using a polygraph recorder (Gould 2400, Oxnard, CA), calibrated at each use with a pressure manometer. A total of nine separate groups of rats were used in this study (n Å 5 in each group). Four groups of rats, a cirrhotic (25-day BDL), cholestatic (5-day BDL), and their respective sham-operated controls were not subjected to any volume challenge; their femoral veins and arteries were cannulated, but they were neither hemorrhaged nor volume-infused. Three groups were subjected to a graded hemorrhage (a cirrhotic, a cholestatic, and a 25-day sham-operated control group). After a baseline blood pressure recording was obtained, a graded hemorrhage was induced by manually withdrawing 12 mL blood per kilogram of body weight (estimated 15% blood volume), at 2 mL/min through the femoral vein catheter. Two groups of rats, cirrhotic and 26-day sham-operated, received an infusion of isotonic saline (12 mL/kg body weight at 2 mL/min) into the femoral vein. For the hemorrhage and volume-infusion protocols, the rats were observed for 90 minutes after the volume manipulation, during which their blood pressure and heart rate were recorded immediately after treatment (3 minutes), and every 30 minutes thereafter. After 90 minutes, the rats were injected with an excess intravenous dose of 60 mg/kg sodium pentobarbital through the femoral catheter. This injection induced cessation of respiration, but the heart continued beating. The rats were then perfused transcardially with 1 L/kg body weight cold phosphate-buffered saline (PBS) at pH 7.2, followed by 1 L/kg cold, filtered, 4% paraformaldehyde in 0.1 mol/L phosphate buffer at pH 7.4. Preparation of the brainstem sections and Fos immunohistochemistry were performed as previously described.22 The brainstems were removed and postfixed in 4% paraformaldehyde overnight at 47C. The brainstems were then cryoprotected in 30% sucrose in 0.1 mol/L phosphate buffer overnight to 3 days at 47C. Serial 30-mm sections of brainstem were cut with a cryostat at 0197C and collected in PBS. Every fourth section was used for Fos immunohistochemical staining, and every other fourth section was used for preabsorption staining to control for antibody specificity. Fos Immunohistochemistry. Sections were processed for Fos immunohistochemical detection using rabbit anti-Fos polyclonal antisera, directed against residues 7 to 14 of human Fos (Oncogene Science, Manhasset, NY). Sections were incubated in a blocking serum, consisting of 1.5% normal goat serum (Vector Labs, Burlingame, CA), diluted in PBS containing 0.4% Triton X-100, for 1 hour at room temperature. The blocking serum was removed and the sections were incubated with the primary antibody, rabbit anti-Fos polyclonal antisera diluted 1:20,000 with the blocking serum, for 48 to 72 hours at 47C. For the preabsorption staining, 1:20,000 primary antibody was preabsorbed overnight (47C) with a 10-fold concentration (100 mg/mL Ab) of the Fos peptide, residues 7 to 14 (Oncogene Science), before being incubated with the brainstem sections. Sections were then washed with PBS for 10 minutes at room tempera-
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ture, then incubated with secondary antibody, biotinylated goat anti-rabbit immunoglobulin G (1:200, Vector) for 30 minutes at room temperature. After the sections were washed in PBS for 10 minutes, they were incubated in Vecastain Elite ABC Reagent (Vector) for 30 minutes at room temperature. The sections were then washed in PBS for 10 minutes and incubated in diaminobenzidinenickel peroxidase substrate (Vector) until the reaction was complete. The sections were then washed three times with PBS and mounted on chrome-alum (Sigma Chemicals, St. Louis, MO)– coated slides. The slides were left to air-dry overnight, and were then dehydrated and cover-slipped. The diaminobenzidine-nickel–stained Fos cells were identified by their size, shape, and black color, discernible from background stain. The brainstem regions were defined by the atlas of stereotaxic coordinates by Paxinos and Watson.23 Preliminary analysis of the sections with the largest degree of Fos staining was performed, after which, to standardize the counting procedures, Fos-immunostaining cells were counted at specific sections according to the stereotaxic coordinates: nucleus tractus solitarius (NTS) at interaural 05.30 mm, bregma 014.30 mm; ventrolateral medulla (VLM) and lateral reticular nucleus at interaural 05.08 mm, bregma 014.08 mm; inferior olivary nucleus at interaural 04.80 mm, bregma 013.80 mm. Counting was performed visually with a 1400 magnification light microscope. All slides were analyzed together, and at two separate times, to decrease the variability in counting error. The final value used was the mean of the two counts. There was always less than 10% discordance between the two counts. Statistical Analysis. All data are presented as means { SEM. Repeat-measures ANOVA was used to analyze blood pressures and heart rates within a group of animals. Nonparametric Kruskal-Wallis ANOVA with a post hoc Dunn’s test was used to compare differences between groups. Significance was set at P õ .05. RESULTS Hemodynamic Measurements. None of the unchallenged groups of rats displayed any significant change in mean arterial blood pressure or heart rate throughout the 90-minute experiment (data not shown). In the saline-infused groups, the arterial pressure initially dropped slightly in both sham and cirrhotic animals, but this did not reach statistical significance (Fig. 1A). Similarly, the heart rates in both the sham and cirrhotic rats dropped slightly, but showed no significant change throughout the 90 minutes (Fig. 1C). The mean arterial pressures in all three groups of hemorrhaged rats initially declined by a similar amount, and then gradually returned toward the baseline values (Fig. 1B). However, whereas the cholestatic and sham rats’ pressures increased by 30 minutes to a value not significantly different from the baseline, the cirrhotic rats’ pressures increased more slowly, and after 90 minutes, were still slightly but significantly decreased compared with the basal value. There was no statistically significant change in heart rate in any group of hemorrhaged rats (Fig. 1D). As previously documented in other studies, baseline heart rates and blood pressures were significantly lower in the cirrhotic compared with the sham groups (data not shown). Fos Expression. Fos immunoreactivity was found in various areas of the medulla, including, most prevalently, the NTS, the VLM, the inferior olivary nucleus, and the lateral reticular nucleus. The greatest staining was located, however, in the NTS and the VLM. Preabsorption controls were performed to control for primary antibody specificity to residues 7-14 of the Fos peptide. No Fos immunoreactivity was detected in these sections. Although Fos immunoreactivity was variably detected in the inferior olivary and the lateral reticular nuclei,
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), cirrhotic FIG. 1. Hemodynamic measurements in 25-day sham ( (- - -), and cholestatic (rrrr) rats. Data are represented as mean { SEM, n Å 5 for each group. Cardiovascular challenge begins at 0 minutes and is complete at 3 minutes. (A) Mean arterial pressure before and after saline infusion. (B) Mean arterial pressure before and after hemorrhage. Mean arterial pressures after hemorrhage are significantly different from baseline in all three groups, and then return to baseline except in the cirrhotic group. *Significantly different from cirrhotic, t Å 0 minutes (P õ .05). (C) Heart rate before and after saline infusion. (D) Heart rate before and after hemorrhage. There were no significant differences in heart rates before and after saline or hemorrhage.
there was no consistent trend in the counts in these areas for the different groups of animals. A representative photomicrograph of NTS staining in unchallenged 25-day sham and cirrhotic rats is shown in Fig. 2, and Fig. 3 depicts a schematic diagram of the NTS region of the brainstem. As noted in previous studies, Fos counts were low in the NTS of unchallenged 25-day sham rats (27.8 { 3.3), but increased significantly with saline infusion (95.2 { 8.5) or hemorrhage (86.0 { 3.7) (Fig. 4). Five-day sham and 5-day BDL (cholestatic) rats showed similar low NTS Fos staining, and this did not change in the cholestatic rats after hemorrhage. In the cirrhotic rats, however, there was a high basal level of Fos-immunoreactive cells in the NTS of unchallenged animals (154.6 { 27.0), significantly greater than the unchallenged sham animals (P õ .01). In the NTS, the number of Fos-immunoreactive cells did not significantly increase from this high basal expression following cardiovascular challenges of hemorrhage (198.4 { 17.9) or saline infusion (158.4 { 32.2). The VLM showed a similar pattern of Fos expression (Fig. 5). The unchallenged sham rats displayed the least Fos immunoreactivity in the VLM (30.6 { 2.7), and this compares with a value of 48.6 { 6.5 in the saline-infused shams (P Å .08), and 61.4 { 6.0 in the hemorrhaged shams (P õ .05).
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FIG. 2. Fos expression in the NTS region of (A) an unchallenged 25day sham-operated rat and (B) an unchallenged cirrhotic rat. (Original magnification 1125.) Darkly-stained oval or round cells indicate the Fos-immunoreactive neurons.
All three of the 5-day animal groups (the shams, cholestatic, and cholestatic-hemorrhaged) displayed low levels of VLM Fos staining. The unchallenged cirrhotic rats again showed a higher Fos activity (54.8 { 12.3 cells; P õ .05) than their unchallenged sham counterparts, and again it was clear that there was also absolutely no effect of volume manipulation in cirrhotic animals; hemorrhaged and saline-infused rats showed remarkably similar numbers of Fos-immunoreactive cells in the VLM. Because blood pressure homeostasis is thought to involve both the NTS and VLM, we thought it reasonable to calculate the total NTS / VLM fos activity to obtain an idea of the overall medullary cardiovascular neural activation (Fig. 6). Fos immunoreactivity was lowest in the sham control rats
FIG. 3. Schematic diagram of Fig. 2B, showing the NTS region (dashed line). CC Å central canal.
