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(Circulation. 1997;96:1654-1659.)
© 1997 American Heart Association, Inc.

Articles

Blockade of Brain `Ouabain' Prevents the Impairment of Baroreflexes in Rats After Myocardial Infarction

Bing S. Huang, MD, PhD; Baoxue Yuan, MD; ; Frans H. H. Leenen, MD, PhD, FRCPC

From the University of Ottawa (Ontario) Heart Institute, Canada.

Correspondence to Frans H.H. Leenen, MD, PhD, FRCPC, Hypertension Unit, H360, Division of Cardiology, University of Ottawa Heart Institute, 1053 Carling Ave, Ottawa, Ontario, Canada, K1Y 4E9. E-mail fleenen{at}ohi-net.heartinst.on.ca

	Abstract

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Background The purpose of this study was to test whether increased brain "ouabain" contributes to impairment of both arterial and cardiopulmonary baroreceptor reflexes in congestive heart failure (CHF).

Methods and Results Two to 5 days after coronary artery ligation (MI) or sham surgery in male Wistar rats, chronic intracerebroventricular (ICV) infusion was started with either antibody Fab fragments, which bind ouabain and related steroids with high affinity, or -globulins as control (200 µg · 12 µL^-1 · d^-1 for both) with osmotic minipumps implanted subcutaneously. After 8 weeks of infusion, in conscious rats, mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), and renal sympathetic nerve activity (RSNA) were recorded at rest and in response to ramp changes in blood pressure (BP) induced by intravenous phenylephrine and nitroprusside and to changes in CVP elicited by acute volume expansion with 5% dextrose. Compared with sham rats, in MI rats with ICV -globulins, resting MAP was significantly lower and CVP increased, and both arterial and cardiopulmonary baroreflex control of RSNA and HR were attenuated. ICV Fab fragments prevented the decrease in resting BP and largely prevented impairment of arterial and cardiopulmonary baroreflex control of both RSNA and HR.

Conclusions These data indicate that increased brain ouabain plays a major role in the impairment of baroreflexes in rats with CHF after myocardial infarction.

Key Words: heart failure • nervous system, autonomic • ouabain, brain • baroreceptors

	Introduction

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In humans with CHF, cardiopulmonary baroreflex control of both efferent sympathetic activity and HR as well as arterial baroreflex control of HR are clearly impaired,^{1 2 3} but impairment in arterial baroreflex control of sympathetic activity is less clear.^{2 4} Impairment of arterial and cardiopulmonary baroreflexes has also been shown in several experimental animal models of CHF.^{5 6 7 8} It is generally assumed that in CHF, reduced afferent input from the baroreceptors is mainly responsible for this impairment.⁹ However, abnormalities in the central components of the baroreflex arc in CHF may contribute as well. No changes were noted in the central control of the arterial baroreflex in dogs with pacing-induced heart failure¹⁰ or rats with CHF after coronary ligation,⁵ both under barbiturate anesthesia. In contrast, both desensitized afferent pathways and abnormal central control of the arterial baroreflex were found in rats with CHF after coronary artery ligation in either the conscious state¹¹ or under urethane anesthesia.¹² Mechanisms leading to central impairment have received little attention thus far. DiBona et al¹¹ reported that in conscious rats with CHF, ICV injection of the AT₁ blocker losartan improves the attenuated arterial baroreflex control of RSNA, suggesting that brain Ang II contributes to baroreflex attenuation.

