NF-κB-mediated inflammatory pathway

brain research 1594 (2015) 245–255

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Baincalein alleviates early brain injury after experimental subarachnoid hemorrhage in rats: Possible involvement of TLR4/NF-κB-mediated inflammatory pathway Chun-xi Wang, Guang-bin Xie, Chen-hui Zhou, Xiang-sheng Zhang, Tao Li, Jian-guo Xu, Ning Li, Ke Ding, Chun-hua Hang, Ji-xin Shin, Meng-liang Zhoun Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, Jiangsu Province, China

art i cle i nfo

ab st rac t

Article history:

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) largely contributes to

Accepted 9 October 2014

unfavorable outcomes. Hence, effective therapeutic strategies targeting on EBI have recently

Available online 17 October 2014

become a major goal in the treatment of SAH patients. Baicalein is a flavonoid that has been

Keywords:

shown to offer neuroprotection in kinds of brain injury models. This study investigated the

Subarachnoid hemorrhage

effects of baicalein on EBI in rats following SAH. SAH was inducted in male Sprauge-Dawley rats

Early brain injury

by injection of fresh non-heparinized arterial blood into the prechiasmatic cistern. Baicalein (30

Baicalein

or 100 mg/kg) or vehicle were administrated 30 min after injury. Neurological deficit, brain

Inflammation

edema, blood–brain barrier (BBB) permeability and neural cell apoptosis were assessed. To

Toll-like receptor 4

explore the further mechanisms, the change of toll-like receptor 4 (TLR4) and nuclear factor-κB

Nuclear factor-κB

(NF-κB) signaling pathway and the levels of apoptosis associated proteins were also examined. Our study showed that treatment with baicalein (30 mg/kg) significantly improved neurological function at 24 h after SAH and reduced brain edema at both 24 h and 72 h after SAH. Baicalein also significantly reduced neural cell death, BBB permeability. These changes were associated with the remarkable reductions of TLR4 expression, IκB-α degradation, NF-κB translocation to nucleus, as well as the expressions of matrix metalloproteinase-9, tight junctions protein, interleukin-1β and tumor necrosis factor- ɑ. These findings suggest that baicalein may ameliorate EBI after SAH potentially via inhibition of inflammation-related pathway. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Aneurismal subarachnoid hemorrhage (SAH) is a devastating stroke subtype with more than 50% combined morbidity and n

mortality (Macdonald, 2014). In the past decades, cerebral vasospasm after SAH was considered as the most important cause of high mortality and poor outcomes (Pluta et al., 2009) and has been long studied and treated, but the outcome is not

Corresponding authors. Fax: þ86 25 51805396. E-mail addresses: [email protected] (J.-x. Shi), [email protected] (M.-l. Zhou).

http://dx.doi.org/10.1016/j.brainres.2014.10.014 0006-8993/& 2014 Elsevier B.V. All rights reserved.

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analysis. No statistical differences were observed among groups with regard to physiological parameters (data not shown). The mortality rates within 72 h in each group were as follows: sham group 0% (0 of 27 rats), SAH group 18.2% (6 of 33 rats), SAHþDMSO group 20.6% (7 of 34 rats), SAHþbaicalein 30 mg/kg group 15.5% (5 of 32 rats), SAHþbaicalein 100 mg/kg group 25.0% (2 of 8 rats). Fig. 1 – Chemical structure of baicalein.

improved even if angiographic vasospasm is reversed (Macdonald et al., 2011; Vajkoczy et al., 2005). Early brain injury (EBI) has recently been coined and refers to the immediate injury to the brain as a whole, involving brain cell death, blood –brain barrier (BBB) disruption, brain edema, and microvascular dysfunction within the first 72 h of the onset, secondary to SAH (Cahill et al., 2006), and may contribute to the high mortality and morbidity rates of SAH (Cahill et al., 2006; Macdonald, 2014). Therefore, effective EBI treatment has recently become a major goal in SAH patients care (Sehba et al., 2012). The pathophysiological mechanisms of EBI are complicated. A number of clinical and animal studies have highlighted a strong contribution of inflammation to EBI after SAH (Sehba et al., 2012). It was reported that toll-like receptors 4 (TLR4), nuclear factor-κB (NF-κB), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) take part in the damaging inflammatory processes after SAH. It was also reported that inhibiting TLR4/NF-κB can down-regulate inflammation and be against brain injury after SAH (Ma et al., 2009; You et al., 2012, 2013). Therefore, TLR4/NF-κB signaling pathway may be a therapeutic target for EBI after SAH. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4one) is a flavonoid with a defined chemical structure (Fig. 1), which is isolated from the roots of Scutellaria baicalensis (Jianjun and Huiru, 2008). Evidence indicates that baicalein has multiple biological activities, like anti-inflammation (Shen et al., 2003), anti-oxidative stress (Gao et al., 2001) and inhibition of platelet aggregation (Huang et al., 2005). The neuroprotective effects of baicalein have been well documented. It has been reported that baicalein reduces inflammatory cytokines after experimental traumatic brain injury (Chen et al., 2008), attenuates cerebral cortex apoptosis (Lebeau et al., 2001) and prevents neurotoxicity induced by hydrogen peroxide (Zhang et al., 2010). All of these properties indicate that baicalein may be a potential agent for prevention and treatment of brain injury. Nevertheless, it is still unknown whether baicalein has the protective effects on EBI after SAH till now. Therefore, the present study was conducted to examine the effects of baicalein on EBI after SAH and whether baicalein can influence TLR4/NF-κB pathway in the brain after SAH.

2.

Results

2.1.

General observations and mortality rate

Total 151 rats were used in this study. Among them 12 rats died during the operation, which were excluded from further

2.2. Baicalein significantly alleviated brain edema at both 24 h and 72 h and improved neurologic function only at 24 h but not at 72 h after SAH Global edema is an independent risk factor for mortality and poor outcome after SAH (Claassen et al., 2002). In our study, baicalein was given at two different concentrations to SAH rats. Brain edema and neurological deficit were evaluated at 24 h and 72 h after SAH. No differences were noted in the water content in cerebellum or brain stem both at 24 h and 72 h after SAH. At 24 h after SAH, treatment with baicalein at both 30 mg/kg and 100 mg/kg dramatically reduced brain water content in the cerebrum and significantly attenuated neurological deficits (#Po0.05, ##Po0.01; Fig. 2A and C). At 72 h after SAH, baicalein (30 mg/kg) significantly reduced brain water content (#Po0.05; Fig. 2B) and had a tendency to reduce neurologic deficit without statistical differences (P ¼0.104; Fig. 2D). Based on these results, the optimal treatment regimen of baicalein (30 mg/kg, 30 min post-SAH) was used in the subsequent studies.

2.3.

Baicalein reduced BBB permeability at 24 h after SAH

BBB permeability was tested at 24 h after SAH in our study (Fig. 3). The amount of extravasated Evans blue dye in the brain was significantly higher in the SAH group as compared to that of the sham group (Po0.01). The levels of extravasated dye did not differ significantly between the SAH group and SAHþDMSO groups (P¼ 0.907). Baicalein treatment resulted in reduced dye extravasation into brain as compared to the SAHþDMSO group (Po0.05).

2.4. Baicalein reduced TLR4 protein expression at 24 h after SAH Western blot (Fig. 4) showed that TLR4 was expressed at a low level in brains in the sham group. The level of TLR4 was significantly increased in the cortex in the SAH group as compared to that of the sham group (Po0.01). The protein expression did not differ significantly between the SAH group and SAHþDMSO groups (P ¼0.951). The expression of TLR4 in the brains of baicalein treatment SAH group were significantly lower than that of the SAHþDMSO group (Po0.05).

2.5. Baicalein blocked the degradation of IκB-α and decreased the p65 translocation to nucleus at 24 h after SAH Next, we investigated the effects of baicalein on NF-κB activity. A decrease of total protein level of IκB-α, an indicator of NF-κB activation, was detected at 24 h after SAH. Baicalein

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Fig. 2 – Bar graphs showing effects of baicalein treatment on the changes in the percentage of brain water content ((A) and (B)) and neurological score ((C) and (D)) at 24 h ((A) and (C)) and 72 h ((B) and (D)) after surgery (n¼6 rats/group). The water content has no difference among groups in cerebellum or brain stem both at 24 h and 72 h after SAH. Both 30 mg/kg and 100 mg/kg of baicalein intraperitoneally at 30 min after SAH significantly reduced the cerebrum water content at 24 h after SAH. 30 mg/kg but not 100 mg/kg of baicalein dramatically alleviated neurological score at 24 h after SAH. Moreover, baicalein at the concentration of 30 mg/kg significantly reduced the cerebrum water content but did not alleviate neurological score at 72 h after SAH. Data are expressed as a mean and SEM. nPo0.01 and nnnPo0.001 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþDMSO. Bai¼ baicalein.

