brain research 1585 (2014) 63–71

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Research Report

Protective effect of stellate ganglion block on delayed cerebral vasospasm in an experimental rat model of subarachnoid hemorrhage Na Hua, Yun Wua, Bai-Zhao Chenb, Jin-Feng Hana, Mai-Tao Zhouc,n a

Jiangsu Province Key Laboratory of Anesthesiology & Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical College, Xuzhou, 209 Tongshan Road, 221004, JS, China b Department of Anesthesiology, Jiangxiaqu No. 1 People's Hospital, Wuhan 430000, HB, China c Department of Anesthesiology, the 101st Hospital of CP.L.A., Xuzhou Medical College, 101 Xing Yuan North Road, Wuxi 214044, JS, China

art i cle i nfo

ab st rac t

Article history:

Stellate ganglion block (SGB) is a blockade of sympathetic ganglia innervating the head and

Accepted 7 August 2014

neck, and is known to function through vasodilation of the target region. However, the

Available online 13 August 2014

effectiveness of SGB in relieving cerebral vasospasm (CVS) through dilation of intracerebral

Keywords:

vessels has not been evaluated. The aim of the present study is to investigate the

Cerebral vasospasm

therapeutic effects of SGB in a rat model of subarachnoid hemorrhage (SAH) complicated

Experimental subarachnoid

by delayed CVS, and explore the underlying mechanisms. The SAH model was established

hemorrhage

by double injection of autologous arterial blood into the cisterna magna. We simulated SGB

Stellate ganglion block

by transection of the cervical sympathetic trunk (TCST), and measured changes in the

Rat

diameter, perimeter and cross-sectional area of the basilar artery (BA) and middle cerebral artery (MCA) to evaluate its vasodilatory effect. To investigate the underlying mechanisms, we determined the expression level of vasoactive molecules endothelin-1 (ET-1) and calcitonin gene-related peptide (CGRP) in the plasma, and apoptotic modulators Bcl-2 and Bax in the hippocampus. We found a significant increase in the diameter, perimeter and cross-sectional area of the BA and right MCA in SAH rats subjected to TCST. Application of SGB significantly reduced the expression of ET-1 while increasing that of CGRP in SAH rats. We also found a significant increase in the expression of Bcl-2 and decrease in the expression of Bax in the hippocampus of SAH rats subjected to TCST, when compared to untreated SAH rats. The mechanism of action of SGB is likely mediated through alterations in the ratio of ET-1 and CGRP, and Bax and Bcl-2. These results suggest that SGB can alleviate the severity of delayed CVS by inducing dilation of intracerebral blood vessels, and promoting anti-apoptotic signaling. Our findings provide evidence supporting the use of SGB as an effective and well-tolerated approach to the treatment of CVS in various clinical settings. & 2014 Elsevier B.V. All rights reserved.

n

Corresponding author. Tel./fax: þ86 0510 85142312. E-mail addresses: [email protected] (N. Hu), [email protected] (M.-T. Zhou).

http://dx.doi.org/10.1016/j.brainres.2014.08.012 0006-8993/& 2014 Elsevier B.V. All rights reserved.

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1.