We showed recently¹³ that in two animal models of CHF, ie, rats after coronary artery ligation and cardiomyopathic hamsters, ouabain-like activity ("ouabain") in the hypothalamus was increased by 100% to 200%. In conscious rats with CHF,¹³ this increase in brain ouabain was associated with increases in plasma catecholamines as well as enhanced sympathoexcitatory response to air stress and sympathoinhibitory response to ICV ₂-adrenergic receptor agonist guanabenz. This neurohormonal excitation could be reversed¹³ by ICV antibody Fab fragments (Digibind), which bind in vitro and in vivo ouabain,^{13 14 15} brain ouabain,^{13 15} and related steroids¹⁶ with high affinity. Like sodium-sensitive hypertension,^{16 17} increased brain ouabain appears to mediate sympathetic hyperactivity in CHF. In SHR and Dahl salt-sensitive rats on high sodium intake, the arterial as well as cardiopulmonary baroreflexes are also impaired, and increased brain ouabain appears to mediate this impairment.^{18 19}

In the present study, we examined whether in rats with CHF induced by coronary artery ligation, increased brain ouabain contributes to the impairment of arterial and cardiopulmonary baroreflex control of RSNA and HR. For this, for 8 weeks after MI, rats received an ICV infusion of the above-noted Fab fragments (or as control, -globulins), followed by assessment of baroreflex function in the conscious state.

	Methods

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Male Wistar rats (Charles River Breeding Laboratories, Montreal, Canada) weighing 250 to 300 g were housed on a 12-hour light/dark cycle and were allowed a 5-day acclimatization on normal rat chow and tap water. All procedures were carried out according to the guidelines of the University of Ottawa Animal Care Committee for the use and care of laboratory animals. Coronary artery ligation was performed as described by Pfeffer et al.²⁰ Briefly, under halothane inhalation anesthesia, rats were connected to a respirator, the left thorax was opened, and the left coronary artery was ligated at 2 to 3 mm from its origin. In sham rats, the same thoracotomy without artery ligation was done.

Between 2 and 5 days after the heart surgery, under pentobarbital sodium anesthesia (65 mg/kg IP), a 23-gauge, stainless steel, right-angled cannula was implanted into the left lateral ventricle and fixed on the skull with dental cement.¹⁶ Coordinates were 0.5 mm posterior and 1.4 mm lateral to the bregma. The shorter arm of the cannula was inserted into the ventricle (3.8 mm from the dura), and the longer arm was connected to an osmotic minipump (model 2002, 12 µL/d, ALZA Corp), which was filled with either antibody Fab fragments (Digibind, Glaxo Wellcome Inc) or -globulins as control (Sigma Chemical Co). Both compounds were dissolved in 0.9% saline and infused at a rate of 200 µg/d. The rationale for this rate for the Fab fragments has been described previously.¹⁶ The osmolarities for Fab fragments and -globulin infusates were 3.46 and 2.98 osm/L and the pH 6.44 and 6.90, respectively. The pump was implanted subcutaneously on the back of the rat. Penicillin G (30 000 IU IM; Derapen, Ayerst Laboratories) was given. Every 2 weeks after the surgery, under halothane anesthesia, the pumps were replaced with new ones filled with the same compound. Fab fragments or -globulins were therefore infused for 8 weeks.

At the end of the 8-week infusion, under halothane anesthesia, the right femoral artery and vein were catheterized with polyethylene tubing. A polyethylene catheter was inserted into the right jugular vein so that its tip was located at the level of the right atrium. With additional methohexital sodium anesthesia (30 mg/kg IV, supplemented with 10 mg/kg as needed; Brevital, Eli Lilly Canada Inc), a pair of platinum electrodes (A-M System, Inc) was placed around the left renal nerve through a flank incision and secured with silicone rubber (SilGel 604, Wacker) as described previously.¹⁶ The catheters and electrodes were tunneled subcutaneously and externalized on the back of the neck.