Fig. 3 – The effect of baicalein on Evans blue dye at 24 h after SAH. Evans blue detected in the SAH group showed significantly higher BBB permeability as compared with that in the sham group. There was no difference between the SAH group and SAHþDMSO groups. Baicalein treatment significant decreased SAH induced BBB permeability (n¼6 rats/group). Data are expressed as a mean and SEM. nnPo0.01 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþ DMSO. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 – Representative Western blots and quantitative analysis of TLR4 in cortex sample at 24 h after SAH. Expression levels of protein are expressed as a ratio of the βactin levels. It shows that TLR4 expression in the SAH and SAHþDMSO groups were significant higher than that in the sham group. There was no difference between the SAH and SAHþDMSO groups. Treatment with baicalein (30 mg/kg) markedly attenuated the SAH upregulation of TLR4 (n ¼ 6 rats/group). Data are expressed as a mean and SEM. nn Po0.01 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþDMSO.

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sham group. They were dramatically increased after SAH. However, they were largely reduced after treating with baicalein (Po0.05; Fig. 6).

2.7. Baicalien decreased matrix metalloproteinase-9 (MMP9) protein expression and ZO-1 degradation at 24 h after SAH MMP-9, an important downstream molecular of TLR4/NF-κB signaling pathway, can degrade interendothelial tight junction proteins and basal lamina proteins of the BBB, leading to BBB breakdown. Significant increases in MMP-9 were observed in both the SAH group and the SAHþDMSO group (Po0.01; Fig. 7A), and baicalein treatment dramatically inhibited the MMP-9 protein expression compared with the SAHþDMSO group (Po0.05; Fig. 7A). ZO-1 was significantly degraded after SAH in the SAH group compared with the sham group (Po0.01; Fig. 7B), and baicalein treatment significantly reduced the degradation of ZO-1 compared with the SAHþDMSO group (Po0.05; Fig. 7B). The MMP-9 immunoreactivities had an inverse relation with the ZO-1 immunoreactivities, and the administration of baicalein seemed to reverse this trend, with the conservation of ZO-1 immunoreactivities and remarkable reduction of MMP-9 immunoreactivities in the brain at 24 h after SAH.

Fig. 5 – Representative Western blots and quantitative analysis of IκB-α (A) and p65 translocation to nucleus (B) in cortex sample at 24 h after SAH. Expression levels of protein are expressed as a ratio of the β-actin or Histone 3 levels. It shows that IκB-α expression in the SAH and SAHþDMSO groups were significant lower than that in the sham group. There was no difference between the SAH and SAHþDMSO groups. Treatment with baicalein (30 mg/kg) attenuated the SAH induced the decrease of IκB-α expression (n ¼ 6 rats/ group). More p65 was translocated to nucleus in the SAH and SAHþDMSO groups compared to the sham group. There was no difference between the SAH and SAHþDMSO groups. Treatment with baicalein (30 mg/kg) decrease p65 translocation to nucleus (n ¼ 6 rats/group). Data are expressed as a mean and SEM. nPo0.05 and nnPo0.01 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþDMSO.

inhibited the degradation of IκB-α (Po0.05; Fig. 5A). Activation of NF-κB promotes nuclear translocation p65 subunits. In the present study, we observed that the nuclear p65 subunit was significantly increased 24 h after SAH (Po0.01; Fig. 5B), which was attenuated by baicalein treatment (Po0.05; Fig. 5B).

2.6. Baicalein reduced production of pro-inflammatory cytokines at 24 h after SAH To study the effects of baicalein on the inflammatory process after SAH, TNF-ɑ and IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 6, the concentrations of TNF-ɑ and IL-1β were relatively lower in the

2.8. Baicalein increased Bcl-2 protein expression, suppressed Bax and caspase-3 protein expressions at 24 h after SAH To investigate the potential effect of baicalein on neural cell survival, we evaluated the expression of the apoptosisrelated proteins Bcl-2, Bax and cleaved caspase-3. Western blot analysis indicated that compared with the sham group, level of Bcl-2 was decreased (Po0.05; Fig. 8A and C) while Bax and cleaved caspase-3 were significantly increased in the cortex in the SAH group (Po0.01; Fig. 8A, B, D, F). Those protein expressions did not differ significantly between the SAH group and SAHþDMSO groups (P40.05; Fig. 8A–D,F). The reduced Bcl-2 and the increased Bax and cleaved caspase-3 were all blocked by baicalein treatment (Po0.05; Fig. 8A–D, F). Moreover, the ratio of Bcl-2 and Bax proteins was increased after baicalien administration compared with the SAHþ DMSO group (Po0.05; Fig. 8A and E).

