Involvement of the Paraventricular Nucleus in the Occurrence of Arrhythmias in Middle Cerebral Artery Occlusion Rats Shuwei Jia, MD,* Qing Xia, MS,† Benping Zhang, MD,‡ and Ling Wang, MD, PhD*

Background: Ischemic stroke complicating with arrhythmia is one of the main causes of sudden death. To investigate the association between ischemic stroke–induced arrhythmia and the activity of paraventricular nucleus (PVN), we used Fos protein as an objective indicator to illustrate the functional state of PVN neurons in middle cerebral artery occlusion (MCAO) rats, in single intracerebroventricular injection of L-glutamate rats and in application of MK-801 before L-glutamate injection and MCAO rats. Methods: The standard limb II electrocardiography was continuously recorded by a biological signal collecting and processing system. The experimental cerebral ischemic animal model was established by occluding the right middle cerebral artery. The Fos protein expression was detected by immunohistochemistry and Western blot. Results: The incidence of arrhythmia was significantly higher than that of controls (75.89% versus 0%), and Fos protein expression in the PVN also increased significantly in MCAO rats; both of them could be blocked by prior application of MK-801. Intracerebroventricular injection of L-glutamate induced changes in Fos protein expression and arrhythmia similar to that in the stroke, which could also be blocked by prior application of MK-801. Conclusions: It was concluded that activation of the PVN in MCAO rats is likely mediated by glutamate via activation of N-methyl-D-aspartic acid (NMDA) receptors, which causes arrhythmias. Key Words: Arrhythmia—glutamate—ischemic stroke—NMDA receptor—paraventricular nucleus. Ó 2015 by National Stroke Association

Introduction Ischemic stroke is a common life-threatening disease. Its high mortality is related to the lethal arrhythmia during the initial period of ischemic stroke.1 Our previous From the *Department of Physiology, Harbin Medical University, Harbin; †Institute of Acupuncture, Tianjin Chinese Medical University, Tianjin; and ‡Department of Neurology of 2nd Affiliated Hospital, Harbin Medical University, Harbin, China. Received November 4, 2014; revision received November 20, 2014; accepted November 24, 2014. This study was supported by Science Research Foundation of Heilongjiang Province Education Department of China (grant no.:12541349); Science Research Foundation of Science and Technology Department of Harbin City, Heilongjiang Province, China (Grant No. 2009RFXXS020). Address correspondence to Ling Wang, MD, PhD, Department of Physiology, School of Basic Medical Science, Harbin Medical University, 157 Baojian Rd, Nangang, Harbin 150081, China. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2015 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.11.025

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studies proved that inward Na1 current, transient outward K1 current, and L-type calcium current channelopathy, and the Ca21 overload in ventricular myocytes isolated from cerebral ischemic rats are likely the major ionic mechanisms underlying cerebrogenic cardiac arrhythmias.2 However, what caused these changes in ventricular myocytes during ischemic stroke remains unknown. Arrhythmias are frequently seen in stroke patients, even in those without history or signs of primary heart diseases,3 which suggest a central nervous system origin of these arrhythmias. Compelling evidence has revealed a close correlation between brain activation of unbalanced autonomic function and the onset of lifethreatening cardiac arrhythmias, particularly sympathetic hyperactivity.4 Recent studies further showed that the imbalance between sympathetic and parasympathetic activities does occur in those patients with cardiovascular disturbances in cerebrovascular diseases.5 However, specific electrocardiographic (ECG) abnormality correlated with a localized intracranial pathologic change has not been established.

Journal of Stroke and Cerebrovascular Diseases, Vol. 24, No. 4 (April), 2015: pp 844-851

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Hypothalamic paraventricular nucleus (PVN) is a crucial region in brain control of autonomic functions and cardiovascular activity. In most instances, stimulation of the PVN could activate sympathetic output and induce arrhythmia.6,7 In the heart, long-term activation of the sympathetic system can lead to uneven sympathetic innervations and the ensuing arrhythmia, even sudden cardiac death. The PVN receives glutamate innervations from forebrain and expresses large amount of N-methyl-D-aspartic acid (NMDA) receptors as well.8 Microinjection of L-glutamate into the PVN could increase sympathetic nerve activity and cause positive cardiovascular response.9 Earlier studies have also shown that ischemic stroke could increase levels of extracellular glutamate in the brain and sympathetic activity.10,11 However, whether glutamatergic activation of the PVN is involved in the occurrence of arrhythmias after ischemic stroke and whether this activation is mediated by NMDA receptors remain to be determined. For this purpose, we used Fos protein as an objective indicator to illustrate the functional state of PVN neurons in rats with middle cerebral artery occlusion (MCAO). We also compared the effects of single intracerebroventricular injection of L-glutamate on Fos expression and arrhythmias. Then we examined prior application of MK-801 on glutamate- and MCAO-evoked responses. The results indicated that activation of the PVN in MCAO rats is likely mediated by glutamate via activation of NMDA receptors, which causes arrhythmias.