Four hours after recovery from the anesthesia and surgery, the rat was placed in a small cage in which it could move back and forth. The catheters inserted into the femoral artery and jugular vein were connected to a Grass 7E polygraph and a Grass 7E44 tachograph and the electrodes to a Grass P511 band-pass amplifier for continuous recording of BP, HR, CVP, and RSNA. RSNA (spikes per second) was counted through a nerve traffic analyzer (model 706C, University of Iowa Bio-engineering). The actual activity was determined by subtracting noise from the total activity.¹⁶

After a 30-minute rest and measurements of baseline BP, HR, and RSNA, BP was raised progressively to achieve a gradual ramp increase in MAP with a maximum of 50 mm Hg over a 3-minute period by intravenous infusion of phenylephrine at increasing rates (5 to 50 µg · kg^-1 · min^-1 dissolved in 5% dextrose) with a Sage 355 infusion pump.²¹ In some rats with MI, maximum MAP increases of only 40 to 45 mm Hg could be reached even at a dose of 100 µg · kg^-1 · min^-1. Ten minutes after the MAP, RSNA, and HR responses to phenylephrine had returned to baseline levels, nitroprusside was infused at gradually increasing rates (5 to 100 µg · kg^-1 · min^-1 IV in 5% dextrose) to induce a ramp MAP decrease with a maximum of -50 mm Hg over 3 minutes. MAP, HR, and RSNA were recorded and converted into digital data via an IBM microcomputer. Infusion speed was <0.08 mL/min for both.

Subsequently, assessment of the cardiopulmonary baroreflex function started after a 30-minute stabilization period. Two doses of 5% dextrose solution (3.3 and 10.0 mL/kg body wt IV over 30 seconds) were infused at an interval of 5 minutes. Changes in CVP, MAP, RSNA, and HR were monitored throughout.

The rats were killed with a overdose of pentobarbital, and the background noise of RSNA was recorded for 20 minutes. The heart was removed, the ventricles were dissected and weighed, and the infarct size of the left ventricle was determined.¹³ Data from rats with infarct size <25% (n=1 and 4 for rats with Fab fragments and -globulins, respectively) were excluded from the analysis.

Responses of RSNA were expressed as percent changes from the baseline levels. To evaluate the sensitivity of arterial baroreflex control of RSNA and HR, the percent changes of RSNA (RSNA) or changes of HR (HR, bpm) at 5 mm Hg incremental increases and decreases in MAP were analyzed together as a logistic model²² with the logistic equation RSNA=P₁+P₂/[1+e^P3(MAP-P4)], where P₁ is lower RSNA plateau, which represents the maximum decrease in RSNA; P₂ is RSNA range; P₃ is a curvature coefficient; and P₄ is MAP₅₀, ie, the MAP at half the RSNA range. The average gain (G) or slope of the curve between the two inflection points is given by G=-P₂xP₃/4.56 and the upper plateau is P₁+P₂, which represents the maximum increase in RSNA. The curve of best fit was obtained via the microcomputer program Sigmoid, provided by the Baker Medical Research Institute, Victoria, Australia. Calculated parameters for each rat were used for statistical analysis. When calculated maximum decreases in RSNA were <100%, -100% was used as the maximum decrease. Sensitivity of the cardiopulmonary baroreflex control of RSNA and HR was evaluated by means of the gain of the reflex, ie, the slope of the relations between RSNA or HR and corresponding CVP (at increments of 0.5 mm Hg) analyzed by linear regression. Because the slopes for the two rates of volume expansion were similar, data from the two were pooled together for the analysis. With SAS software (SAS Institute Inc), two-way ANOVA was performed for all data. When F ratios were significant, a Duncan multirange test was followed. Statistical significance was defined as P<.05.

	Results

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General Characteristics
The baseline MAP was significantly lower and CVP higher in MI rats with -globulins versus sham rats (Table 1). In MI rats, ICV Fab fragments prevented the decrease in baseline MAP but had little effect on baseline CVP. There was no significant difference in baseline HR among the three groups of rats.

View this table: [in this window] [in a new window]   Table 1. General Characteristics at the End of 8-Week Infusion

There was no difference in infarct size of the left ventricle between MI rats treated with Fab fragments or -globulins. Both groups of MI rats showed similar increases in left and right ventricular wet weight compared with sham rats. The body weight gain and the weight at the time of hemodynamic assessment were significantly less in MI rats with ICV -globulins than in sham rats treated with -globulins (Table 1).