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FIG. 4. Number of Fos-like immunoreactive (FLI) cells in the NTS. Data are represented as mean { SEM, n Å 5 for each group. *Significantly different from its corresponding sham control (P õ .05).
(58.4 { 5.4), and significantly different from shams receiving a saline infusion (143.8 { 11.3) and hemorrhage challenge (147.4 { 7.5). The combined NTS / VLM Fos staining patterns for the cholestatic and cirrhotic rats again showed the same profile noted for the NTS and VLM individually; cholestatic rats had no baseline increased expression and no response to hemorrhage, whereas the cirrhotic rats had baseline increased counts, and also no changes with saline infusion or hemorrhage. DISCUSSION
Cardiovascular perturbations in cirrhosis have been extensively documented, and, in particular, evidence of abnormal regulation of parameters such as blood pressure, volume, and flow has been well reported. Because neural input, predominantly the sympathetic nervous system, is involved in this regulation, attention has been focused on this area. It has been assumed, based on indirect evidence, that the sympathetic system is activated in cirrhosis. For example, plasma catecholamine levels have been found to be increased in both patients and animal models with cirrhosis,10-12 and direct measurement of skeletal muscle sympathetic nerve activity in patients with cirrhosis has shown increased spike discharge
FIG. 5. Number of Fos-like immunoreactive (FLI) cells in the ventrolateral medulla. Data are represented as mean { SEM, n Å 5 for each group. *Significantly different from its corresponding sham control (P õ .05).
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FIG. 6. Number of Fos-like immunoreactive (FLI) cells in the medullary cardiovascular centers (NTS / VLM). Data are represented as mean { SEM, n Å 5 for each group. BDL Å bile duct ligated. *Significantly different from its corresponding sham control (P õ .05).
activity, suggestive of increased central sympathetic trafficking.14 In the present study, we directly document for the first time clearcut evidence of central neuronal activation, using Fos expression in the medullary cardiovascular control centers. The NTS receives direct vagal sensory innervation, and bidirectional pathways exist between the NTS and the rostral and caudal VLM.24,25 Accordingly, it is thought that these two areas of the medulla are dominantly involved in regulation of blood pressure homeostasis.25,26 The present results therefore suggest that the manifestations of increased peripheral sympathetic activity are directly caused by increased central neuronal activity, rather than other proposed factors such as decreased extraction of norepinephrine from the neurovascular junction27 and other peripheral nervous abnormalities. The increases in both the NTS and VLM Fos staining with cardiovascular challenge in the sham-operated rats agrees with values previously reported in the literature.18-20,28,32 As another ‘‘internal control,’’ the lack of significant change in the inferior olivary or lateral reticular nuclei in the controls of cirrhotic rats is reassuring, because neither of these areas is thought to be important in blood pressure/volume homeostasis.25,26 Moreover, several previous studies have also found that acute hemorrhage activates Fos immunoreactivity in the NTS and VLM, and generally not other medullary nuclei.18-20,28 We were surprised by the marked elevation in Fos activation in the unchallenged cirrhotic rats. Because c-fos is an early-response gene, one might have expected that chronic long-standing sympathetic activation would lead to downregulation of baseline Fos expression, with a ‘‘resetting’’ of the central cardiovascular control centers. This is clearly not the case, and the reasons for this remain obscure. Interestingly, a similar lack of down-regulation was noted with c-fos staining in the brainstem of chronically salt-loaded (and thus, presumably chronically volume-overloaded) rats.28 Whatever the mechanism, the Fos overexpression in our unchallenged cirrhotic rats is very much in keeping with the chronically activated sympathetic system, with high plasma catecholamines, and evidence of end-organ ‘‘overdrive’’ with peripheral vasodilatation, increased heart rate, and cardiac output. The peculiar pattern of the Fos expression with baseline increased staining, and complete failure to show further acti-