2.9. SAH

Baicalein decreased neural cell apoptosis at 24 h after

In the present study, we use terminal deoxynucleotidyl transferase-mediated uridine 50 -triphosphate-biotin nick end-labeling (TUNEL) staining to observe the neural cell apoptosis. Significant increases in apoptotic index were detected in the cortex of the SAH group and the SAHþDMSO group at 24 h after blood injection when compared with rats in the sham group (Po0.001; Fig. 9). The number of positive TUNEL cells in the brain was decreased by baicalein admission (Po0.05; Fig. 9).

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Fig. 6 – Results of ELISA analysis of the levels IL-1β and TNF-α protein at 24 h following SAH. IL-1β and TNF-α protein increased after SAH, but were suppressed in the baicalein treated group. Data are expressed as a mean and SEM. nPo0.05 and nnop0.01 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþDMSO.

Fig. 7 – Representative Western blots and quantitative analysis of MMP-9 (A) and ZO-1�B) in cortex sample at 24 h after SAH. Expression levels of protein are expressed as a ratio of the β-actin levels. It shows that MMP-9 expression in the SAH and SAHþDMSO groups were significant higher than that in the sham group. There was no difference between the SAH and SAHþDMSO groups. Treatment with baicalein (30 mg/kg) significantly attenuated the SAH induced increase of MMP-9 expression (n ¼6 rats/group). ZO-1 in the SAH and SAHþDMSO groups were significant lower than that in the sham group. There was no difference between the SAH and SAHþDMSO groups. Treatment with baicalein (30 mg/kg) markedly reduced the SAH induced ZO-1 degradation (n ¼ 6 rats/group). Data are expressed as a mean and SEM. nPo0.05 and nnPo0.01 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþDMSO.

3.

Discussion

The present study shows for the first time that baicalein administration could significantly attenuate EBI (including brain edema, neurological deficit, BBB disruption, and neural cell apoptosis) after SAH in rats. Furthermore, we found that the potential mechanisms responsible for the neuroprotective effects of baicalein were associated with the inhibition of TLR4/NF-κB pathway. SAH is a severe clinical problem associated with high mortality and morbidity. More recently, accumulating studies indicate that EBI rather than vasospasm is the main cause of poor outcomes in patients with SAH (Cahill et al., 2006; Sehba et al., 2012). Therefore, EBI has emerged as a new frontier and requires a better understanding and consideration in devising therapeutic strategy for improving SAH outcomes. The pathophysiological mechanisms of EBI are complicated and

increasing evidences suggest that inflammatory mechanisms are involved in the progression of EBI after SAH (Cahill et al., 2006; Kooijman et al., 2014; Sehba et al., 2012). Following SAH, glial cells are activated (Kooijman et al., 2014), BBB permeability is increased (Cahill et al., 2006) and peripheral immune cells infiltrate into the brain (Sehba et al., 2012), leading to the production of a great number of inflammatory cytokines and chemokines, which induce brain edema and neuronal injury. Therefore, inhibiting inflammatory response can significantly alleviate EBI after SAH. Baicalein, an extract of S. baicalensis Georgi, penetrates BBB 20–30 min after administration (Tsai et al., 2002) and has been proved to exert potential anti-inflammatory and neuroprotective effects in recent studies (Chen et al., 2008; Lebeau et al., 2001; Shen et al., 2003; van Leyen et al., 2006). The neuroprotective effects of baicalein have been shown on several brain injury animal models, including stroke,