Arterial Baroreflex Control of RSNA and HR
An increase or decrease in BP induced inhibitory or excitatory response of RSNA (Fig 1). Compared with sham rats, in MI rats with -globulins both the maximal increase and decrease in RSNA, reflected by the first and second plateaus of the RSNA-MAP reflex curve, were significantly attenuated (Table 2), leading to a significant decrease in the range of the RSNA. Moreover, the maximal slope of the curve was significantly less in MI rats with -globulins than in sham rats (Table 2), indicating an attenuation of arterial baroreflex control of RSNA in MI rats. In MI rats treated with -globulins, the curve also shifts to the left. These differences in ranges, maximal responses, slopes, and curve positions between sham and MI rats were not observed when Fab fragments were administered ICV for 8 weeks in MI rats (Fig 1, Table 2).

View larger version (28K): [in this window] [in a new window]   Figure 1. Arterial baroreflex control of RSNA analyzed as a logistic model. Each point represents mean±SEM of changes in RSNA (% baseline) in response to changes in MAP at increments of 5 mm Hg elicited by intravenous infusion of nitroprusside or phenylephrine. CHF indicates coronary artery ligation and infusion of -globulins; CHF+Fab, coronary artery ligation and infusion of antibody Fab fragments; and sham, sham operation and infusion of -globulins.

View this table: [in this window] [in a new window]   Table 2. Estimated Parameters of the Arterial Baroreflex

No significant differences in the low or high plateaus of HR responses were observed between the three groups (Table 2). Compared with sham rats, in MI rats with -globulins, the maximal slope was significantly decreased, and the curve shifted to the left. In MI rats with Fab fragments, in contrast, the maximal slope was not significantly different versus sham. The left shift of the curve observed in MI rats with -globulins was no longer present in MI rats with ICV Fab fragments (Fig 2).

View larger version (30K): [in this window] [in a new window]   Figure 2. Arterial baroreflex control of HR analyzed as a logistic model. Each point represents mean±SEM of changes in HR (bpm) in response to changes in MAP at increments of 5 mm Hg elicited by intravenous infusion of nitroprusside or phenylephrine. CHF indicates coronary artery ligation and infusion of -globulins; CHF+Fab, coronary artery ligation and infusion of antibody Fab fragments; and sham, sham operation and infusion of -globulins.

Cardiopulmonary Baroreflex
Volume expansion at both rates significantly increased CVP and decreased HR and RSNA (Figs 3 and 4). For the same rates of volume expansion, the peak increase in CVP in MI rats with -globulins (6.7±0.3 mm Hg) was significantly larger than in sham rats (4.9±0.4 mm Hg) or MI rats with Fab fragments (5.2±0.4 mm Hg) (P<.05 for both) (Fig 3). Volume expansion caused a minor increase (all <5 mm Hg, P=NS) in MAP. In MI rats treated with -globulins, the gain of RSNA in response to increases in CVP was significantly less than in both sham rats and MI rats with Fab fragments (6.2±0.7% versus 12.3±1.2% or 12.8±2.5% resting per mm Hg, P<.05 for both). In MI rats with -globulins, the gain of HR in response to increases in CVP was also significantly smaller than in sham (5.3±0.6 versus 7.2±0.5 bpm/mm Hg, P<.05). After ICV Fab fragments in CHF rats, the difference in gains of HR between MI and sham rats became minor and insignificant (6.9±0.5 versus 7.2±0.5 bpm/mm Hg).

View larger version (29K): [in this window] [in a new window]   Figure 3. Line graph obtained by plotting changes in RSNA against CVP recorded during volume expansion in CHF rats with -globulins (CHF), CHF with Fab fragments (CHF+Fab), and sham-operated rats with -globulins (sham). Each point is mean±SEM of RSNA (% baseline) response at 0.5 mm Hg increments in CVP (mm Hg).