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vation with severe cardiovascular stimulation, generally agrees with the pattern of cirrhotic cardiovascular physiological phenomena previously reported. For example, cirrhotic cardiomyopathy is also manifested by increased baseline cardiac output and blunted contractile responsiveness to stimuli.4 A similar pattern has been extensively documented, with attenuation of cardiovascular reflexes in patient and animal models with liver disease (reviewed in Thuluvath29 and Lee30). Indeed, the present results of completely blunted neuronal responsiveness are strikingly similar to the results of peroneal nerve sympathetic activity measured by microneurography, in patients with decompensated cirhosis.14 In that study, baseline tonic nervous electrical activity was significantly increased in cirrhotic patients, but, whereas a Valsalva maneuver induced a significant increase in the nervous electrical activity in normal subjects, in cirrhotic patients, a completely blunted response was observed.14 Up to now, it had been assumed that such hyporesponsiveness was mediated by peripheral humoral factors or peripheral end-organ failure, i.e., defects in the blood vessel per se, but the present results are the first clear demonstration of what had long been suspected: central cardiovascular regulatory mechanisms are also disordered. Why the cirrhotic rats failed to exhibit any neuronal activation to either volume depletion or infusion remains unclear. One might question whether the magnitude of the cardiovascular challenges were equivalent in the cirrhotic and its sham-operated control groups, because plasma volume is known to be augmented in cirrhosis. Accordingly, it may have been possible that normalizing the volume hemorrhaged or infused by means of body weight produced an alteration of a proportionately smaller ratio of total blood volume in the cirrhotic rats, and thus not enough to activate central neuronal cardiovascular pathways. However, we think this possibility is extremely unlikely because changes in blood pressure, especially with hemorrhage, were dramatic and of generally similar magnitudes in the cirrhotic and control rats. Indeed, if anything, the hemorrhagic stimulus may have been greater in the cirrhotic rats, because, even at 90 minutes, the blood pressure had not returned to baseline values in this group, whereas, in the sham-operated and cholestatic rats, the pressures had been restored. A blunting of neuronal activation in the hypothalamic region has recently been reported by Swain and Maric in response to restraint stress in the 5-day BDL cholestatic rat.31 Our results showing that this acutely cholestatic model has no baseline increase in NTS and VLM Fos staining, and no discernible neuronal response to a hemorrhagic stimulus, agrees remarkably well with their data. Therefore, these results suggest that at least part of the blunted neuronal responsiveness may be caused by the presence of jaundice per se. We also cannot definitively rule out a confounding effect of pentobarbital anesthesia on the results. The anesthetic induced some baseline depression of heart rate and arterial pressure, as reported previously.32 Moreover, the lack of significant heart rate changes with volume manipulation indicates blunting of baroreflex activity, even in the control sham-operated rats, and the pentobarbital may be responsible for this. However, the protocol involved a large volume depletion or infusion over a short period, and we felt that attempting this degree of volume manipulation in a conscious animal would result in severe stress, which is known
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to dramatically activate brainstem Fos staining.28,31,33 Accordingly, the use of a general anesthetic, while making the interpretation of the results more complicated, was felt to be unavoidable. Significant stress just at the time of pentobarbital injection might also have affected the results. We tried to avoid this by eliminating from further study any rats that had struggled or squealed during the injection. The general lack of Fos activation in the control rats not subjected to any volume manipulation would compellingly argue that we were indeed successful in avoiding major stress. In conclusion, central medullary nuclei involved in cardiovascular regulation of cirrhotic rats showed neuronal activation, but volume manipulation failed to induce any further activation. Rats with acute cholestasis but not cirrhosis also had blunted responses, suggesting that at least part of the central hyporesponsiveness may be related to jaundice. These results for the first time confirm a prominent central role of disordered cardiovascular regulation in chronic liver disease. Acknowledgment: We are grateful to Lorraine Oland for helpful advice on the Fos assay; Yikun Zhang for technical assistance; and Dr. Stefan Urbanski for taking the photomicrograph of the brainstem section. REFERENCES 1. Abelmann WH, Kowalski HJ, McNeely WF. The hemodynamic response to exercise in patients with Laennec’s cirrhosis. J Clin Invest 1955;34: 690-695. 2. Kontos HA, Shapiro W, Mauck HP, Patterson JP. General and regional circulatory alterations in cirrhosis of the liver. Am J Med 1964;37:526535. 