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Introduction

Delayed cerebral vasospasm (CVS) remains an unpredictable and inadequately treated complication of subarachnoid hemorrhage (SAH). CVS begins at day 3, reaches peak severity at days 6–8, and usually lasts 2–3 weeks following the occurrence of SAH. It is characterized by sustained narrowing of the arteries, causing a reduction in cerebral blood flow, leading to ischemia and infarction in the brain. The outcome and prognosis of SAH is poor (Hong et al., 2012; Kolias et al., 2009; Nolan and Macdonald, 2006). In patients developing CVS after SAH, the risk of mortality is increased 1.5–3 fold in the first 2 weeks (Biller et al., 1988). Current management modalities for this condition include hypervolemic–hemodilution–hypertensive (HHH) therapy (Hunt and Bhardwaj, 2007), interventional neuroradiological procedures like transluminal angioplasty (Newell et al., 1989), and administration of drugs such as calcium channel antagonists, HMG-CoA reductase inhibitors (or statins) and endothelin-1 antagonists (Athar and Levine, 2012; Siasios et al., 2013). Despite the limited success achieved by these approaches, 26–38% patients either develop sequelae or die of severe symptomatic vasospasm (Dorsch, 1995; Newell et al., 1989). The success of the current treatment strategies against delayed CVS after SAH remain inconsistent, and seem to have variable effects on the outcome (Rabinstein et al., 2003) Therefore, continuous efforts are being made to improve the management of delayed CVS by developing strategies to enhance cerebral blood flow more effectively. The human stellate ganglion (SG) is composed of the inferior cervical ganglion and the superior thoracic ganglion of the sympathetic nerve trunk. Stellate ganglion block (SGB) is a technique used to temporarily block sympathetic innervation of the head, neck and upper extremities, inducing peripheral vasodilation in these regions, that causes complete inactivation of the sympathetic nervous system in this region, and helps relieve pain, swelling and other distressing symptoms (Elias, 2000). Based on the knowledge that rats do not possess ganglia analogous to the human SG, but that the middle and inferior cervical sympathetic ganglia perform functions similar to the human SG (Hanamatsu et al., 2002), we simulated SGB in a rat SAH model by performing TCST (transection of the cervical sympathetic trunk). Several reports have demonstrated that TCST in rats could result in the same outcome as long-term repetitive SGB (Okada et al., 1996; Xiong et al., 2008; Zhang et al., 2004), indicating that rat TCST is an ideal model for simulating human SGB. Although several reports have provided evidence in favor of SGB as a tool for relieving CVS and improving intracerebral blood flow following SAH, the reliability of its therapeutic effects and the precise mechanism underlying its function remain unclear (Bindra et al., 2011; Jain et al., 2011; Prabhakar et al., 2007; Treggiari et al., 2003). In this study, we investigated the molecular mechanisms mediating and regulating the effect of SGB on delayed CVS following hemorrhage. First, we assessed the severity of vasospasm by measuring neurological function, and the diameter, perimeter and crosssectional area of the basilar artery (BA) and middle cerebral artery (MCA). Next, we examined alterations in two pathways

associated with cardiovascular pathologies: (1) the relative expression level of the vasoconstrictor endothelin-1 (ET-1) and the vasodilator calcitonin gene-related peptide (CGRP); and (2) the ratio of pro-apoptotic protein Bax and antiapoptotic Bcl-2, indicative of the strength of apoptotic signaling. Our observations indicate that changes in the relative expression of aforementioned genes could play a significant role in facilitating the therapeutic effect of SGB during CVS.

2.

Results

2.1. TCST treatment improves neurological function in SAH rats We conducted tests to assess neurological function in rats at 48 h following induction of SAH. In comparison to the sham group, the neurological score in SAH rats was significantly higher (median score 10 [8–12] versus 0 [0–1], po0.05). Following TCST, these rats displayed a significant reduction in motor and behavioral deficits compared to the SAH group (median score 5 [3–7 versus 10[8–12], po0.05).

2.2.

TCST treatment reduces vasospasm in SAH rats

As shown in Figs. 1 and 2, we found significant differences in the morphology of BA and MCA between the four experimental groups on day 7 after SAH. Examination under the brightfield microscope showed that the BA and MCA were regularly aligned with endothelial cells and elastic lamina in both control groups, sham and normal. However, in the SAH group, prominent morphological vasoconstriction was observed, characterized by a corrugated internal elastic lamina, thickened vessel wall, and contracted smooth muscle cells. Interestingly, vasospasm of the BA and right MCA was clearly attenuated in rats subjected to TCST. Here, the endothelial vessel lining, elastic lamina and smooth muscle cells were found to be intact and comparable to the control groups. Next, we analyzed the images further and quantified the aforementioned anatomical features. We found a marked reduction in the diameter, perimeter and cross-sectional area of the BA in SAH group compared with the sham group (diameter: 189.70721.95 versus 237.86721.50 mm, po0.01; perimeter: 786.33739.29 versus 848.37739.97 mm, po0.01; area: 30229.7976432.62 versus 41726.8472037.30 mm2, po0.001). Treatment with TCST led to a significant increase in these characteristics of the BA compared with the SAH group (diameter: 225.88726.09 versus 189.70721.95 mm, po0.05; perimeter: 827.14730.55 versus 786.33739.29 mm, po0.05; 39964.3876190.03 versus 30229.7976432.62 mm2; po0.01). No significant differences were found between normal and sham groups (p40.05) Fig. 3. As shown in Fig. 4, we found a significant reduction in the diameter, perimeter and cross-sectional area of the right MCA in the SAH group compared to the sham group (po0.01, po0.01 and po0.001, respectively). As seen before, TCST treatment caused a significant increase in these parameters in comparison to the untreated SAH group (po0.05, po0.05 and po0.01, respectively). These observations suggest that