Materials and Methods Experimental Animals Male Wistar rats (Experimental Animal Centre of Harbin Medical University, Harbin, China. Certificate No.09-2-1), 220-250 g, were used in this study. All rats were bred in an animal room with controlled temperature (23 6 1 C), humidity (55 6 5%), and food and water available ad libitum. All experimental protocols were preapproved by the Experimental Animal Ethic Committee of Harbin Medical University, China.

Experimental Groups All rats were monitored the ECG for 20 minutes, and those with normal ECG were used in the following experiments. The standard limb II ECG was continuously recorded by a recorder (BL420; Chengdu TME Technology Co, Ltd, China). To observe the changes of the activity of PVN neurons after MCAO, 144 rats were randomly divided into the following 3 groups: (1) control (n 5 16), (2) sham-operated (n 5 16), and (3) right MCAO (n 5 112). These rats in the third group were further randomly and equally divided into different time groups (n 5 16 per group), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 16 hours after MCAO. The rats in the

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control and sham-operated groups were sacrificed at 2 hours after anesthesia or operation, and the rats in MCAO groups were sacrificed at the corresponding time points after MCAO. The right (ischemic side) brain of each rat was used to detect Fos protein expression using immunohistochemistry and Western blot. To observe the effects of glutamate on arrhythmia induced by MCAO, 32 rats were randomly and equally divided into 4 groups (n 5 8 per group): (1) saline (10 mL), (2) L-glutamate (.5 mmoles, 10 mL), (3) MK-801 (4 nmoles, 10 mL) before L-glutamate, and (4) MK-801 preceding MCAO. All rats were sacrificed at 30 minutes after single intracerebroventricular injection or surgery, and the right brain of each rat was used to detect Fos protein expression by Western blot. In determining the application amount of glutamate, we first calculated that in a total intracranial cerebrospinal fluid (CSF) of 200 mL in an adult rat,12 injection of .5 mmoles of L-glutamate in 10 mL into the lateral ventricle would create a glutamate concentration of 2500 mmol/L after its even distribution in the CSF, which is much higher than 8.2 mmol/L, a positive predictive value for progressing stroke13 and ensures sufficient glutamate diffused into the brain parenchyma from the CSF.

Preparation of Cerebral Ischemia Model by MCAO Following an overnight fast, each rat was anesthetized with chloral hydrate (350 mg/kg, intraperitoneally). After exposure of the right common carotid artery, internal carotid artery, and external carotid artery surgically, a paraffin wax–coated fishing thread (diameter .28 mm) was aseptically introduced into the right common carotid artery, in an anterograde fashion toward the carotid bifurcation. It was then directed distally up the right internal carotid artery to a distance of 17.5 6 .5 mm from the carotid bifurcation to occlude the origin of the MCA. In sham-operated rats, the thread was immediately removed as soon as the origin of the MCA was reached.

Identification of Cerebral Infarction and Evaluation of Neurologic Deficit after MCAO The neurologic deficits were evaluated by counting cumulative scales from scale 0-4, no visible neurologic deficits as the scale 0, forelimb flexion as the scale 1, contralateral forelimb grips weakly as the scale 2, if the animal was allowed to move around freely, it circled to the paretic side only when pulled by the tail as the scale 3, and spontaneous circling as the scale 4, respectively. For identification of the cerebral infarction, the brain was removed out and dissected into coronal sections of 2-mm thick after the animal was tested for neurologic deficits, then immersed into a saline solution containing 2% of 2, 3, 5-triphenyltetrazolium chloride at 37 C for 30 minutes. If the scale was over 2 and 2, 3, 5-triphenyltetrazolium chloride staining was significant, the model was successful (Fig 1).

