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Estrogen in the parabrachial nucleus attenuates the sympathoexcitation following MCAO in male rats
Brain Research 1066 (2005) 187 – 195 www.elsevier.com/locate/brainres
Research Report
Estrogen in the parabrachial nucleus attenuates the sympathoexcitation following MCAO in male rats Tarek M. Saleh a,b,*, Barry J. Connell a,c, Alastair E. Cribb a,b,c a
Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3 b Prince Edward Island Health Research Institute, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3 c Laboratory of Comparative Pharmacogenetics, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3 Accepted 21 October 2005 Available online 1 December 2005
Abstract Recent investigations have provided evidence to suggest systemic estrogen administration prevented or reversed the sympathoexcitation observed following middle cerebral artery occlusion (MCAO) in male rats. The present investigation sought to determine the role of estrogen injected directly into the parabrachial nucleus (PBN) on the MCAO-induced sympathoexcitation as well as the role of the rostral ventrolateral medulla (RVLM) in mediating the sympathoexcitatory response. Male Sprague – Dawley rats were anesthetized with sodium thiobutabarbitol (100 mg/kg) and were instrumented to continuously record blood pressure, heart rate and renal sympathetic nerve activity (RSNA). Following occlusion of the middle cerebral artery, there was a significant increase in RSNA (from 3.8 T 0.4 to 8.3 T 0.6 AV/s; P < 0.05) which was significantly attenuated by the prior bilateral injection of estrogen (0.5 AM in 200 nl) into the PBN. Pre-injection of lidocaine (5% in 200 nl) directly into the RVLM resulted in only a slight reduction in the magnitude of the MCAO-induced sympathoexcitation (P > 0.05). Extracellular electrophysiological recordings from RVLM neurons demonstrated that MCAO did not produce any significant change in neuronal activity over the experimental time course (P > 0.05). Also, bilateral injection of estrogen into the PBN prior to MCAO or sham conditions did not result in any significant change in RVLM neuronal activity. These results indicate that estrogen receptors in the PBN play a major role in modulating the sympathoexcitatory response from ischemic forebrain nuclei, and that the pathway from the PBN to sympathetic preganglionic nuclei may not involve a synapse in the RVLM. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Autonomic tone; Rostral ventrolateral medulla; Stroke; Electrophysiology
1. Introduction The majority of stroke patients in which significant autonomic and cardiovascular changes occur are usually the result of an occlusion of the middle cerebral artery (MCAO). In the clinical scenario, the autonomic dysfunction observed within 1 – 2 h of stroke is in the form of sympathoexcitation [13,14] and this has been replicated in * Corresponding author. Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3. Fax: +1 902 566 0832. E-mail address: [email protected] (T.M. Saleh). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.062
an animal model following permanent MCAO [7,33]. In both humans and rats, occlusion of the right MCAO produces an ischemic lesion which includes the insular cortex [13,14], a forebrain nucleus involved in autonomic and cardiovascular regulation [16]. Thus, the therapeutic potential of drugs to recover or prevent autonomic dysfunction following MCAO is of considerable interest. Estrogen is well established as a cardioprotective and neuroprotective hormone [1,4,8,34], particularly as it relates to the central modulation of autonomic nervous system function [17,18,22,23]. In general, experiments in both male and female rats have demonstrated that estrogen administered either systemically or directly into various central
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autonomic nuclei, resulted in significantly decreased blood pressure and basal sympathetic tone and/or enhanced parasympathetic tone within 30 min of injection ([3,9,17 – 23]). In addition to this ability of estrogen to decrease basal sympathetic tone, our lab showed that estrogen was also able to reduce the approximately 3 fold increase in renal sympathetic nerve activity (RSNA) observed following MCAO in male rats, and that this estrogen-mediated effect was via an action within the CNS [24,25]. Of the central autonomic nuclei involved in cardiovascular regulation, the parabrachial nucleus (PBN) in particular has been demonstrated to be an important synapse in the modulation of neurotransmission between the insular cortex and subcortical autonomic nuclei [10]. The PBN has a direct, reciprocal connection with the insula [30,32] and has been shown to be involved in relaying visceral afferent information to this region [2] as well as modulate changes in autonomic tone evoked by insular stimulation and/or lesion [5,7,14]. Therefore, to determine the role of estrogen receptors in the PBN in modulating the sympathoexcitatory response to MCAO, direct injection of the potent and selective estrogen receptor antagonist, ICI 182,780 into the PBN was used, resulting in a significant attenuation in the ability of systemically administered estrogen to attenuate the MCAOinduced sympathoexcitation [25]. Further, extracellular levels of estrogen within the PBN of male rats have been shown to increase immediately following MCAO [28]. Also, estrogen has been shown to almost completely block the increase in extracellular activity of PBN neurons observed following MCAO [28]. Although these investigation indirectly suggest that estrogen plays an important role in modulating descending sympathetic control, no study to date has determined the effect of estrogen injection directly into the PBN in modulating the sympathoexcitatory response to MCAO. The PBN in turn projects to various medullary autonomic regulatory nuclei, including the rostral ventrolateral medulla (RVLM) [35], as well as directly to sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord [6,31]. Changes in RSNA as well as extracellular neuronal activity within the RVLM have been shown to be reciprocally related to alterations in baroreceptor activation indicating the importance of the RVLM in mediating acute changes in both blood pressure and sympathetic tone [11]. This, as well as numerous other neuroanatomical and neurophysiological evidence has given the RVLM the reputation of being a ‘‘presympathetic’’ nucleus [11], it is unknown if the RVLM is involved in mediating the sympathoexcitatory response of the ischemic insular cortex induced following MCAO. Therefore, the current investigation was undertaken to determine the role of estrogen in the PBN in modulating the MCAO-induced increase in RSNA as well as the role of the RVLM in mediating the sympathoexcitatory response following MCAO. These results are necessary to increase our understanding of the role of estrogen in CNS in
preventing the cardiovascular consequences of MCAO as well as the neuroanatomical pathway involved in relaying the autonomic dysfunction observed following MCAO.