View larger version (28K): [in this window] [in a new window]   Figure 4. Line graph obtained by plotting changes in HR against CVP recorded during volume expansion in CHF rats with -globulins (CHF), CHF with Fab fragments (CHF+Fab), and sham-operated rats with -globulins (sham). Each point is mean±SEM of the HR (bpm) response at 0.5 mm Hg increments in CVP (mm Hg).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
As a major new finding, the present study demonstrates that in rats with CHF after MI, chronic blockade of brain ouabain by ICV antibody Fab fragments prevents, to a large extent, the impairment of arterial as well as cardiopulmonary baroreflex control of both HR and RSNA.

Desensitization of Arterial Baroreflex
In rats, a low-output heart failure can be induced by acute coronary artery ligation, which causes an MI in the range of 40% to 50% of the LV.^{5 6} An MI of similar size was associated with a significant increase in CVP (Table 1) and, in our previous studies¹³ in rats 4 weeks after MI, with a marked increase in LV end-diastolic pressure versus sham rats (10 to 20 versus 0 to 5 mm Hg), indicating the development of moderate CHF. As in most previous studies (eg, References 5, 6, and 12^{5 6 12} ), in the present study, arterial baroreflex control of HR as well as sympathetic nerve activity is impaired in rats with CHF after MI. This impairment of the arterial baroreflex could be the result of attenuated afferent neural pathways, including baroreceptors, altered central integration, impaired efferent neural pathways, or dysfunction of end organs. Whether a dysfunction of the central control is involved in the impairment of baroreflexes in CHF is still controversial. In conscious rats with CHF, acute blockade of brain Ang II improved the attenuated arterial baroreflex regulation of RSNA, suggesting that changes in central control contribute to the impairment of the reflex.¹¹ In the present study, in conscious rats with CHF, arterial baroreflex control of HR and RSNA are both attenuated to similar degrees. This attenuation in reflex control of HR and RSNA is prevented by ICV antibody Fab fragments, which bind ouabain and related steroids with high affinity.^{13 14 15} Fab fragments at the dose used are ineffective when administered peripherally.²³ Moreover, because ouabain and related steroids sensitize baroreceptors peripherally,²⁴ Fab fragments would have attenuated the arterial baroreflex further if they had acted peripherally. Thus, the present results indicate that changes in central control of the baroreflex play a major role in the impairment of the arterial baroreflex in rats with CHF after MI.

Relevance of Anesthesia
Using sinus nerve stimulation and renal nerve recording, Wang et al¹⁰ reported no changes in central control of the arterial baroreflex in pentobarbital-anesthetized dogs with pacing-induced heart failure. In rats with CHF after MI, under methohexital anesthesia the central gain for RSNA, as obtained by simultaneously recording responses of aortic depressor and RSNA to changes in MAP, was normal versus control rats.⁵ In contrast, under light urethane anesthesia in rats with CHF after MI,¹² in addition to abnormal afferent mechanisms, the central gain for lumbar sympathetic nerve activity, as obtained by stimulation of the aortic depressor nerve, was impaired as well. Different experimental models of CHF, in particular the anesthetics used,²⁵ may contribute to the different results regarding the central control of the arterial baroreflex. Compared with the anesthesia induced by barbiturates as used by DiBona and Sawin⁵ and Wang et al,¹⁰ anesthesia induced by a low dose of urethane as used by Jung et al¹² appears to have less effect on the central nervous system, sympathetic activity, and baroreflex function.^{25 26} However, the conscious state is clearly the preferable approach to prevent confounding effects of anesthetics.

Cardiopulmonary Baroreflex
Previous studies have established that development of CHF is associated with impairment of cardiopulmonary baroreflex function in humans^{3 4} and animals.^{5 6 27} In the present study, in conscious rats with CHF after MI, cardiopulmonary baroreflex control of HR and RSNA were attenuated, although to different degrees (75% for HR versus 50% for RSNA of the gains in sham rats). Changes in central control of the cardiopulmonary baroreflex in CHF appear to play a major role in this attenuation because centrally administered Fab fragments prevent the attenuation of the reflex control of both RSNA and HR.