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Figure 1. Representative diagrams of cerebral infarction in the rats with right middle cerebral artery occlusion. Coronal brain sections stained with 2% of 2, 3, 5-triphenyltetrazolium chloride. Note that the areas with red represents normal tissue, with white represents infarct tissue (black arrow) (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article).

Intracerebroventricular Injection The head of rat was fixed on the stereotaxic instruments (SN-3; Narishige, Japan) and oriented according to the B coordinate system of Pellegrino’s atlas after anesthesia. A stainless steel tube with an external diameter of .8 mm was inserted into the right lateral ventricle (Anterior to the Bregma point, 0.1 mm; right to the midline, 1.5 mm; and 3.0 mm from the surface of the skull.) for injection. All injections were completed over 1 minute with a Hamilton syringe, and the syringe was left in situ for 5 minutes.

Immunohistochemistry Rats were overdosed with chloral hydrate (700 mg/kg, intraperitoneally) and transcardially perfused with 300 mL of phosphate-buffered saline (PBS, pH 7.4) followed by 500 mL of cold 4% paraformaldehyde in PBS (pH 7.4). Then, the brains were removed and postfixed in the same fixative at 4 C overnight. The hypothalamus with surrounding brain tissues was dissected and embedded in paraffin. Coronal brain sections (50 mm in thickness) throughout the rostrocaudal extent of the hypothalamus were obtained on a Manual Rotary Microtome (Leica RM2016, Shanghai, China). A total of 10 hypothalamic sections were taken from each animal, according to Paxinos & Watson atlas, which contain the PVN (plane of sections relative to bregma: P1.8P2.12 mm). These sections were rehydrated and microwaved in citric acid buffer to retrieve antigens. After inhibition of endogenous peroxidases with 10% H2O2, serial sections were incubated with blocking buffer (5% bovine serum albumin [BSA]). Next, all sections were reacted with the primary polyclonal rabbit anti-Fos antibody (dilution, 1:2000 in .01 M PBS; pH, 7.4; sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight and then were reacted with alkaline phosphatase goat anti-rabbit IgG (dilution, 1:500 in .1% BSA; pH, 7.4; ZB-2308; ZSGB, China.) at 37 C for 30 minutes. The antigen–antibody reaction sites were visualized with DAB-kit (ZLI-9031; ZSGB, China) and the reaction was stopped in PBS. These sections were then stained with hematoxylin, dehydrated through graded alcohols, cleared in xylene, and finally, cover-slipped with Distyrene Plasticizer Xylene. The images were visually examined with conventional microscopy. The positive immune substance of

Fos protein in the PVN appeared yellow brown and located in the nuclei. In high magnification visions (3400), the number of positive neurons was counted in 5 areas and the reported numbers of each sample were the average of these 5. The examiner was blinded to the treatment. To eliminate the possibility of nonspecific staining, controls of no-primary and no-secondary antibody reactions were also performed.

Western Blot The PVN was quickly rinsed in ice-cold PBS buffer (pH, 7.4) containing protease inhibitors and homogenized with 200 mL ice-cold Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitors (1 mM phenylmethylsulfonylfluoride) in a homogenizer. The homogenate was then centrifuged at 10000 rpm for 10 minutes at 4 C and the supernatant was collected. Protein concentration of the collected supernatant was determined by BCA protein assay kit (Beyotime Biotechnology, China). Equal amount of total lysate was denatured at 95 C for 5 minutes and then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinyl difluoride membrane (Millipore, Bedford, MA), and the blots were blocked with 3% BSA dissolved in Tris-buffered saline containing .1% Tween 20 for 30 minutes at room temperature. Membranes were then incubated overnight at 4 C with the primary antibodies against Fos (sc-52) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Kangcheng, Shanghai, China), respectively. The GAPDH was used as an internal control for equal input of protein samples. Then the membranes were incubated with alkaline phosphatase–conjugated secondary antibodies (ZB-2308) at 37 C for 60 minutes and the blots were visualized using the alkaline phosphatase color development kit (Beyotime Biotechnology). Alphaview 3.1.1.0 software was used to quantify the band intensities. Relative levels of Fos protein were attained by normalizing the band densities to those of GAPDH, and then, compared with that of controls.