2. Results Prior to any central drug administration, baseline mean arterial pressure (MAP) and heart rate (HR) were measured (109 T 11 mm Hg and 367 T 33 beats/min, respectively) and no significant differences were found between any group of animals (P > 0.05; n = 36) for either parameter. MAP and HR were measured at 5 min post-drug injection (immediately prior to MCAO (n = 28) or sham (n = 7)). The MAP and HR values were not statistically different from pre-drug values (P > 0.05). In addition, over the time course of the experiment, no significant changes in either parameter were observed (P > 0.05; data not shown) in sham or MCAO groups except the group in which lidocaine was injected into the RVLM. Lidocaine in the RVLM resulted in a decrease in MAP and HR of 34 T 11 mm Hg and 47 T 23 beats/min, respectively. The average duration for this decrease in MAP and HR was 44 T 11 min. Also in these animals, when baroreceptors were unloaded using sodium nitroprusside, the decrease in MAP (22 mm Hg) and reflex tachycardia (9 beats/min) were sustained for an average duration of 47 min. For all animals, rectal temperatures were 36 T 1.0 -C throughout the time course of the study. 2.1. Effect of estrogen injection in the PBN on RSNA At all time points measured following MCAO and the prior bilateral injection of saline into the PBN, renal sympathetic nerve activity (RSNA) was significantly elevated from a baseline value of 3.8 T 0.5 AV/s to a peak value of 8.1 T 0.7 AV/s at 90 min post-MCAO (Figs. 1A and C; P < 0.05). Prior to the termination of the experiment 4 h post-MCAO, RSNA remained significantly elevated (6.5 T 0.6 AV/s; P < 0.05; Fig. 1C). In contrast, following MCAO and the prior bilateral injection of estrogen into the PBN resulted in no significant change in RSNA at any time point measured post-MCAO (P > 0.05; Figs. 1B and C). The specificity of this estrogenmediated effect was supported by the co-injection of estrogen with the estrogen receptor antagonist ICI 182,780 into the PBN. In these animals, the RSNA was significantly elevated from the baseline value of 3.4 T 0.5 AV/s to a peak value of 8.3 T 0.7 AV/s at 90 min post-MCAO (P < 0.05; Fig. 1C). As observed in saline pretreated animals, the RSNA remained significantly elevated at the termination of the experiment (240 min post-MCAO (6.9 T 06 AV/s; P < 0.05; Fig. 1C). 2.2. Effect of lidocaine injection in the RVLM on RSNA At all time points measured following sham surgery with prior bilateral saline injections into the RVLM, RSNA was
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Fig. 1. Cardiovascular and autonomic responses to bilateral saline or estrogen injections into the parabrachial nucleus (PBN) following middle cerebral artery occlusion (MCAO). Representative physiograph tracings showing baseline blood pressure, heart rate and renal sympathetic nerve activity (RSNA) following bilateral saline (A) or estrogen (B) injection into the PBN prior to (preMCAO) and following (60 min and 120 min) MCAO. (C) Plot of average MCAOinduced changes in RSNA before and after the bilateral injection of saline, estrogen or estrogen/ICI 182,780 into the PBN (* indicates P < 0.05 compared to baseline; @ indicates P < 0.05 compared to saline and estrogen/ICI 182,780 group).
not significantly different from a baseline value of 3.6 T 0.5 AV/s (P > 0.05; Figs. 2A and C). Middle cerebral artery occlusion resulted in a significant increase in RSNA of similar magnitude and duration as was observed above when saline was injected into the PBN (P < 0.05; Figs. 2A and C). In the group of animals in which the bilateral injection of lidocaine into the RVLM had been made prior to MCAO, RSNA was significantly increased from 3.6 T 0.6 AV/s to 7.0 T 1.2 AV/s by 90 min post-MCAO (P < 0.05: Figs. 2B and C). Although this peak RSNA increase following MCAO was less than that observed in saline injected animals, the values were not statistically different.
2.3. Electrophysiological changes in RVLM neuronal activity following MCAO In all animals, baseline excitability of RVLM neurons was approximately 20 T 4 spikes/bin prior to the direct micro-injection of drug into the right PBN. In shamoperated animals, the prior injection of estrogen into the PBN (Fig. 3B) did not significantly alter RVLM neuronal excitability at any time point compared to pre-injection values (P > 0.05 for each time point in each group). The injection of either estrogen or saline into the right PBN did not produce significant changes in RVLM neuronal
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Fig. 2. Cardiovascular and autonomic responses to bilateral saline or lidocaine injections into the rostral ventrolateral medulla (RVLM) and following either middle cerebral artery occlusion (MCAO) or sham surgery. Representative physiograph tracings showing baseline blood pressure, heart rate and renal sympathetic nerve activity (RSNA) following bilateral saline (A) or lidocaine (B) injection into the RVLM prior to (preMCAO) and following (60 min and 120 min) MCAO. (C) Plot of average MCAO or sham surgery-induced changes in RSNA before and after the bilateral injection of saline or lidocaine into the RVLM (* indicates P < 0.05 compared to baseline).
excitability (P > 0.05; Figs. 3A and B). MCAO did not alter RVLM neuronal excitability in either the estrogen or saline group measured at any time point over the 4 h post-MCAO (P > 0.05; Figs. 3A and B). 2.4. Histological verification of electrode/dialysis probe placement In all experiments, recording electrode and syringe placements were verified to be located within the RVLM and PBN respectively. Syringe tip sites in the PBN were
predominantly located in the lateral subdivision distributed within the dorsal, central and external and internal lateral subnuclei between bregma 9.0 and 9.7 mm [15]. Injection and recording sites in the RVLM were also verified to be predominantly located at the level of bregma 11.9 mm, with a distribution between bregma 11.8 to 12.7 mm. Injections located dorsally in the region of the nucleus ambiguus or caudally in the caudal ventrolateral medulla were excluded from analysis. Interestingly, recordings from ambiguus neurons following MCAO resulted in very different results compared to
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Fig. 3. (A) Representative sample of running rate meter response of extracellular multi unit neuronal activity from the right rostral ventrolateral medulla (RVLM) following saline injection into the right parabrachial nucleus (PBN) prior to (preMCAO) and following MCAO (30 min and 120 min). (B) MCAO and sham surgery-induced changes in extracellular multi unit neuronal activity in the RVLM before and after the injection of saline or estrogen into the PBN.
RVLM recordings and will be the subject of our next investigation.