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Fig. 1 – Effect of TCST on the morphology of the basilar artery in SAH rats. HE staining of the BA( 100) A–D: Representative brightfield images of the BA in all four experimental groups, procured with a light microscope. (A) Normal group, showing the endothelium with an elastic lamina and muscle cell nucleus; (B) sham group showing the endothelium with an elastic lamina and muscle cell nucleus; (C) SAH group showing a vasoconstricted artery with contracted endothelium, corrugated elastic lamina, and interstitial edema; and (D) SAHþTCST group showing a slightly constricted artery, with the endothelium, elastic lamina, and muscle cell nucleus.

application of SGB can successfully attenuate the vasoconstriction induced by SAH in the BA and right MCA.

that SGB induces a decrease in the ratio of ET-1/CGRP, in favor of vasodilation.

2.3. SGB reduces ET-1 and increases CGRP concentration in plasma

2.4. SGB inhibits Bax and upregulates Bcl-2 expression in the hippocampus

To determine the effect of SGB on vasoactive substances, we performed ELISA on the plasma samples collected from animals of all four groups, to examine changes in the protein concentration of the vasoconstrictor ET-1 and the vasodilator CGRP. As shown in Fig. 5, in comparison to the sham groups, ET-1 expression was markedly increased in all SAH rats (SAH group: 53.6876.68 versus 27.9974.02 pg/ml, po0.001; SAHþTCST group: 36.7074.85 versus 27.9974.02 pg/ml; po0.01). Application of SGB significantly lowered the elevation of plasma ET-1 concentration in SAH rats (36.7074.85 versus 53.6776.68 pg/ml, po0.001). In contrast, the level of CGRP was significantly reduced after SAH, when compared with the sham group (SAH group: 633.32755.61 versus 877.14788.48 pg/ml, po0.001; SAHþTCST group: 746.16784.53 versus 877.14788.48 pg/ml, po0.01). Application of SGB elevated this depletion of plasma CGRP (746.16784.53 versus 633.32755.61 pg/ml, po0.01). These observations suggest

We performed immunohistochemical analyses to quantify the number of cells per visual field, expressing the pro-apoptotic protein Bax in the hippocampus of sham, SAH and SAHþTCST groups (Fig. 6). We found a significant upregulation of Bax in all SAH rats when compared to the sham group (SAH group: 26.8372.29 versus 5.1471.17, po0.001; and SAHþTCST group: 16.2071.22 versus 5.1471.17, po0.001). This elevation in Bax expression was partially, but significantly rescued in the SAHþTCST group (16.2071.22 versus 26.8372.29, po0.001). The expression of the pro-survival protein Bcl-2 in the hippocampus of SAH group was significantly lower than in the sham group (po0.001). However, in contrast to Bax, treatment with TCST caused a larger increase in Bcl-2 expression, compared to the SAH group (po0.001). These observations suggest that SGB induces a decrease in the Bax/Bcl-2 ratio in the hippocampus, in favor of anti-apoptotic signaling.

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3.

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Discussion

To the best of our knowledge, the present study is the first to report the therapeutic effects of stellate ganglion block on cerebral vasospasm following the induction of experimental subarachnoid hemorrhage in rats. Based on examination and quantification of the changing morphology of the basilar and

middle cerebral arteries, we have demonstrated that SGB can reduce CVS, by triggering dilation of these blood vessels. The double-hemorrhage model used in this study has been shown to effectively generate a higher degree of vasospasm with lower mortality, and hence seems more appropriate than the traditional model of subarachnoid hemorrhage (Gules et al., 2002; Lee et al., 2008; Meguro et al., 2001). The SAH rat models generated by this method follow the same time course

Fig. 2 – Effect of TCST on the morphology of the middle cerebral artery in SAH rats. HE staining of the MCA( 200). A–D: Representative brightfield images of the MCA in all four experimental groups, procured with a light microscope. (A) Normal group, (B) sham group, (C) SAH group, and (D) SAHþTCST group.