Statistical Analysis All experiments data were presented as mean 6 standard error. Student paired t test with 2-tails was used for

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Figure 2. ECG changes in MCAO Rats. (A) Normal ECG; (B) examples of bigeminy were recorded by an ECG detector from most MCAO rats; (C) examples of ventricular tachyarrhythmia from a few MCAO rats and most of them died. Abbreviations: ECG, electrocardiography; MCAO, middle cerebral artery occlusion.

comparisons between 2 groups (control and experimental groups). Statistical comparisons among multiple groups were performed using analysis of variance. Differences were considered to be statistically significant when P , .05.

Results ECG Changes in MCAO Rats To link arrhythmias with ischemic stroke, we first observed ECGs of rats from different groups. The ECGs of sham-operated rats (n 5 16) were all normal. The incidence of arrhythmia with right MCAO was observed as high as 75.89% (85 of 112), which is significantly higher than that of sham-operated rats (P , .001, chi-square test). There were 2 episodes of arrhythmias, the peak times of which occurred at 15.63 6 1.58 minutes and 238.36 6 14.32 minutes after MCAO and lasted 31.02 6 1.85 minutes and 26.28 6 1.36 minutes, respectively. The type of arrhythmia was mainly premature ventricular contraction (especially ventricular bigeminy) and occasionally ventricular tachyarrhythmia (Fig 2). The incidence of arrhythmia with single intracerebroventricular injection of L-glutamate was observed in 87.5% (7 of 8) of the rats, similar to those with MCAO, which is significantly higher than that of saline group (P , .001, chi-square test). The incidence of arrhythmia with single intracerebroventricular injection of MK-801 with or without L-glutamate was both zero (0 of 8). In the MCAO rats with prior application of MK-801, the incidence of arrhythmia was also zero (0 of 8). These data strongly indicate that cerebral ischemia could induce arrhythmia and that the NMDA receptors mediated the arrhythmogenic effect of glutamate.

Fos Immunohistochemical Staining after MCAO To establish a correlation of the arrhythmias with PVN neuron activity, we further observed expressions of Fos proteins in immunostaining. Fos protein is an objective indicator reflecting the functional state of neurons. In the control group and vehicle/sham group, only a small amount of lightly stained positive nuclei could be seen in the PVN, whereas in the MCAO groups, the color of positive nuclei in the PVN became dark and the number of positive nuclei increased (Fig 3, A). Compared with control group, the number of Fos positive nuclei in the PVN at 15 minutes (P , .05), 30 minutes (P , .01), and 4 hours group (P , .01) was all significantly higher than control (Fig 3, B), respectively.

The Expression of Fos Protein in the PVN after MCAO To further quantitate Fos expressions, we performed Western blots of the PVN lysates (Fig 3, C). After MCAO, the expression of Fos protein in the PVN (Fig 3, D) increased significantly at 15 minutes (P , .05), 30 minutes (P , .01), and 4 hours (P , .01). These findings indicate that the PVN neurons were activated twice after MCAO, a result consistent with those of immunohistochemical observations.

Fos Protein Expressions in the PVN and Glutamatergic Transmission Ischemic stroke could increase levels of extracellular glutamate in the brain10; microinjection of L-glutamate into the PVN could increase sympathetic nerve activity and cause positive cardiovascular response.9 In this work, we further examined if this glutamatergic action

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Figure 3. Fos expressions in the PVN in different experimental groups. (A) Typical immunohistochemical images of the PVN. (a), Control group, (b), sham group, (c-i) MCAO group sampled at15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 16 hours after MCAO, respectively. (B) Summary graph showing the number of Fos positive nuclei in the PVN in different groups. *P , .05, **P , .01 versus control group in analysis of variance. (C and D) Exemplary bands of Fos protein levels in Western blots (C) and the bar graph (D) summarizing relative changes in Fos protein levels at different times after MCAO. Abbreviations: MCAO, middle cerebral artery occlusion; PVN, paraventricular nucleus.

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Figure 4. Fos protein levels at different glutamatergic activities in Western blots. (A) Exemplary bands of Western blot in blank control and intracerebroventricular microinjections of saline (10 mL), L-glutamate (.5 mmoles, 10 mL), MK-801 (4 nmoles, 10 mL) before glutamate, and MK-801 before MCAO. (B) Bar graphs summarizing relative changes in Fos protein levels at different conditions specified above. *P ,.05 versus control group in analysis of variance. Abbreviation: MCAO, middle cerebral artery occlusion.

was mediated by NMDA receptor. We first verified that microinjection of L-glutamate into the lateral ventricle caused a significant increase in Fos protein expression in the PVN (P , .05) and typical arrhythmia, whereas the same amount of saline injection had no effect (P . .05). Microinjection of MK-801 into the lateral ventricle alone did not influence Fos expression in the PVN; however, prior application of MK-801 blocked the increases in Fos levels after glutamate injection (P . .05) or MCAO (P . .05; Fig 4). These findings support a hypothesis that during ischemic stroke the increased glutamate in the brain10 activates the PVN neurons via NMDA receptors.