3. Discussion This study supported previous findings from our laboratory and others that in male rats, MCAO induced a significant elevation in RSNA. This study further showed that the MCAO-induced sympathoexcitatory response could be significantly attenuated by the prior injection of estrogen into the PBN. Further, this study added to our knowledge regarding the role of the RVLM in mediating the sympathoexcitatory response observed following MCAO. Blocking neurotransmission through the RVLM using lidocaine only slightly attenuated the sympathoexcitatory response to MCAO. Also, direct recordings from neurons in the RVLM revealed no significant change in RVLM neuronal activity at any time point measured post-
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MCAO. Taken together, these results showed that the relay of sympathoexcitatory information from the insular cortex could be modulated by the activation of estrogen receptors in the PBN and that the RVLM was only minimally involved in the transmission of MCAO-induced changes in RSNA. The pontine parabrachial nucleus (PBN) has been demonstrated to be an important modulatory site in the relay of both ascending and descending visceral and autonomic information [2,10,12]. In particular, the PBN plays an important role in the central regulation of autonomic and cardiovascular function [10,12]. Under normal conditions, many beneficial cardiovascular effects of estrogen have been described by examining the consequences of estrogen receptor activation within the rat PBN, including the enhancement of baroreceptor sensitivity, increase in parasympathetic tone (vagal efferent nerve activity) and decrease in sympathetic tone ([17,18,22,23]; [19 – 21]). In the current set of experiments, estrogen injected directly into the PBN completely blocked the sympathoexcitation observed following MCAO in rats. The observation that the co-injection of the selective estrogen receptor antagonist, ICI 182,780, completely reversed this effect, indicated that estrogenic action was mediated by estrogen receptors in the PBN. Since bilateral estrogen injections into the PBN completely blocked the MCAO-induced increase in RSNA, we can conclude that the PBN represents a mandatory synapse for descending sympathoexcitatory information originating in the ischemic insular cortex. The insula has a strong direct reciprocal connections with the PBN [30,32]. It is therefore possible that following MCAO, the hypoglycemia- and hypoxia-induced cell death and subsequent anoxic depolarization of cortical neurons may result in excessive activation of subcortical autonomic nuclei to which the insula projects. Studies in our lab have shown that both increases in visceral afferent activation [26] or excessive cortical stimulation (such as that observed following MCAO; [28]) result in a significant elevation in the extracellular level of estrogen within the PBN. We hypothesized that this pathologyinduced estrogen release in vivo into the PBN may result in modulating neurotransmission through this nucleus. Evidence from our laboratory indicated that estrogen strongly inhibited vagal afferent stimulation-induced activation of thalamic neurons via an interaction with GABAA receptors in the PBN [21]. Further, systemic estrogen administration has been shown to decrease PBN neuronal excitability, resulting in both a decrease in extracellular glutamate and an increase in extracellular GABA levels [19]. The presence of ICI 182,780 in the PBN, prior to either the intravenous administration of estrogen or MCAO, blocked the action of estrogen to decrease the sympathoexcitation observed following MCAO [19,27], suggesting an acute, receptor-mediated effect of estrogen in this nucleus.
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There is neuroanatomical and electrophysiological evidence indicating that the PBN may exert its cardiovascular and autonomic effects via axonal connections with the rostral ventro-lateral medulla (RVLM). The RVLM provides a tonic excitatory drive to sympathetic preganglionic neurons that maintain both tonic and phasic control of arterial pressure and vasomotor tone [11]. There is a direct glutamatergic projection from the PBN to the RVLM suggesting that activation of the PBN would result in excitation of RVLM neurons resulting in an increase in RSNA [35]. The second and third experiments of this investigation tested the role of the RVLM in the MCAOinduced changes in RSNA. Blocking all neurotransmission in the RVLM with the reversible anesthetic lidocaine did not significantly reduce the MCAO-induced increase in RSNA. However, any potential role of the RVLM in this paradigm cannot be completely discounted as there was a minor, albeit non-significant, attenuation in the MCAOinduced RSNA level. Although a minor attenuation of the MCAO-induced sympathoexcitation was observed, we were unable to measure any change in the extracellular activity of these neurons following MCAO. We therefore conclude that the RVLM would only have a minor role, if any, in relaying the autonomic consequences following MCAO (Fig. 4). Also, the results of this study showed that injection of estrogen into the PBN did not alter RVLM activity (preMCAO). If estrogen activated a dormant projection to the RVLM, then we would have seen a change in RVLM neuronal activity. However, if estrogen inhibited PBN neurons thus preventing activation of an RVLM projection pathway, we would not see any change in RVLM activity, which is supported by the results presented here. This is consistent with an inhibitory role of estrogen in the PBN as previously determined [19,21]. Also, the fact that RVLM activity was unchanged following MCAO is also consistent with the fact that no significant changes in mean arterial blood pressure were observed following MCAO. This lack of significant cardiovascular changes following MCAO in this rat model [1,7,8,24,25,27 –29,33,34] or those in humans following a stroke [13,14] is consistent with our current observations. Taken together with the strong participatory role of the PBN in mediating the sympathoexcitatory response to MCAO, our data suggest that the pathway mediating the sympathoexcitatory response from the insula is through a direct projection from the PBN to sympathetic preganglionic neurons located in the intermediolateral cell column of the spinal cord (IML; Fig. 4). In conclusion, further detailed examination of the role of the RVLM in mediating the autonomic dysfunction following MCAO as well as an experiment designed to determine if other nuclei between the PBN and IML participate in mediating this response is warranted. These experiments are important to help us understand where estrogen acts in the CNS to protect
Fig. 4. Schematic diagram illustrating the neuronal pathway suggested to be activated following middle cerebral artery occlusion (MCAO) which leads to enhanced sympathetic outflow. Information from the ischemic IC is relayed through the PBN and then to sympathetic preganglionic neurons in the IML. A minor pathway (dashed lines) may also relay information from the IC and/or PBN to the RVLM. (IC, insular cortex; PBN, parabrachial nucleus; RVLM, rostral ventrolateral medulla; IML, intermediolateral cell column; RSNA, renal sympathetic nervous activity.)
against the autonomic dysfunction observed following MCAO.