3. Abelmann WH. Hyperdynamic circulation in cirrhosis: a historical perspective. HEPATOLOGY 1994;20:1356-1358. 4. Ma Z, Lee SS. Cirrhotic cardiomyopathy: getting to the heart of the matter. HEPATOLOGY 1996;24:451-459. 5. Bomzon A, Blendis LM. The nitric oxide hypothesis and the hyperdynamic circulation in cirrhosis. HEPATOLOGY 1994;20:1343-1350. 6. Benoit JN, Barrowman JA, Harper SL, Kvietys PR, Granger DN. Role of humoral factors in the intestinal hyperemia associated with chronic portal hypertension. Am J Physiol 1984;247:G486-G493. 7. Pak JM, Lee SS. Glucagon in portal hypertension. J Hepatol 1994;20: 825-832. 8. Pak JM, Lee SS. Vasoactive effects of bile salts in cirrhotic rats: in vivo and in vitro studies. HEPATOLOGY 1993;18:1175-1181. 9. Sitzmann JV, Li SS, Adkinson NF. Evidence for a role of prostacyclin as a systemic hormone in portal hypertension. Surgery 1991;109:149153. 10. Henriksen JH, Ring-Larsen H, Kanstrup IL, Christensen NJ. Splanchnic and renal elimination and release of catecholamines in cirrhosis. Evidence of enhanced sympathetic nervous activity in patients with decompensated cirrhosis. Gut 1984;25:1034-1043. 11. Willet I, Esler M, Burke F, Leonard P, Dudley F. Total and renal sympathetic nervous system activity in alcoholic cirrhosis. J Hepatol 1985;1: 639-648. 12. Gaudin C, Ruget G, Braillon A, Selz F, Cuche JL, Lebrec D. Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats. Life Sci 1989;45:1333-1339. 13. Moreau R, Lee SS, Hadengue A, Braillon A, Lebrec D. Hemodynamic effects of a clonidine-induced decrease in sympathetic tone in patients with cirrhosis. HEPATOLOGY 1987;7:149-154. 14. Floras JS, Legault L, Morali GA, Hara K, Blendis LM. Increased sympathetic outflow in cirrhosis and ascites: direct evidence from intraneural recordings. Ann Intern Med 1991;114:373-380. 15. Morgan JL, Curran T. Role of ion flux in the control of c-fos expression. Nature 1986;22:552-555. 16. Sheng M, Greenberg ME. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 1990;4:477-485. 17. Dragunow M, Faull R. The use of c-fos as a metabolic marker in neuronal pathway tracing. J Neurosci Methods 1989;29:261-265. 18. Erickson JT, Millhorn DE. Fos-like protein is induced in neurons of
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the medulla oblongata after stimulation of the carotid sinus nerve in awake anesthetized rats. Brain Res 1991;567:11-24. Dun NJ, Dun SL, Chiaia NL. Hemorrhage induces Fos immunoreactivity in rat medullary catecholaminergic neurons. Brain Res 1993;608:223232. Morgan JL, Curran T. Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci 1989;12:459-462. Murphy AZ, Ennis M, Shipley MT, Behbehani MM. Directionally specific changes in arterial pressure induce differential patterns of fos expression in discrete areas of the rat brainstem: a double-labeling study for fos and catecholamines. J Comp Neurol 1994;349:36-50. Fraser KA, Davison JS. Meal-induced c-fos expression in brainstem is not dependent on cholecystokinin release. Am J Physiol 1993;265: R235-R239. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd ed. San Diego: Academic Press, 1986. Sumal KK, Blessing WW, Joh TH, Reis DJ, Pickel VM. Synaptic interactions of vagal afferents and catecholaminergic neurons in the rat nucleus solitarius. Brain Res 1983;277:31-40. Spyer KM. The central nervous organization of reflex circulatory control. In: Loewy AD, Spyer KM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press, 1990:168-188. Guyenet PG. The role of the ventral medulla oblongata in blood pressure
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regulation. In: Loewy AD, Spyer KM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press, 1990:129-149. Westfall TC, Meldrum MJ. Alterations in release of norepinephrine at the vascular neuroeffector junction in hypertension. Annu Rev Pharmacol Toxicol 1985;25:621-641. Chan RKW, Brown ER, Ericsson A, Kovacs KJ, Sawchenko PE. A comparison of two immediate-early genes, c-fos and NGFI-B, as markers for functional activation in stress-related neuroendocrine circuitry. J Neurosci 1993;13:5126-5138. Thuluvath PJ, Triger DR. Autonomic neuropathy and chronic liver disease. Q J Med 1989;72:737-747. Lee SS. Cardiac abnormalities in liver cirrhosis. West J Med 1989;151: 530-535. Swain MG, Maric M. Impaired stress and interleukin-1b induced hypothalamic expression of the neuronal activation marker FOS in cholestatic rats. HEPATOLOGY 1996;24:914-918. Lee SS, Hadengue A, Girod C, Braillon A, Lebrec D. Hemodynamic characterization of bile duct ligated cirrhotic rats: effects of pentobarbital sodium. Am J Physiol 1986;251:G176-G180. Umemoto S, Noguchi K, Kawai Y, Senba E. Repeated stress reduces the subsequent stress-induced expression of Fos in rat brain. Neurosci Lett 1994;167:101-104.
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