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Fig. 8 – Representative Western blots and quantitative analysis of Bcl-2, Bax and cleaved caspase-3 in the cortex at 24 h after SAH. The expression of Bcl-2 protein was significantly raised by baicalein administration compared with the SAH and SAHþDMSO groups ((A) and (C)). The Bax protein level in the baicalein group showed a significantly down-regulated compared with the SAH and SAHþDMSO groups ((A) and (D)). Moreover, the ratio of Bcl-2 and Bax protein was increased after baicalein treatment (E). The expression of cleaved caspase-3 protein showed a similar trend as the Bax protein ((B) and (F)) (n¼ 6 rats/group). Data are expressed as a mean and SEM. nPo0.05 and nnPo0.01 compared to sham, ns compared to SAH, # Po0.05 compared to SAHþDMSO.

traumatic brain injury and Parkinson’s disease (Chen et al., 2008; Mu et al., 2011; van Leyen et al., 2006). Importantly, early baicalein treatment attenuates oxidative stress, cerebral vasospasm and finally improves neurological function following SAH (Kuo et al., 2013). In the present study, we explored the protective effects of baicalein on EBI after SAH. We demonstrated that baicalein treatment intraperitoneally 30 min after SAH induced remarkably improvement of EBI events such as brain edema, clinical scale, BBB disruption and neural cell apoptosis. These results suggest that baicalein has protective effects on EBI after SAH, but the mechanisms is unclear. We explore the potential mechanisms of its protective effects as well in this study. Toll-like receptors (TLRs), a family of transmembrane proteins, belong to pattern-recognition receptor family that could recognize exogenous pathogen-associated molecular patterns and endogenous damage-associated molecular patterns. They are believed to play a key role in the induction of innate and adaptive immunity (Akira et al., 2001). TLR4 was the first mammalian TLR recognized. NF-κB, the downstream of TLR4 is an important transcription factor associated with

innate immune responses and inflammation. Activation of TLR4 stimulates IκB phosphorylation and degradation, leading to nuclear translocation of NF-κB, which initiates transcription of genes associated with innate immune responses and inflammation (Akira et al., 2001; Anderson, 2000). A major and the most studied IκB subtype is IκB-ɑ. There is abundant evidence show the critical role of TLR4/NF-κB pathway in the initiation of brain inflammation in several central nervous system (CNS) diseases, such as infectious and autoimmune CNS diseases, ischemic brain injury and intracerebral hemorrhage (Kong and Le, 2011; Ziai, 2013). The previous studies from our group and also from others indicated that TLR4/NF-κB signaling pathway was activated in the brain after SAH and inhibiting NF-κB could protect against delayed brain injury after SAH (Ma et al., 2009; You et al., 2012, 2013). Furthermore, previous studies showed that progesterone, melatonin and tamoxifen have neuroprotective effects in EBI after SAH by inhibiting TLR4/NF-κB inflammatory pathway (Sun et al., 2013; Wang et al., 2011; 2013). All those results demonstrate that TLR4/NF-κB is involved in EBI following SAH and TLR4/NF-κB signaling pathway may be the

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Fig. 9 – Effect of baicalein on TUNEL staining (A) at two different magnifications (A1–D1:  200; A2–D2:  400) and neural cell death quantification (B) at 24 h after surgery. Administration of baicalein significantly decreased the apoptotic index in the rat brain following SAH (n ¼3 rats/group). Data are expressed as a mean and SEM. nnnPo0.001 compared to sham, ns compared to SAH, #Po0.05 compared to SAHþDMSO.

therapeutic target for SAH. Studies from other groups showed that baicalein could exert anti-inflammatory via inhibiting NF-κB signaling pathway in several disease models (Fan et al., 2013; Kang et al., 2003; Lee et al., 2011). Therefore, we hypotheses that baicalein may be protective against EBI after SAH by inhibiting TLR4/NF-κB mediated inflammatory signaling pathway. In this study, we observed that SAH was associated with a significant increase of TLR4 expression and a decrease of IκB-α degradation, combined with more nuclear translocation of p65 in the cortex at 24 h after SAH, which were consistent with the previous studies (Ma et al., 2009; You et al., 2012, 2013). Treatment with baicalein dramatically reduce the TLR4 expression, IκB-α degradation and nuclear translocation of p65. Meanwhile, baicalein reduced the production of pro-inflammatory cytokines (TNF-a and IL1β) at 24 h after SAH. These data suggeste that baicalein can inhibit TLR4/NF-κB mediated inflammatory signaling pathway at 24 h after SAH. However, the further mechanism of baicalein suppressing TLR4/NF-κB signaling pathway after SAH remains unclear and more studies are needed. Brain edema is an independent risk factor for mortality and poor outcome after SAH (Claassen et al., 2002). MMP-9 belongs to matrix metalloproteases, a family of zinc