4. Experimental procedures All experiments were carried out in accordance with the guidelines of the Canadian council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee (protocol #00-023). 4.1. General surgical procedures Experiments were performed on a total of 36 Sprague – Dawley male rats (Charles River; Montreal, PQ) weighing
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250 –350 g. For all animals, food and tap water were available ad libitum. Rats were anaesthetized with sodium thiobutabarbitol (Inactin; RBI, Natick, MA; 100 mg/kg; ip) providing a stable plane of anesthesia for the full experiment. A polyethylene catheter (PE-50; Clay Adams; Parsippany, NJ) was inserted into the right femoral artery to monitor blood pressure and heart rate. Arterial blood pressure was measured with a pressure transducer (Gould P23 ID; Cleveland, OH) connected to a Gould model 2200S physiograph. Heart rate was determined from the pulse pressure using a Gould tachograph (Biotach). These parameters were displayed and analyzed using PolyviewPro/32 data acquisition software (Grass; Warwick, RI). In one rat group (n = 4), a polyethylene catheter (PE-10; Clay Adams; Parsippany, NJ) was inserted into the right femoral vein for the intravenous administration of sodium nitroprusside. All animals had an endotracheal tube inserted within the trachea and body temperature was monitored with a digital rectal thermometer and maintained at 36 T 1 -C. 4.2. Middle cerebral artery occlusions Animals were placed in a David Kopf stereotaxic frame (Tujunga, CA) and the right middle cerebral artery (MCA) approached through a rostro-caudal incision of the skin and frontalis muscle at the level of bregma. The frontalis and temporalis muscles were then reflected anteriorly and posteriorly to expose the squamosal bone to the point where the zygoma and the squamosal bone fuse. A hole was made in the rostro-dorsal part of the squamosal bone which was then removed to expose the MCA. The bent tip of a 25gauge hypodermic needle was used to cut and retract the dura mater over the MCA. The MCA was permanently occluded at 3 points along the MCA by passing current between forceps connected to a bipolar electrical coagulator (Cameron-Miller Inc.; Chicago, IL). The first occlusion was made just dorsal to the rhinal fissure, the second just ventral to the bifurcation of the MCA to the frontal and parietal cortices, and the third was made just before the bifurcation of the MCA along the parietal cortex. This 3-point occlusion protocol has been shown to result in reproducible lesion volumes [24,25,27– 29]. Sham-operated animals had the dura removed and the MCA isolated, but no current was allowed to pass between the forceps connected to the coagulator. 4.3. Autonomic nerve isolation and recording A group of animals (n = 24) were instrumented to record changes in efferent sympathetic nerve activity. To record efferent sympathetic nerve activity, the right kidney was exposed through a retroperitoneal incision. With the aid of an operating stereomicroscope, a renal nerve branch was isolated and a bipolar platinum recording electrode secured in place. The multi-unit renal nerve activity was amplified by a Grass model P-55 preamplifier with a 100 Hz to a 3-
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kHz bandpass and 60 Hz notch filter, displayed and analyzed using Polyview Pro 32 data acquisition and analysis software (Grass; Warwick, RI). Raw nerve activity was sampled at a rate of 2000/s. Animals were allowed to stabilize for 30 min following nerve isolation prior to drug injection or nerve activity measurements. 4.4. Electrophysiological experiments Extracellular single and multi-unit recordings of RVLM neurons (n = 11) were obtained using a 3.0 M NaCl-filled glass recording microelectrode lowered stereotaxically into the right RVLM. Extracellular signals were amplified and displayed on a digital oscilloscope (BK Precision Instruments; Chicago, IL, USA) and fed into a window discriminator (Digitimer; IBIS Instruments, Mississauga, ON). The electrical activity was then forwarded to a microcomputer for display as a continuous time histogram and analyzed with use of the Integrated Program for Electrophysiological Experiments (IPEE) software program (Conrad Yim Software, Etobicoke, ON, Canada). Discriminated neuronal activity with amplitudes 1 standard deviation above baseline were acquired and quantified by the IPEE program as the number of spikes per bin (1 second bin width). All animals were allowed to stabilize for a minimum of 30 min prior to any data recording. 4.5. Central recordings and microinjections All rats were placed in a stereotaxic frame (David Kopf, Tujunga, CA) and holes were drilled through the skull to permit the stereotaxic insertion of a 1.0-Al Hamilton microsyringe (CSC, Montreal, Quebec) or a glass recording electrode using coordinates obtained from a rat atlas [15]. Glass recording microelectrodes were lowered stereotaxically into the right RVLM. In the first experiment, blood pressure, heart rate and RSNA recordings were made immediately prior to drug injection, and then at 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, 210 and 240 min following MCAO. Immediately prior to MCAO, bilateral injections of 17h-estradiol (0.5 AM in 200 nl; n = 5; Sigma Aldrich, St. Louis, MO), co-injection of 17h-estradiol and the selective estrogen receptor antagonist, ICI 182,870 (2 AM in 200 nl; Tocris, Bollwin, MO; n = 4), or physiological saline (0.9% in 200 nl; n = 4) were made into the PBN 5 min prior to MCAO. The doses of estrogen or ICI 182,780 used in this study were previously determined from dose – response relationship as the optimal dose resulting in changes in of PBN neuronal excitability and blockade of this estrogeninduced effect, respectively [21]. In the second experiment, extracellular multi-unit neuronal activity was recorded from the RVLM immediately prior to the unilateral injection of either estrogen (0.5 AM in 200 nl; n = 8; Sigma Aldrich, St. Louis, MO) or saline (0.9% in 200 nl; n = 3) into the right PBN. This central injection was made 5 min prior to either MCAO (n = 4 of the estrogen
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group and n = 3 for the saline group) or sham surgery (n = 4 for the estrogen group). Blood pressure, heart rate and neuronal activity measurements were made immediately prior to drug injection, immediately prior to MCAO or sham, and then at 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, 210 and 240 min following MCAO or sham. In the third experiment, blood pressure, heart rate and RSNA were recorded prior to and following MCAO or sham, and the bilateral injection of the reversible anesthetic lidocaine (5.0% in 200 nl; n = 4 for MCAO; Vetoqinol Canada, Joliette, PQ) or physiological saline (0.9% in 200 nl; n = 4 for MCAO and n = 3 for sham) into the RVLM. The bilateral injection of drug into the RVLM preceded MCAO or sham by 5 min and cardiovascular and autonomic measurements were done at the same times mentioned above. 4.6. Inhibition of RVLM neuronal activity To determine the effect of bilateral injection of lidocaine (5.0% in 200 nl/side) into the RVLM on the reflex tachycardia following baroreceptor deactivation, a bolus intravenous injection of the vasodilator sodium nitroprusside (0.01 mg/kg in 0.1 ml; Sigma; St. Louis, MO) was used. This allowed us to determine the duration of inhibition of the RVLM by measuring the duration of the depressor response until recovery of the baroreflex-induced increase in sympathetic tone mediated by the RVLM was attained. 4.7. Histological procedures Four hours following MCAO or sham surgery, the animals were transcardially perfused with 0.9% saline followed by 10% formalin. The brains were removed and stored in 10% formalin. The location of the tip of the glass recording electrode and the Hamilton microsyringe were verified histologically in thionin-stained coronal sections (T100 Am) according to landmarks and coordinates identified in the rat stereotaxic atlas [15]. In all cases in which an MCAO or sham were made, infarct sizes were measured by taking digital photomicrographs of the relevant cortical sections and quantified as previously described [24,25]. 4.8. Data analysis All data are presented as mean T standard error of the mean (S.E.M.). Changes from baseline in mean arterial pressure, heart rate, RVLM extracellular activity and RSNA were analyzed with one way analysis of variance (ANOVA) for repeated measures and when necessary, followed by a Student –Newman –Keul’s post hoc analysis (SigmaStat and SigmaPlot. Jandel Scientific). Differences between groups at identical time points were determined using unpaired student t tests with a Bonferonni correction for multiple comparisons. In all cases, differences were considered significant only if P 0.05.
Acknowledgment This work was supported by a grant from the Canadian Institute for Health Research (CIHR; MOP 50095). AEC is a Canada Research Chair.