Fig. 3 – Quantification of the anatomical features of the basilar artery. Bar graphs represent averaged values of (A) crosssectional area, (B) perimeter, and (C) diameter of the BA in all four experimental groups. SAH versus sham **po0.01, ***po0.001; SAHþTCST versus SAH #po0.05, ##po0.01.

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Fig. 4 – Quantification of the anatomical features of the middle cerebral artery. Bar graphs represent averaged values of (A) cross-sectional area, (B) perimeter, and (C) diameter of the right MCA in all four experimental groups. Data are represented as mean7SD, n ¼ 8 rats per group. SAH versus sham **po0.01, ***po0.001; SAHþTCST versus SAH #po0.05, ##po0.01.

Fig. 5 – ELISA-based examination of the effect of TCST on the concentration of ET-1 and CGRP in the plasma. Concentrations of (A) ET-1 and (B) CGRP in normal, sham, SAH and SAHþTCST groups 120 h after SAH induction. Data are represented as mean7SD, n¼ 8 rats per group. SAH versus sham **po0.01, ***po0.001; SAHþTCST versus SAH ## po0.01, ###po0.001. of pathological progression as humans, with maximum narrowing of blood vessels at day 7, and therefore were used in this study (Meguro et al., 2001). We examined the effects of SGB on the morphology of constricted BA and MCA vessels following induction of SAH. Our results clearly show that SGB can lead to a significant increase in the diameter, perimeter, and cross-sectional area of the MCA and BA, and improve the gross anatomical appearance of these arteries. This suggests that SGB could alleviate, and possibly even prevent the occurrence of CVS following SAH.

Fig. 6 – Immunohistochemical analysis of the effect of TCST on the expression of Bax and Bcl-2 in the hippocampus. Box plot quantification of the number of cells (per visual field) stained positive for (A) Bax and (B) Bcl-2, in sham, SAH and SAHþTCST groups. SAH versus sham ***po0.001; SAHþTCST versus SAH ###po0.001. SGB is an established therapeutic procedure used routinely for treating patients diagnosed with disorders of the sympathetic nervous system, characterized predominantly by pain and/or circulatory deficiencies, but also including conditions such as post-herpetic neuralgia and complex regional pain syndrome (reflex sympathetic dystrophy) (Yucel et al., 2009). The concept of using SGB for relieving CVS is not novel (Jain et al., 2011). Jain et al. demonstrated that SGB can lead to a significant increase in cerebral perfusion by relieving

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symptomatic CVS. They also demonstrated its efficacy in two patients who failed to respond to papaverine administration (Prabhakar et al., 2007). In addition, SGB can also improve cerebral blood circulation and increasing oxygen supply in certain cerebrovascular complications (Bindra et al., 2011; Moore, 2006). These findings further corroborate the observation that SGB triggers vasodilation and improves the pathology of SAH in our model system. Umeyama et al. reported that SGB can increase intracerebral blood flow and improve the blood flow in the retino-choroidal arteries, a branch of the internal carotid artery, by 20% (Umeyama et al., 1993, 1995). Furthermore, Zhang et al. reported that SGB performed before operation could significantly reduce the incidence of CVS after clipping of intracranial aneurysm. Altogether, these reports demonstrate that SGB has high potential to mitigate CVS following the induction of SAH (Zhang et al., 2011). However, some studies have reported contradictory results where SGB was not found to have a significant impact on cerebral and extracerebral blood flow. One report performed digital subtraction angiography (DSA), and noted that while cervical sympathectomy (simulation of SGB) could improve cerebral perfusion and increase cerebral circulation time, it did not change the caliber of major vessels (Treggiari et al., 2003). Another report used magnetic resonance angiography to conclude that while that SGB could increase the diameter of extracranial vessels of healthy volunteers, the intracranial vessels remained unaffected (Kang et al., 2010). Our study, however, demonstrates that SGB does have a vasodilatory effect on the intracranial vessels, MCA and BA, in a rat SAH model, proving its clinical potential as a treatment strategy to reverse CVS in patients suffering from subarachnoid hemorrhage. The stellate ganglion is composed of cell bodies of the inferior cervical and first thoracic sympathetic ganglia. The associated cerebral vasculature receive noradrenergic sympathetic input mainly through the fibers that originate in the cervical ganglion, accompany the carotid artery, and project into the ipsilateral cerebral hemisphere (Edvinsson, 1975; Tuor, 1990; Umeyama et al., 1995). Intracerebral vessels constrict in response to cervical sympathetic stimulation and dilate when these fibers are interrupted (Edvinsson, 1975; Tuor, 1990). A blockage of the stellate ganglion induces a definite ipsilateral increase in cerebral blood flow, as determined by cerebral scintigraphy (Umeyama et al., 1995). The mechanism underlying the physiological consequences of blockage of sympathetic nerve activity or reversal of overactivity, irrespective of whether the sympathetic disorder causes a critical reduction in cerebral blood flow, may be explained by the subsequent dilation of intracerebral vessels and improvement of cerebral blood flow. Studies have shown that SGB can induce a significant decrease in zero flow pressure, a surrogate marker of cerebral vascular tone, thus improving cerebral perfusion pressure (Gupta et al., 2005). One study used transcranial Doppler ultrasound to demonstrate an increase in carotid and vertebral blood flow after SGB (Ohinata et al., 1997). These findings, together with our own results, further support the suggested clinical use of SGB to relieve CVS following SAH. To explore the molecular mechanisms underlying SGB, we studied changes in the expression of two major factors that