endopeptidases, can degrade interendothelial tight junction proteins and basal lamina proteins of the BBB, leading to BBB breakdown and brain edema. As an important downstream mediator within TLR4/NF-κB signaling pathway, a body of evidences have indicated that MMP-9 participates in the BBB disruption and brain edema formation after SAH (Feiler et al., 2011). Recently, some studies suggested that baicalein can inhibit MMP-9 (Chao et al., 2013; Lee and Lee, 2012). In this study we assessed the effect of baicalein on MMP-9 expression. It was found that baicalein inhibited the SAH induced increase of MMP-9 and concomitantly restored level of ZO-1 (one of tight junction proteins). These findings demonstrated that baicalein can block the increase of MMP-9 and the degradation of tight junction protein after SAH, ultimately leading to the BBB protection. However, the present study has limitations. First, our data regarding the improvement of EBI after SAH could be explained partly by beneficial effects of baicalein through suppressing TLR4/ NF-κB inflammatory signaling pathway. However, we have not explored the effects of baicalein on oxidative stress (Zhang et al., 2010) and platelet aggregation (Huang et al., 2005) after SAH, which also play the important role in brain injury following SAH (Huang et al., 2005). Second,

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we have not further studied the dose and time window of baicalein for SAH. Hence, future studies are warranted. In addition, treatment with baicalein at dose 100 mg/kg at 24 h after SAH and at dose of 30 mg/kg at 72 h after SAH could improved molecular/anatomical outcomes but without significant difference in neurological score. There are fewer neurological tests suitable for rats prechiasmatic injection SAH model, and the difference in model and the scoring system could bring inconsistent results. We extrapolate that the scoring system we used in this study is not enough to discriminate the neurological difference in different groups. So more sensitive neurobehavior tests are needed in our future studies. In conclusion, baicalein treatment reduce brain edema, BBB disruption, neural apoptosis and improve neurological function at 24 h or 72 h after SAH in rats, demonstrating that administration of baicalein has protective effects against EBI in this rat model of SAH. In addition, our results imply that the neuroprotective effects of baicalein on EBI are depended, at least partly, on inhibition of TLR4/ NF-κB-mediated inflammatory signaling pathway.

4.

Experimental procedures

4.1.

Animal preparation

4.3.

Experimental groups and baicalein treatment

Baicalein (Sigma, St. Louis, MO, USA, 98% pure) was prepared in dimethyl sulfoxide (DMSO), diluted immediately before injection in 0.9% saline (the final concentration of baicalein was 10 mg/ml and DMSO was 10%). Baicalein solution was administered via intraperitoneal injection and there were two sets of this experiments. In the first set, two concentrations of baicalein (low: 30 mg/kg; high: 100 mg/kg) were used to test the neuroprotection of baicalein on EBI after SAH. We chose those dose based on the study of traumatic brain injury in rats (Chen et al., 2008). Baicalein or vehicle (n ¼6 rat/group) was injected intraperitoneally at 30 min after SAH, and then baicalein or vehicle was administered every 24 h. In the vehicle treated group, an equal volume of 10% DMSO was administered. After assessing the neurological deficits, rats were deeply anesthetized at 24 h or 72 h after SAH and brain water content was measured. In the second set, rats were treated with baicalein (30 mg/ kg) or vehicle at 30 min after SAH. Rats were euthanized for Western blot analysis, ELISA (n ¼6 rats/group) and histochemistry study (n ¼3 rats/group) at 24 h after SAH.

4.4.

Neurologic scoring

All procedures including animal use and all surgical procedures were approved by the Animal Care and Use Committee of Jinling Hospital and accorded to Guide for the Care and Use of Laboratory Animals by National Institutes of Health. Male Sprague-Dawley rats (280–320 g) were purchased from Animal Center of Jinling Hospital, Jiangsu, China. The rats were raised on a 12 h dark–light cycle circumstance with free access to food and water.

Three behavioral activity examinations (Table 1) were evaluated in a blinded fashion at 24 h and 72 h after SAH. Grading of neurologic deficits was as follows: (1) no neurologic deficit (scores¼ 0); (2) minimum or suspicious neurologic deficit (scores¼ 1); (3) mild neurologic deficit (scores¼ 2–3); (4) severe neurologic deficit (scores¼ 4–6).