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[28] T.M. Saleh, B.J. Connell, C. Legge, A.E. Cribb, Stroke-induced changes in estrogen release and neuronal activity in the parabrachial nucleus of the male rat, J. Stroke Cerebrovasc. Dis. 13 (2004) 24 – 34. [29] T.M. Saleh, B.J. Connell, C. Legge, A.E. Cribb, Estrogen attenuates neuronal excitability in the insular cortex following middle cerebral artery occlusion, Brain Res. 1018 (2004) 119 – 129. [30] C.B. Saper, Reciprocal parabrachial – cortical connections in the rat, Brain Res. 242 (1982) 33 – 40. [31] C.B. Saper, The spinoparabrachial pathway: shedding new light on an old path, J. Comp. Neurol. 353 (1995) 477 – 479. [32] M.T. Shipley, M.S. Sanders, Special senses are really special: evidence for a reciprocal, bilateral pathway between the insular cortex and nucleus parabrachialis, Brain Res. 8 (1982) 493 – 501. [33] K.E. Smith, V.C. Hachinski, C.J. Gibson, J. Ciriello, Changes in plasma catecholamine levels after insula damage in experimental stroke, Brain Res. 375 (1986) 182 – 185. [34] T. Toung, R. Traystman, P. Hurn, Estrogen-mediated neuroprotection after experimental stroke in male rats, Stroke 29 (1998) 1666 – 1670. [35] L. Wen-Bin, J.Y.H. Chan, Glutamatergic projection to RVLM mediates suppression of reflex bradycardia by parabrachial nucleus, J. Am. Physiol. 45 (1999) H1482 – H1492.
Research Report
Estrogen in the parabrachial nucleus attenuates the sympathoexcitation following MCAO in male rats Tarek M. Saleh a,b,*, Barry J. Connell a,c, Alastair E. Cribb a,b,c a
Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3 b Prince Edward Island Health Research Institute, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3 c Laboratory of Comparative Pharmacogenetics, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3 Accepted 21 October 2005 Available online 1 December 2005
Abstract Recent investigations have provided evidence to suggest systemic estrogen administration prevented or reversed the sympathoexcitation observed following middle cerebral artery occlusion (MCAO) in male rats. The present investigation sought to determine the role of estrogen injected directly into the parabrachial nucleus (PBN) on the MCAO-induced sympathoexcitation as well as the role of the rostral ventrolateral medulla (RVLM) in mediating the sympathoexcitatory response. Male Sprague – Dawley rats were anesthetized with sodium thiobutabarbitol (100 mg/kg) and were instrumented to continuously record blood pressure, heart rate and renal sympathetic nerve activity (RSNA). Following occlusion of the middle cerebral artery, there was a significant increase in RSNA (from 3.8 T 0.4 to 8.3 T 0.6 AV/s; P < 0.05) which was significantly attenuated by the prior bilateral injection of estrogen (0.5 AM in 200 nl) into the PBN. Pre-injection of lidocaine (5% in 200 nl) directly into the RVLM resulted in only a slight reduction in the magnitude of the MCAO-induced sympathoexcitation (P > 0.05). Extracellular electrophysiological recordings from RVLM neurons demonstrated that MCAO did not produce any significant change in neuronal activity over the experimental time course (P > 0.05). Also, bilateral injection of estrogen into the PBN prior to MCAO or sham conditions did not result in any significant change in RVLM neuronal activity. These results indicate that estrogen receptors in the PBN play a major role in modulating the sympathoexcitatory response from ischemic forebrain nuclei, and that the pathway from the PBN to sympathetic preganglionic nuclei may not involve a synapse in the RVLM. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Autonomic tone; Rostral ventrolateral medulla; Stroke; Electrophysiology
1. Introduction The majority of stroke patients in which significant autonomic and cardiovascular changes occur are usually the result of an occlusion of the middle cerebral artery (MCAO). In the clinical scenario, the autonomic dysfunction observed within 1 – 2 h of stroke is in the form of sympathoexcitation [13,14] and this has been replicated in * Corresponding author. Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., Canada C1A 4P3. Fax: +1 902 566 0832. E-mail address: [email protected] (T.M. Saleh). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.062
an animal model following permanent MCAO [7,33]. In both humans and rats, occlusion of the right MCAO produces an ischemic lesion which includes the insular cortex [13,14], a forebrain nucleus involved in autonomic and cardiovascular regulation [16]. Thus, the therapeutic potential of drugs to recover or prevent autonomic dysfunction following MCAO is of considerable interest. Estrogen is well established as a cardioprotective and neuroprotective hormone [1,4,8,34], particularly as it relates to the central modulation of autonomic nervous system function [17,18,22,23]. In general, experiments in both male and female rats have demonstrated that estrogen administered either systemically or directly into various central
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autonomic nuclei, resulted in significantly decreased blood pressure and basal sympathetic tone and/or enhanced parasympathetic tone within 30 min of injection ([3,9,17 – 23]). In addition to this ability of estrogen to decrease basal sympathetic tone, our lab showed that estrogen was also able to reduce the approximately 3 fold increase in renal sympathetic nerve activity (RSNA) observed following MCAO in male rats, and that this estrogen-mediated effect was via an action within the CNS [24,25]. Of the central autonomic nuclei involved in cardiovascular regulation, the parabrachial nucleus (PBN) in particular has been demonstrated to be an important synapse in the modulation of neurotransmission between the insular cortex and subcortical autonomic nuclei [10]. The PBN has a direct, reciprocal connection with the insula [30,32] and has been shown to be involved in relaying visceral afferent information to this region [2] as well as modulate changes in autonomic tone evoked by insular stimulation and/or lesion [5,7,14]. Therefore, to determine the role of estrogen receptors in the PBN in modulating the sympathoexcitatory response to MCAO, direct injection of the potent and selective estrogen receptor antagonist, ICI 182,780 into the PBN was used, resulting in a significant attenuation in the ability of systemically administered estrogen to attenuate the MCAOinduced sympathoexcitation [25]. Further, extracellular levels of estrogen within the PBN of male rats have been shown to increase immediately following MCAO [28]. Also, estrogen has been shown to almost completely block the increase in extracellular activity of PBN neurons observed following MCAO [28]. Although these investigation indirectly suggest that estrogen plays an important role in modulating descending sympathetic control, no study to date has determined the effect of estrogen injection directly into the PBN in modulating the sympathoexcitatory response to MCAO. The PBN in turn projects to various medullary autonomic regulatory nuclei, including the rostral ventrolateral medulla (RVLM) [35], as well as directly to sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord [6,31]. Changes in RSNA as well as extracellular neuronal activity within the RVLM have been shown to be reciprocally related to alterations in baroreceptor activation indicating the importance of the RVLM in mediating acute changes in both blood pressure and sympathetic tone [11]. This, as well as numerous other neuroanatomical and neurophysiological evidence has given the RVLM the reputation of being a ‘‘presympathetic’’ nucleus [11], it is unknown if the RVLM is involved in mediating the sympathoexcitatory response of the ischemic insular cortex induced following MCAO. Therefore, the current investigation was undertaken to determine the role of estrogen in the PBN in modulating the MCAO-induced increase in RSNA as well as the role of the RVLM in mediating the sympathoexcitatory response following MCAO. These results are necessary to increase our understanding of the role of estrogen in CNS in
preventing the cardiovascular consequences of MCAO as well as the neuroanatomical pathway involved in relaying the autonomic dysfunction observed following MCAO.