contribute to CVS after SAH, ET-1 and CGRP. Endothelin-1 is the most potent known endogenous vasoconstrictor expressed during ischemic insult. It binds to specific receptors on smooth muscle cells and causes constriction of blood vessels and proliferation of endothelial cells (Chow et al., 2002). Increased levels of ET-1 are found in the plasma and cerebral spinal fluid (CSF) of SAH patients, suggesting that it might directly contribute to the vasospasm that often follows SAH (Juvela, 2000; Seifert et al., 1995). CGRP is the most potent vasoactive substance known. It is an intrinsic vasodilatory peptide found in the perivascular nerve fibers of intracranial arteries. It acts by binding to G-protein coupled receptors on blood vessels, Schwann cells and mononuclear cells, leading to vascular dilation in an endothelium- and NO (nitrous oxide)-independent manner (Lennerz et al., 2008). Several studies have reported changes in CGRP expression in perivascular nerve fibers after SAH. CGRP alone can significantly attenuate vasospasm in SAH, and greatly reduce cell death in the brain and endothelial lining (Sun et al., 2010). Some have suggested that intrathecal injection of CGRP into the cisterna magna could significantly dilate the spastic basilar artery (Imaizumi et al., 1996). In this study, we found that SGB significantly decreased the expression of ET-1 while increasing that of CGRP, possibly resulting in a molecular cascade favoring vasodilation. This finding suggests that SGB might alleviate CVS by regulating plasma concentrations of ET-1 and CGRP. The precise mechanism of SGB action needs to be studied further. It has been suggested that apoptotic cell death plays an important role in not only inducing the long-term morbidity associated with SAH, but also in the etiology of CVS, which in turn results in hypoperfusion and possibly cell death in related brain areas (Zhou et al., 2011). Apoptotic regulators of the Bcl family are widely distributed in the outer mitochondrial membrane and endoplasmic reticulum of cells, playing a key role in the apoptotic cascade. Bax plays a central role in the mitochondria-dependent apoptotic pathway. In response to a death signal, Bax is transported to the surface of the mitochondrial membrane, where it promotes influx of Ca2þ and release of cytochrome C. This causes a decrease in membrane potential, lack of ATP formation and activation of downstream caspase enzymes leading to apoptotic cell death (Danial et al., 2003). Alternately, the antiapoptotic regulator Bcl-2 can directly inhibit the function of Bax, prevent the accumulation of intracellular Ca2þ, and subsequent activation of caspase-mediated apoptosis (He et al., 1997; Scorrano and Korsmeyer, 2003). There is additional evidence from previous studies that in response to SAH, major mediators of the apoptotic pathway are suppressed, resulting in a significant upregulation of antiapoptotic Bcl-2, and downregulation of pro-apoptotic molecules, including Bax (He et al., 2012). In the present study, we performed immunohistochemical assays to identify, label and quantify the number of cells expressing Bax and Bcl-2 in the hippocampus of our SAH rat models with and without application of the stellate ganglion block. We show that in response to SGB, the expression of Bcl-2 is significantly enhanced concomitant with a decrease in Bax, in the SAH rats. This suggests that SGB might activate anti-apoptotic pathways by suppressing Bax and activating