Previous studies on central regulation of cardiopulmonary baroreflex function in CHF were all performed under anesthesia. Atrial baroreceptor sensitivity is decreased in anesthetized dogs with high-output CHF.²⁷ In rats with CHF under methohexital anesthesia, DiBona and Sawin⁵ measured the multifiber as well as single-unit activity from the cut peripheral end of the right vagus nerve in response to volume expansion and demonstrated that in rats with CHF, the attenuated cardiopulmonary baroreflex control of RSNA is due to impairment at peripheral afferent levels of the reflex but not to changes in the central nervous system. As discussed above, the effects of (barbiturate) anesthesia on central regulation may lead to suppression of central changes in cardiopulmonary baroreflex control in rats with CHF.

For the same volume expansion, CVP increased more in CHF rats with -globulins than in sham rats or CHF rats with Fab fragments. Together with the observation that ICV Fab fragments increased resting BP in CHF rats, it appears that blockade of brain ouabain decreases preload and/or improves cardiac function in rats after MI. However, more comprehensive hemodynamic assessments are clearly needed to substantiate this speculation.

Brain Ouabain and Impairment of Baroreflexes
We showed previously¹³ that in rats with CHF 4 weeks after MI, brain ouabain is significantly increased in brain areas such as the hypothalamus, pons, and pituitary. Moreover, acute blockade of brain ouabain reversed increased sympathoexcitatory and decreased sympathoinhibitory responses, decreased RSNA, and normalized plasma catecholamines.¹³ Further studies are still needed to establish the actual time course of changes in brain ouabain versus sympathetic activity during both the short-term and more chronic phases of CHF after MI.

In the present study, we show that in rats with CHF after MI, arterial and cardiopulmonary baroreflex control of HR and RSNA become attenuated and that blockade of brain ouabain prevents, to a large extent, the impairment of both the arterial and cardiopulmonary baroreflexes. Thus, as in salt-sensitive hypertension,^{16 17 18 19} in rats with CHF an increase in brain ouabain appears to be responsible for sympathetic hyperactivity as well as the impairment of baroreflex control.

DiBona et al showed¹¹ that in conscious rats with CHF, the attenuated arterial baroreflex control of RSNA was improved by acute ICV injection of a low dose of losartan. Because increased brain Ang II can desensitize the arterial baroreflex,²⁸ an increase in brain Ang II may contribute to the impairment of the arterial baroreflex in CHF. We showed previously that the sympathoexcitatory and pressor effects of acute²⁹ or chronic (Huang and Leenen, unpublished observation) ICV hypertonic saline in normotensive rats and of high dietary sodium in SHR²³ can be prevented by blockade of either brain ouabain by the Fab fragments or of AT1 receptors by losartan. Sympathoexcitatory responses to brain ouabain appear to be mediated through activation of the brain renin-angiotensin system. Further studies are needed to reveal the relationship between brain ouabain and brain Ang II in the central regulation of baroreflexes in CHF.

Afferent pathways of cardiopulmonary receptors that cause excitatory sympathetic responses have been demonstrated in dogs,³⁰ cats,³¹ and SHRs.³² In cats during coronary artery occlusion, ventricular bulging after coronary artery occlusion may activate cardiac mechanoreceptors and increase the discharge rate of vagal afferents.³³ Elevation of the LV end-diastolic pressure may increase excitatory afferent signals in cats via ventricular vagal C fibers.³⁴ In sinoaortic denervated SHRs on high sodium intake, volume expansion increased lumbar sympathetic nerve activity.³² This response was eliminated by bilateral vagotomy, suggesting that the response originates from vagal afferents.³² Chronic heart failure represents an absolute or relative volume-expanded state. As CHF progresses, excitatory input increases and inhibitory input decreases from cardiopulmonary as well as arterial mechanoreceptors.³⁵ It is intriguing to speculate that an increase in cardiac filling pressures activates sympathetic excitatory afferents and leads to an increase in brain ouabain. The latter appears to integrate central changes leading to both an increase in resting sympathetic activity and blunting of baroreflex control of HR and RSNA, thereby also maintaining higher sympathetic activity during increases in hemodynamic load.