2. Results Prior to any central drug administration, baseline mean arterial pressure (MAP) and heart rate (HR) were measured (109 T 11 mm Hg and 367 T 33 beats/min, respectively) and no significant differences were found between any group of animals (P > 0.05; n = 36) for either parameter. MAP and HR were measured at 5 min post-drug injection (immediately prior to MCAO (n = 28) or sham (n = 7)). The MAP and HR values were not statistically different from pre-drug values (P > 0.05). In addition, over the time course of the experiment, no significant changes in either parameter were observed (P > 0.05; data not shown) in sham or MCAO groups except the group in which lidocaine was injected into the RVLM. Lidocaine in the RVLM resulted in a decrease in MAP and HR of 34 T 11 mm Hg and 47 T 23 beats/min, respectively. The average duration for this decrease in MAP and HR was 44 T 11 min. Also in these animals, when baroreceptors were unloaded using sodium nitroprusside, the decrease in MAP (22 mm Hg) and reflex tachycardia (9 beats/min) were sustained for an average duration of 47 min. For all animals, rectal temperatures were 36 T 1.0 -C throughout the time course of the study. 2.1. Effect of estrogen injection in the PBN on RSNA At all time points measured following MCAO and the prior bilateral injection of saline into the PBN, renal sympathetic nerve activity (RSNA) was significantly elevated from a baseline value of 3.8 T 0.5 AV/s to a peak value of 8.1 T 0.7 AV/s at 90 min post-MCAO (Figs. 1A and C; P < 0.05). Prior to the termination of the experiment 4 h post-MCAO, RSNA remained significantly elevated (6.5 T 0.6 AV/s; P < 0.05; Fig. 1C). In contrast, following MCAO and the prior bilateral injection of estrogen into the PBN resulted in no significant change in RSNA at any time point measured post-MCAO (P > 0.05; Figs. 1B and C). The specificity of this estrogenmediated effect was supported by the co-injection of estrogen with the estrogen receptor antagonist ICI 182,780 into the PBN. In these animals, the RSNA was significantly elevated from the baseline value of 3.4 T 0.5 AV/s to a peak value of 8.3 T 0.7 AV/s at 90 min post-MCAO (P < 0.05; Fig. 1C). As observed in saline pretreated animals, the RSNA remained significantly elevated at the termination of the experiment (240 min post-MCAO (6.9 T 06 AV/s; P < 0.05; Fig. 1C). 2.2. Effect of lidocaine injection in the RVLM on RSNA At all time points measured following sham surgery with prior bilateral saline injections into the RVLM, RSNA was
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Fig. 1. Cardiovascular and autonomic responses to bilateral saline or estrogen injections into the parabrachial nucleus (PBN) following middle cerebral artery occlusion (MCAO). Representative physiograph tracings showing baseline blood pressure, heart rate and renal sympathetic nerve activity (RSNA) following bilateral saline (A) or estrogen (B) injection into the PBN prior to (preMCAO) and following (60 min and 120 min) MCAO. (C) Plot of average MCAOinduced changes in RSNA before and after the bilateral injection of saline, estrogen or estrogen/ICI 182,780 into the PBN (* indicates P < 0.05 compared to baseline; @ indicates P < 0.05 compared to saline and estrogen/ICI 182,780 group).
not significantly different from a baseline value of 3.6 T 0.5 AV/s (P > 0.05; Figs. 2A and C). Middle cerebral artery occlusion resulted in a significant increase in RSNA of similar magnitude and duration as was observed above when saline was injected into the PBN (P < 0.05; Figs. 2A and C). In the group of animals in which the bilateral injection of lidocaine into the RVLM had been made prior to MCAO, RSNA was significantly increased from 3.6 T 0.6 AV/s to 7.0 T 1.2 AV/s by 90 min post-MCAO (P < 0.05: Figs. 2B and C). Although this peak RSNA increase following MCAO was less than that observed in saline injected animals, the values were not statistically different.
2.3. Electrophysiological changes in RVLM neuronal activity following MCAO In all animals, baseline excitability of RVLM neurons was approximately 20 T 4 spikes/bin prior to the direct micro-injection of drug into the right PBN. In shamoperated animals, the prior injection of estrogen into the PBN (Fig. 3B) did not significantly alter RVLM neuronal excitability at any time point compared to pre-injection values (P > 0.05 for each time point in each group). The injection of either estrogen or saline into the right PBN did not produce significant changes in RVLM neuronal
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Fig. 2. Cardiovascular and autonomic responses to bilateral saline or lidocaine injections into the rostral ventrolateral medulla (RVLM) and following either middle cerebral artery occlusion (MCAO) or sham surgery. Representative physiograph tracings showing baseline blood pressure, heart rate and renal sympathetic nerve activity (RSNA) following bilateral saline (A) or lidocaine (B) injection into the RVLM prior to (preMCAO) and following (60 min and 120 min) MCAO. (C) Plot of average MCAO or sham surgery-induced changes in RSNA before and after the bilateral injection of saline or lidocaine into the RVLM (* indicates P < 0.05 compared to baseline).
excitability (P > 0.05; Figs. 3A and B). MCAO did not alter RVLM neuronal excitability in either the estrogen or saline group measured at any time point over the 4 h post-MCAO (P > 0.05; Figs. 3A and B). 2.4. Histological verification of electrode/dialysis probe placement In all experiments, recording electrode and syringe placements were verified to be located within the RVLM and PBN respectively. Syringe tip sites in the PBN were
predominantly located in the lateral subdivision distributed within the dorsal, central and external and internal lateral subnuclei between bregma 9.0 and 9.7 mm [15]. Injection and recording sites in the RVLM were also verified to be predominantly located at the level of bregma 11.9 mm, with a distribution between bregma 11.8 to 12.7 mm. Injections located dorsally in the region of the nucleus ambiguus or caudally in the caudal ventrolateral medulla were excluded from analysis. Interestingly, recordings from ambiguus neurons following MCAO resulted in very different results compared to
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Fig. 3. (A) Representative sample of running rate meter response of extracellular multi unit neuronal activity from the right rostral ventrolateral medulla (RVLM) following saline injection into the right parabrachial nucleus (PBN) prior to (preMCAO) and following MCAO (30 min and 120 min). (B) MCAO and sham surgery-induced changes in extracellular multi unit neuronal activity in the RVLM before and after the injection of saline or estrogen into the PBN.
RVLM recordings and will be the subject of our next investigation.