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Bcl-2 simultaneously, and thus have a neuroprotective effect on hippocampal neurons in SAH rats. In conclusion, our study provides evidence that stellate ganglion block can be used for the treatment of cerebral vasospasm either supplementary to standard therapeutic procedures, or in cases where traditional approaches have failed or are inapplicable. It is a relatively easy, affordable and minimally invasive procedure. In addition, treatment can be repeated without the requirement of additional cerebral angiography. This study demonstrates that SGB can reverse cerebral vasospasm in SAH rat models by inducing vasodilation of intracerebral arteries. We also show that the increase in the ratio of expression of vasodilator CGRP/vasoconstrictor ET-1 may be partly responsible for the therapeutic effects of SGB. In addition, SGB seems to activate anti-apoptotic signaling pathways (by reducing Bax/Bcl-2 ratio) in the hippocampus. This could suppress neuronal loss and promote cell survival, and might be another mechanism underlying the beneficial physiological consequences of SGB. However, the precise mechanism of action of SGB remains to be investigated further.

4.

Experimental procedures

4.1.

Materials

All experimental protocols were approved by the Committee for Animal Care and Use of Xuzhou Medical College (Xuzhou, China). Experiments were conducted in accordance with animal ethics standards at the Institute of Neurological Diseases of the CP.L.A. no. 101 Hospital of Xuzhou Medical College. We obtained 80 healthy male SD rats weighing 300–350 g from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). ELISA kits were procured from Shanghai Westang Biotech Co., Ltd. (Shanghai, China). Rabbit anti-Bcl-2 and rabbit anti-Bax antibodies were purchased from Bioworld Technology Co., Ltd. (Nanjing, China). Other analytical reagents and equipments were supplied by the Institute of Neurological Diseases of CP.L.A. no. 101 Hospital. The rats included in the study were randomly assigned into four experimental groups: normal group (n¼8), not subjected to any treatment/intervention; sham group (n¼ 24), administered physiological saline into the subarachnoid space after sham surgery; SAH group (n¼24), subjected to SAH induction; SAHþTCST group (n¼24), subjected to SAH induction followed by right TCST. All rats were kept at 22–25 1C, 65–70% humidity, with a 12 h light/dark cycle, and were given sufficient food and water. Neurological examination was performed 48 h after SAH induction in all groups.

4.2.

Methods

4.2.1.

SAH induction

at 37 1C, and all surgeries were performed under aseptic conditions. We induced SAH by double autologous blood injection into the cisterna magna as previously reported, with some modifications (Ersahin et al., 2009; Hänggi et al., 2009; Lee et al., 2008). Briefly, the animal was placed in the supine position, and a catheter was placed in the left femoral artery for removal of blood. After positioning and fixing the animal, the cisterna magna was punctured using a needle with a rounded tip and a side hole, non-heparinized autologous blood (0.3 ml) obtained from the femoral artery was slowly injected into the cisterna magna over a 2 min period to induce the first SAH. The rat was titled 301 with the head down for 30 min to permit blood distribution around the basal arteries. Finally, the incision was sutured and the animal was allowed to recover from the effect of anesthesia before being returned to the cage. A second autologous blood injection was administered 48 h after the first injection (Wu et al., 2011). The day of first SAH induction was designated day 1. The rats in the sham group received two intracisternal injections of physiological saline instead of blood, according to the same surgical procedure. Rats that died during surgery were excluded from the experiment.

4.2.2.

Establishment of the TCST model

To simulate SGB, we preformed TCST in animals of the SAHþTCST group. Following the second injection of autologous blood, the right cervical sympathetic trunk was dissected 3 mm below the superior cervical ganglion on the dorsal surface of the right common carotid artery. A small (1–2 mm) piece of the nerve trunk was removed to minimize any occurrence of regeneration, and the incision was sutured (Richard et al., 2007; Xiong et al., 2008). Blockage was performed in the right cervical sympathetic trunk in all rats. In the sham and SAH group, the cervical sympathetic trunk was exposed but not transected. All rats were kept alive for 5 days following surgery under appropriate conditions.

4.2.3.