In animals with CHF,⁸ perfusing the carotid sinus with ouabain improves or normalizes the baroreflex function, suggesting that in CHF the impairment of arterial baroreflex function results at least partially from an exaggerated cellular Na⁺,K⁺-ATPase at the baroreceptor membrane, eg, by aldosterone.³⁶ Peripheral ouabain is increased in CHF in humans³⁷ or rats.¹³ There is so far no evidence for a (patho)physiological role of peripheral ouabain in the actual regulation of baroreflex function. In CHF, an increase in peripheral ouabain may not be sufficiently large to affect the baroreflex, and/or effects of increased brain ouabain may override the effects of peripheral ouabain. Conversely, one cannot exclude that in animals under barbiturate anesthesia, central regulation of both central and peripheral ouabain becomes suppressed,³⁸ leading to a decrease in plasma ouabain, minimizing its effects on baroreceptors if there are any.

Possible Limitations of the Study
Several possible limitations in methodological aspects should be considered. First, saline was used as the vehicle of ICV infusion. Although artificial cerebrospinal fluid is more physiological than saline, at infusion rates of 0.5 µL/h relative to the secretion rate of cerebrospinal fluid in rats (120 to 320 µL/h),³⁹ the impact of saline versus artificial cerebrospinal fluid seems minor. Second, changes in baroreflex function by Fab fragments may relate to nonspecific mechanisms such as acidity, osmolarity, and possible toxic effects on certain brain regions by, eg, antibody-antigen complex. These, however, seem unlikely because of the above-noted low infusion rates, and as we showed previously,¹⁸ long-term infusion of the Fab fragments does not affect arterial baroreflex function or other parameters of autonomic control in conscious normotensive rats or in SHRs on regular sodium intake. In previous studies in salt-sensitive hypertensive rats¹⁶ or rats with CHF,¹³ no evidence for nonspecific effects was noted. Effects of Fab fragments on cardiopulmonary baroreflex in normotensive rats have not yet been assessed, and at present we can therefore not exclude that the gain also would have changed in sham rats. Third, baroreflex assessment 4 hours after anesthesia and surgery may still be influenced by the surgery and (less likely⁴⁰ ) the residual effects of the short-acting anesthetic. The RSNA signal deteriorates over time, and fluid and food intake are often less than optimal the first night after surgery.⁴¹ Thus, the timing of these assessments is a compromise. Finally, only minor changes in MAP were observed during volume expansion, but filling pressures were not measured during changes in BP. The results, therefore, particularly at the higher end, reflect some combination of the arterial and the cardiopulmonary baroreflexes.

In summary, in conscious rats 8 weeks after coronary artery ligation, arterial as well as cardiopulmonary baroreflex control of both HR and RSNA are impaired. Chronic blockade of brain ouabain by ICV infusion of antibody Fab fragments prevents the impairment of baroreflexes. These results indicate that in rats with low-output heart failure after MI, changes in central control appear to play a major role in the attenuation of baroreflex control of both HR and RSNA and that an increase in brain ouabain activity appears to play a major role in the central mechanisms involved.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II BP = blood pressure CHF = congestive heart failure CVP = central venous pressure HR = heart rate ICV = intracerebroventricular LV = left ventricular MAP = mean arterial pressure MI = myocardial infarction RSNA = renal sympathetic nerve activity SHR = spontaneously hypertensive rat

	Acknowledgments

 
This study was supported by operating grant MT-13182 from the Medical Research Council of Canada. Dr Leenen is a Career Investigator of the Heart and Stroke Foundation of Ontario, Canada. Digibind was a generous gift from Glaxo Wellcome Inc, Toronto, Canada. The authors wish to thank Drs Allyn L. Mark and John S. Floras for review of the manuscript and critical comments.

Received February 28, 1997; accepted March 9, 1997.

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