3. Discussion This study supported previous findings from our laboratory and others that in male rats, MCAO induced a significant elevation in RSNA. This study further showed that the MCAO-induced sympathoexcitatory response could be significantly attenuated by the prior injection of estrogen into the PBN. Further, this study added to our knowledge regarding the role of the RVLM in mediating the sympathoexcitatory response observed following MCAO. Blocking neurotransmission through the RVLM using lidocaine only slightly attenuated the sympathoexcitatory response to MCAO. Also, direct recordings from neurons in the RVLM revealed no significant change in RVLM neuronal activity at any time point measured post-
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MCAO. Taken together, these results showed that the relay of sympathoexcitatory information from the insular cortex could be modulated by the activation of estrogen receptors in the PBN and that the RVLM was only minimally involved in the transmission of MCAO-induced changes in RSNA. The pontine parabrachial nucleus (PBN) has been demonstrated to be an important modulatory site in the relay of both ascending and descending visceral and autonomic information [2,10,12]. In particular, the PBN plays an important role in the central regulation of autonomic and cardiovascular function [10,12]. Under normal conditions, many beneficial cardiovascular effects of estrogen have been described by examining the consequences of estrogen receptor activation within the rat PBN, including the enhancement of baroreceptor sensitivity, increase in parasympathetic tone (vagal efferent nerve activity) and decrease in sympathetic tone ([17,18,22,23]; [19 – 21]). In the current set of experiments, estrogen injected directly into the PBN completely blocked the sympathoexcitation observed following MCAO in rats. The observation that the co-injection of the selective estrogen receptor antagonist, ICI 182,780, completely reversed this effect, indicated that estrogenic action was mediated by estrogen receptors in the PBN. Since bilateral estrogen injections into the PBN completely blocked the MCAO-induced increase in RSNA, we can conclude that the PBN represents a mandatory synapse for descending sympathoexcitatory information originating in the ischemic insular cortex. The insula has a strong direct reciprocal connections with the PBN [30,32]. It is therefore possible that following MCAO, the hypoglycemia- and hypoxia-induced cell death and subsequent anoxic depolarization of cortical neurons may result in excessive activation of subcortical autonomic nuclei to which the insula projects. Studies in our lab have shown that both increases in visceral afferent activation [26] or excessive cortical stimulation (such as that observed following MCAO; [28]) result in a significant elevation in the extracellular level of estrogen within the PBN. We hypothesized that this pathologyinduced estrogen release in vivo into the PBN may result in modulating neurotransmission through this nucleus. Evidence from our laboratory indicated that estrogen strongly inhibited vagal afferent stimulation-induced activation of thalamic neurons via an interaction with GABAA receptors in the PBN [21]. Further, systemic estrogen administration has been shown to decrease PBN neuronal excitability, resulting in both a decrease in extracellular glutamate and an increase in extracellular GABA levels [19]. The presence of ICI 182,780 in the PBN, prior to either the intravenous administration of estrogen or MCAO, blocked the action of estrogen to decrease the sympathoexcitation observed following MCAO [19,27], suggesting an acute, receptor-mediated effect of estrogen in this nucleus.
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There is neuroanatomical and electrophysiological evidence indicating that the PBN may exert its cardiovascular and autonomic effects via axonal connections with the rostral ventro-lateral medulla (RVLM). The RVLM provides a tonic excitatory drive to sympathetic preganglionic neurons that maintain both tonic and phasic control of arterial pressure and vasomotor tone [11]. There is a direct glutamatergic projection from the PBN to the RVLM suggesting that activation of the PBN would result in excitation of RVLM neurons resulting in an increase in RSNA [35]. The second and third experiments of this investigation tested the role of the RVLM in the MCAOinduced changes in RSNA. Blocking all neurotransmission in the RVLM with the reversible anesthetic lidocaine did not significantly reduce the MCAO-induced increase in RSNA. However, any potential role of the RVLM in this paradigm cannot be completely discounted as there was a minor, albeit non-significant, attenuation in the MCAOinduced RSNA level. Although a minor attenuation of the MCAO-induced sympathoexcitation was observed, we were unable to measure any change in the extracellular activity of these neurons following MCAO. We therefore conclude that the RVLM would only have a minor role, if any, in relaying the autonomic consequences following MCAO (Fig. 4). Also, the results of this study showed that injection of estrogen into the PBN did not alter RVLM activity (preMCAO). If estrogen activated a dormant projection to the RVLM, then we would have seen a change in RVLM neuronal activity. However, if estrogen inhibited PBN neurons thus preventing activation of an RVLM projection pathway, we would not see any change in RVLM activity, which is supported by the results presented here. This is consistent with an inhibitory role of estrogen in the PBN as previously determined [19,21]. Also, the fact that RVLM activity was unchanged following MCAO is also consistent with the fact that no significant changes in mean arterial blood pressure were observed following MCAO. This lack of significant cardiovascular changes following MCAO in this rat model [1,7,8,24,25,27 –29,33,34] or those in humans following a stroke [13,14] is consistent with our current observations. Taken together with the strong participatory role of the PBN in mediating the sympathoexcitatory response to MCAO, our data suggest that the pathway mediating the sympathoexcitatory response from the insula is through a direct projection from the PBN to sympathetic preganglionic neurons located in the intermediolateral cell column of the spinal cord (IML; Fig. 4). In conclusion, further detailed examination of the role of the RVLM in mediating the autonomic dysfunction following MCAO as well as an experiment designed to determine if other nuclei between the PBN and IML participate in mediating this response is warranted. These experiments are important to help us understand where estrogen acts in the CNS to protect
Fig. 4. Schematic diagram illustrating the neuronal pathway suggested to be activated following middle cerebral artery occlusion (MCAO) which leads to enhanced sympathetic outflow. Information from the ischemic IC is relayed through the PBN and then to sympathetic preganglionic neurons in the IML. A minor pathway (dashed lines) may also relay information from the IC and/or PBN to the RVLM. (IC, insular cortex; PBN, parabrachial nucleus; RVLM, rostral ventrolateral medulla; IML, intermediolateral cell column; RSNA, renal sympathetic nervous activity.)
against the autonomic dysfunction observed following MCAO.