Neurological evaluation

The neurological potential of animals was evaluated 48 h after SAH induction using Bederson's modified neurological examination test, as described previously (Ersahin et al., 2009, 2010). Briefly, a 20-point neurological score was used to assess motor and behavioral deficits. The performance of animals in six different tests designed to assess consciousness, performance on a smooth climbing platform, extremity tonus, walking and postural reflexes, circling, and response to nociceptive stimuli, was evaluated. The motor and behavioral performance was graded within a score range of 0–20, with a higher score representing serious neurological deficits. The sequence in which animals were tested by a given task was determined randomly.

4.2.4. All invasive procedures were performed under anesthesia administered through intraperitoneal (i.p.) injection of chloral hydrate (350 mg/kg) and while the animals were allowed to breathe spontaneously. The body temperature was controlled

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ELISA

At day 7 after surgical intervention, blood samples were collected from anesthetized animals, and analyzed for ET-1 and CGRP expression levels using an ELISA kit specific for rats. To collect the plasma, the homogenates were centrifuged at 3000  g for 15 min, and the supernatant was assayed

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for the protein concentrations of ET-1 and CGRP, in accordance with the manufacturer's instructions. The concentrations (pg/ml) were determined based on a standard curve, prepared using a known set of serial dilutions of standard proteins. The number of animals used in the ELISA and histological study are as follows: normal (n¼ 8), sham (n¼ 8), SAH (n ¼8), and SAHþTCST (n¼ 8).

4.2.5. Histological analysis and measurement of diameter, perimeter and cross-sectional area of the basilar artery and middle cerebral artery At day 7 after surgical intervention, rats were anesthetized, perfused with physiological saline followed by 4% paraformaldehyde (PFA) (pH 7.4) and dissected to harvest the brain with an intact BA and MCA. This tissue was then post-fixed in 4% PFA at 4 1C overnight, dehydrated and embedded in paraffin. To avoid the arterial branches, the BAs were transected at the same middle position each time (2 mm). The right MCAs were transected at the origin of the arteries (2 mm). Every fourth slice sectioned along the coronal plane was stained with hematoxylin and eosin (HE) and observed under the microscope. The diameter, perimeter and cross-sectional area of the BA and MCA were measured using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD) and an Olympus microscope (Olympus, Optical Tokyo, Japan). Histological sections of BA and MCAs were imaged under the microscope and projected as digitized video images. The perimeter of the vessels was measured by tracing the luminal surface of the intima. For each animal (in each group), four slices each of the BA and MCA were analyzed, and the diameter, perimeter and crosssectional area were calculated and reported as the average of four independent measurements. All measurements were made by a technician who was blinded to the experiment.

4.2.6.

Immunohistochemistry

Anesthetized rats were perfused through the heart with physiological saline followed by 4% PFA (pH 7.4) on day 7 following SAH induction. The brain (with an intact hippocampus) was removed, post-fixed in 4% PFA overnight, embedded in paraffin, and sliced along the coronal plane into 5-mm thick sections. The sections were then mounted on glass slides, dewaxed through an alcohol gradient, soaked in antigen retrieval solution (citrate buffer, 0.01 M, pH 6.0), incubated with rabbit anti-Bcl-2 (1:100) and rabbit anti-Bax (1:100) primary antibodies overnight at 4 1C. Next day, slides were rinsed in 0.01 M PBS, incubated with biotinylated goat anti-rabbit IgG at room temperature (RT) for 30 min, followed by peroxidaselabeled streptavidin for 15 min. Finally, the immunostained cells were visualized by developing the sections in an appropriate substrate of peroxidase to generate a colored product. Cells whose cytoplasm stained brown were defined positive, and analyzed using the Image-Pro-Plus 6.0 image analysis system. Eight animals were used for the immunohistochemical study in the sham, SAH and SAHþTCST groups.

4.3.

Statistical analysis

Statistical analysis was performed with the SPSS 13.0 Statistical Software. All data are represented as mean7standard deviation (SD). Statistical differences between the various

groups was assessed by analysis of variance (ANOVA) performed in one-way ANOVA, followed by the LSD test for multiple comparisons. A value of po0.05 was considered statistically significant.

Acknowledgments This work was supported by Jiangsu University Science & Technology Development Foundation (No. JLY20120134).

r e f e r e n c e s

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