Discussion In the present study, we found that MCAO induced arrhythmias and increased PVN neuron activity significantly, which could be simulated by intracerebroventricular injection of L-glutamate and blocked by prior application of MK-801. These results indicate that activating NMDA glutamate receptors is responsible for the occurrence of arrhythmias in ischemic stroke, which is likely mediated by the increase in PVN neuron activity. It is known that cardiovascular disturbances with imbalanced outflows of sympathetic and parasympathetic activities occurred in patients of cerebrovascular diseases.5 Dogan et al3 further identified that arrhythmias occur in 44% of the patients of ischemic stroke without

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history of primary heart diseases. Consistently, we found that the incidence of arrhythmia occurred in 75.89% of the rats with MCAO. The higher incidence of arrhythmias in experimental animals may reflect the uniformity of ischemic stroke in MCAO rats relative to the various degrees of injuries in stroke patients. Nevertheless, our results confirm the previously observed correlation between brain injury and arrhythmias. In most cases, occurrence of arrhythmias is considered to be because of excessive activation of sympathetic output.14 The PVN is a crucial brain region in the control of autonomic functions and cardiovascular activity, and stimulation of the PVN could increase cardiac sympathetic activity and induce arrhythmia.6,7 The PVN mainly contains 2 types of neurons: magnocellular neurons and parvocellular neurons.6 The magnocellular neurons are mainly in the dorsolateral regions and parvocellular neurons are mainly in the ventromedial regions. The main function of magnocellular neurons is synthesis of arginine vasopressin (AVP) and oxytocin. The parvocellular neurons are further divided into parvocellular neuroendocrine neurons and parvocellular preautonomic neurons according to their function and morphology.15 The parvocellular preautonomic neurons send their projections to intermediolateral cell column (IML) and form synapses with sympathetic preganglionic neurons in the IML to excite cardiac sympathetic nerve directly.16 Our results demonstrated that activated PVN neurons in MCAO rats complicating with arrhythmia presented at both the dorsolateral and ventromedial regions. These findings suggest that increases in sympathetic outflow through the PVN-IML pathway are responsible for MCAO-associated arrhythmias although an involvement of magnocellular neurons of the PVN could not be excluded. This proposal is consistent with previous reports that stimulation of the parvocellular preautonomic neurons with an excitatory amino acid increases the renal sympathetic nerve activity and heart rate, and these responses could be blocked by intrathecal applications of AVP receptor antagonists and oxytocin receptor antagonists in thoracic spinal cord, respectively.17 In addition, after the ischemic stroke, plasma AVP levels increase significantly in rats with MCAO,18 which can depress myocardial function.19Taken together, these findings indicate that MCAO-associated arrhythmias are due to activation of peptidergic neurons in the PVN. The PVN receives glutamatergic and GABAergic projections from forebrain and other surrounding nuclei. Neuron activity in the PVN depends on the balance between the glutamatergic and GABAergic inputs on the PVN neurons20; the former activates the PVN and the latter inhibits it. Normally, the tonic inhibitory effect of g-Aminobutyric acid (GABA) dominates activity of the PVN.21 However, once intranuclear glutamate levels increase and surpass GABAergic inhibition, the PVN