4. Experimental procedures All experiments were carried out in accordance with the guidelines of the Canadian council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee (protocol #00-023). 4.1. General surgical procedures Experiments were performed on a total of 36 Sprague – Dawley male rats (Charles River; Montreal, PQ) weighing
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250 –350 g. For all animals, food and tap water were available ad libitum. Rats were anaesthetized with sodium thiobutabarbitol (Inactin; RBI, Natick, MA; 100 mg/kg; ip) providing a stable plane of anesthesia for the full experiment. A polyethylene catheter (PE-50; Clay Adams; Parsippany, NJ) was inserted into the right femoral artery to monitor blood pressure and heart rate. Arterial blood pressure was measured with a pressure transducer (Gould P23 ID; Cleveland, OH) connected to a Gould model 2200S physiograph. Heart rate was determined from the pulse pressure using a Gould tachograph (Biotach). These parameters were displayed and analyzed using PolyviewPro/32 data acquisition software (Grass; Warwick, RI). In one rat group (n = 4), a polyethylene catheter (PE-10; Clay Adams; Parsippany, NJ) was inserted into the right femoral vein for the intravenous administration of sodium nitroprusside. All animals had an endotracheal tube inserted within the trachea and body temperature was monitored with a digital rectal thermometer and maintained at 36 T 1 -C. 4.2. Middle cerebral artery occlusions Animals were placed in a David Kopf stereotaxic frame (Tujunga, CA) and the right middle cerebral artery (MCA) approached through a rostro-caudal incision of the skin and frontalis muscle at the level of bregma. The frontalis and temporalis muscles were then reflected anteriorly and posteriorly to expose the squamosal bone to the point where the zygoma and the squamosal bone fuse. A hole was made in the rostro-dorsal part of the squamosal bone which was then removed to expose the MCA. The bent tip of a 25gauge hypodermic needle was used to cut and retract the dura mater over the MCA. The MCA was permanently occluded at 3 points along the MCA by passing current between forceps connected to a bipolar electrical coagulator (Cameron-Miller Inc.; Chicago, IL). The first occlusion was made just dorsal to the rhinal fissure, the second just ventral to the bifurcation of the MCA to the frontal and parietal cortices, and the third was made just before the bifurcation of the MCA along the parietal cortex. This 3-point occlusion protocol has been shown to result in reproducible lesion volumes [24,25,27– 29]. Sham-operated animals had the dura removed and the MCA isolated, but no current was allowed to pass between the forceps connected to the coagulator. 4.3. Autonomic nerve isolation and recording A group of animals (n = 24) were instrumented to record changes in efferent sympathetic nerve activity. To record efferent sympathetic nerve activity, the right kidney was exposed through a retroperitoneal incision. With the aid of an operating stereomicroscope, a renal nerve branch was isolated and a bipolar platinum recording electrode secured in place. The multi-unit renal nerve activity was amplified by a Grass model P-55 preamplifier with a 100 Hz to a 3-
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kHz bandpass and 60 Hz notch filter, displayed and analyzed using Polyview Pro 32 data acquisition and analysis software (Grass; Warwick, RI). Raw nerve activity was sampled at a rate of 2000/s. Animals were allowed to stabilize for 30 min following nerve isolation prior to drug injection or nerve activity measurements. 4.4. Electrophysiological experiments Extracellular single and multi-unit recordings of RVLM neurons (n = 11) were obtained using a 3.0 M NaCl-filled glass recording microelectrode lowered stereotaxically into the right RVLM. Extracellular signals were amplified and displayed on a digital oscilloscope (BK Precision Instruments; Chicago, IL, USA) and fed into a window discriminator (Digitimer; IBIS Instruments, Mississauga, ON). The electrical activity was then forwarded to a microcomputer for display as a continuous time histogram and analyzed with use of the Integrated Program for Electrophysiological Experiments (IPEE) software program (Conrad Yim Software, Etobicoke, ON, Canada). Discriminated neuronal activity with amplitudes 1 standard deviation above baseline were acquired and quantified by the IPEE program as the number of spikes per bin (1 second bin width). All animals were allowed to stabilize for a minimum of 30 min prior to any data recording. 4.5. Central recordings and microinjections All rats were placed in a stereotaxic frame (David Kopf, Tujunga, CA) and holes were drilled through the skull to permit the stereotaxic insertion of a 1.0-Al Hamilton microsyringe (CSC, Montreal, Quebec) or a glass recording electrode using coordinates obtained from a rat atlas [15]. Glass recording microelectrodes were lowered stereotaxically into the right RVLM. In the first experiment, blood pressure, heart rate and RSNA recordings were made immediately prior to drug injection, and then at 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, 210 and 240 min following MCAO. Immediately prior to MCAO, bilateral injections of 17h-estradiol (0.5 AM in 200 nl; n = 5; Sigma Aldrich, St. Louis, MO), co-injection of 17h-estradiol and the selective estrogen receptor antagonist, ICI 182,870 (2 AM in 200 nl; Tocris, Bollwin, MO; n = 4), or physiological saline (0.9% in 200 nl; n = 4) were made into the PBN 5 min prior to MCAO. The doses of estrogen or ICI 182,780 used in this study were previously determined from dose – response relationship as the optimal dose resulting in changes in of PBN neuronal excitability and blockade of this estrogeninduced effect, respectively [21]. In the second experiment, extracellular multi-unit neuronal activity was recorded from the RVLM immediately prior to the unilateral injection of either estrogen (0.5 AM in 200 nl; n = 8; Sigma Aldrich, St. Louis, MO) or saline (0.9% in 200 nl; n = 3) into the right PBN. This central injection was made 5 min prior to either MCAO (n = 4 of the estrogen
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group and n = 3 for the saline group) or sham surgery (n = 4 for the estrogen group). Blood pressure, heart rate and neuronal activity measurements were made immediately prior to drug injection, immediately prior to MCAO or sham, and then at 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, 210 and 240 min following MCAO or sham. In the third experiment, blood pressure, heart rate and RSNA were recorded prior to and following MCAO or sham, and the bilateral injection of the reversible anesthetic lidocaine (5.0% in 200 nl; n = 4 for MCAO; Vetoqinol Canada, Joliette, PQ) or physiological saline (0.9% in 200 nl; n = 4 for MCAO and n = 3 for sham) into the RVLM. The bilateral injection of drug into the RVLM preceded MCAO or sham by 5 min and cardiovascular and autonomic measurements were done at the same times mentioned above. 4.6. Inhibition of RVLM neuronal activity To determine the effect of bilateral injection of lidocaine (5.0% in 200 nl/side) into the RVLM on the reflex tachycardia following baroreceptor deactivation, a bolus intravenous injection of the vasodilator sodium nitroprusside (0.01 mg/kg in 0.1 ml; Sigma; St. Louis, MO) was used. This allowed us to determine the duration of inhibition of the RVLM by measuring the duration of the depressor response until recovery of the baroreflex-induced increase in sympathetic tone mediated by the RVLM was attained. 4.7. Histological procedures Four hours following MCAO or sham surgery, the animals were transcardially perfused with 0.9% saline followed by 10% formalin. The brains were removed and stored in 10% formalin. The location of the tip of the glass recording electrode and the Hamilton microsyringe were verified histologically in thionin-stained coronal sections (T100 Am) according to landmarks and coordinates identified in the rat stereotaxic atlas [15]. In all cases in which an MCAO or sham were made, infarct sizes were measured by taking digital photomicrographs of the relevant cortical sections and quantified as previously described [24,25]. 4.8. Data analysis All data are presented as mean T standard error of the mean (S.E.M.). Changes from baseline in mean arterial pressure, heart rate, RVLM extracellular activity and RSNA were analyzed with one way analysis of variance (ANOVA) for repeated measures and when necessary, followed by a Student –Newman –Keul’s post hoc analysis (SigmaStat and SigmaPlot. Jandel Scientific). Differences between groups at identical time points were determined using unpaired student t tests with a Bonferonni correction for multiple comparisons. In all cases, differences were considered significant only if P 0.05.
Acknowledgment This work was supported by a grant from the Canadian Institute for Health Research (CIHR; MOP 50095). AEC is a Canada Research Chair.
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