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neuron activity would increase. In the PVN, glutamate mainly acts on the NMDA receptors to activate PVN neurons.8 Several studies have also shown that glutamate levels increased significantly in the blood and the CSF of patients with acute ischemic stroke.22,23 However, Brouns et al24 failed to replicate glutamate elevation in the CSF of stroke patients recently. The controversy could be because of the following facts as the authors pointed out: (1) most glutamate in CSF was measured in the earlier stage after stroke onset, which is too early to let high-level glutamate in the brain parenchyma diffuse into the CSF yet; (2) most samples in controls were from patients with clinical suspicion of diseases, which increased the variance range of glutamate concentration in controls, making potential increases of glutamate in stroke group less clear statistically; (3) in previous studies, high CSF glutamate levels were detected mainly in a group of selected unstable progressing stroke patients13,25; however, in Brouns’ research, the progressing stroke patients only accounted for 15.7% of their study population. Thus, they could not replicate glutamate elevation in CSF of stroke patients. The MCAO model we used in this study likely simulates the unstable progressing stroke and could induce increases in brain glutamate content as reported.26 Therefore, our result supports most of the previous findings that stroke can significantly increase brain CSF glutamate levels. It was also observed that there were 2 profiles of the increases in glutamate levels: the first increase occurred in a few hours after the ischemic stroke and the second increase occurred at about 24 after ischemic stroke.27 In our study, the activity of the PVN neurons also increased twice after MCAO, which is consistent with the glutamate changes in patients with ischemic stroke. We also found that microinjection of L-glutamate into the lateral ventricle caused a significant increase in PVN neuron activity and typical arrhythmia, and that could be blocked by MK-801. Although Fos expression can be induced by many stimuli other than glutamate and the PVN may not be the only mediator of increased glutamate in the brain and CSF during MCAO, the PVN is certainly an important target of MCAO-induced glutamate increase. Together, the present results strongly suggest that the PVN neurons are activated via NMDA receptors by high levels of glutamate in the PVN of patients with acute ischemic stroke. Taken together, our results confirm that activation of the PVN is involved in the arrhythmias during MCAO and that activation of NMDA receptors along the cerebroventricular cavity, particularly the PVN, underlies the arrhythmias. The arrhythmias induced by PVN activation are likely mediated by increasing the sympathetic nerve excitability descending from the parvocellular PVN neurons while involvement of magnocellular neurons remains possible.

Acknowledgments: The authors thank Dr. Yu-Feng Wang for editorial assistance.

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ISCHEMIC STROKE COMPLICATING WITH ARRHYTHMIA 18. Chang Y, Chen TY, Chen CH, et al. Plasma argininevasopressin following experimental stroke: effect of osmotherapy. J Appl Physiol 2006;100:1445-1451. 19. Indrambarya T, Boyd JH, Wang Y, et al. Low-dose vasopressin infusion results in increased mortality and cardiac dysfunction following ischemia-reperfusion injury in mice. Crit Care 2009;13:R98. 20. Herman JP, Tasker JG, Ziegler DR, et al. Local circuit regulation of paraventricular nucleus stress integration: glutamate-GABA connections. Pharmacol Biochem Behav 2002;71:457-468. 21. Li YF, Jackson KL, Stern JE, et al. Interaction between glutamate and GABA systems in the integration of sympathetic outflow by the paraventricular nucleus of the hypothalamus. Am J Physiol Heart Circ Physiol 2006; 291:H2847-H2856. 22. Castillo J, Davalos A, Naveiro J, et al. Neuroexcitatory amino acids and their relation to infarct size and neurological deficit in ischemic stroke. Stroke 1996; 27:1060-1065.

851 23. Skvortsova VI, Raevskii KS, Kovalenko AV, et al. Levels of neurotransmitter amino acids in the cerebrospinal fluid of patients with acute ischemic insult. Neurosci Behav Physiol 2000;30:491-495. 24. Brouns R, Van Hemelrijck A, Drinkenburg WH, et al. Excitatory amino acids and monoaminergic neurotransmitters in cerebrospinal fluid of acute ischemic stroke patients. Neurochem Int 2010;56:865-870. 25. D avalos A, Castillo J, Serena J, et al. Duration of glutamate release after acute ischemic stroke. Stroke 1997; 28:708-710. 26. Yang MX, Yu WT, Hu JK, et al. Effect of Zhongfengkang on cerebral glutamate and calcium of rats with focal cerebral ischemia. Chinese Journal of integrative medicine on cardio/cerebrovascular disease 2005;3: 877-878. 27. Yang Q, Zhang M, Zheng J, et al. Dynamic change of serum glutamic acid and neurologic impairment in patient with ischemic stroke. Chinese Journal of Clinical Rehabilitation 2005;9:96-97.

Involvement of the paraventricular nucleus in the occurrence of arrhythmias in middle cerebral artery occlusion rats.

Ischemic stroke complicating with arrhythmia is one of the main causes of sudden death. To investigate the association between ischemic stroke-induced...
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