4.2.

Rat brains were removed 24 or 72 h after surgery and separated into cerebrum, cerebellum and brain stem. Each part was weighed immediately after removal (wet weight) and after drying in 80 1C for 72 h (dry weight) and the percentage of water content was calculated as [(wet weight dry weight)/wet weight]  100%.

Animal model of SAH

Experimental SAH was produced according to the prechiasmatic injection method has been described before (Jeon et al., 2010). In briefly, Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) (Sigma, Shanghai, China) and then rats were fixed in the stereotactic apparatus. After careful disinfection, a midline scalp incision was made and an about 1 mM hole was drilled 7.5 mM anterior to the bregma in the midline, at an angle of 30E caudally. After that the rats were positioned in supine position. Then carefully disinfecting again, an insulin injection needle (BD Science) was used to get 300 μl of non-heparinized blood from the femoral artery. The needle was advanced about 11 mM into the prechiasmatic cistern through the burr hole, and the 300 μl blood was injected into the prechiasmatic cistern slowly (over 20 s). Rats in the sham group was injected an equal volume of sterile saline in the same manner. The burr hole was sealed with bone wax, and the incision was surgically sutured. When all these procedures were done, 1 ml of 0.9% NaCl solution was injected subcutaneously to prevent dehydration and then the rats were kept in a 30 head-down position. After recovery from anesthesia, the rats were returned to their cages and raised at 2371 1C.

4.5.

Brain water content

Table 1 – Behavior scores. Category

Behavior

Score

Appetite

Finished meal Left meal unfinished Scarcely ate Active, barking or standing Lying down, will stand and walk with some stimulation

0 1 2 0 1

Almost always lying down No deficits Unable walk because of ataxia or paresis Impossible to walk and stand because of ataxia and paresis

2 0 1 2

Activity

Deficits

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4.6.

BBB permeability

Evans blue (EB) extravasation was used to assess BBB permeability. In brief, EB dye (2%; 4 ml/kg) was injected over 2 min into tail vein and allowed to circulate for 1 h. Rats were then reanesthetized and perfused transcardially with 4 1C saline to remove intravascular EB dye. After decapitation, the brains were removed and hemogenized in physiological phosphatebuffered saline (PBS, PH 7.4) and trichloroacetic acid was then added to precipitate the protein. Sample were cooled and contrifuged. The resulting supernatant was measured for absorbance of EB at 620 nm by using a spectrophotometer.

4.7.

Perfusion–fixation and tissue preparation

All rats were anesthetized as above, and perfused through the left cardiac ventricle with ice-cold 0.9% normal saline solutions until the effluent from the right atrium was clear. Animals which had obvious blood clots in the prechiasmatic cistern were selected for further analysis. After blood clots on the brain tissue were cleared carefully, the temporal lobe tissue was harvested on ice and stored in  80 1C for Western blot and ELISA. For TUNEL stain study, the rats were perfused with ice-cold 0.9% normal saline solutions followed by 4% buffered paraformaldehyde; the brain was immersed in 4% buffered paraformaldehyde overnight and then embedded in paraffin for TUNEL stain study.

4.8.

Cruz, CA, USA), rabbit anti-P65 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti- IκB-α (1:1000; Anbo Biotechnology, San Francisco, CA, USA), rabbit anti-Cleaved Caspase-3 (1:500; Anbo Biotechnology, San Francisco, CA, USA), rabbit anti-Bax (1:1000; Abcam), rabbit anti-Bcl-2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Histon 3 (1:1000; Cell Signaling, Beverly, MA) and rabbit anti-β-Actin (1:5000; Bioworld Technology, Minneapolis, MN, USA). After being washed with TBST (3  15 min), the membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG (1:5000, Bioworld Technology, Minneapolis, MN, USA) for 2 h at room temperature. Bands were visualized using the enhanced chemiluminescence reagent kit (Biyuntian) and quantification was performed by optical density methods using ImageJ software (NIH). Results are expressed as a relative density to β-actin or Histon 3 subsequently normalized to the mean value of the sham group.

4.10.

ELISA

Total protein was determined using a bicinchoninic acid assay kit (Pierce Biochemicals). The levels of inflammatory cytokines of the brain tissue were quantified using ELISA kits specific for rats according to the manufacturers’ instructions (TNF- a, from Diaclone Research, France; IL-1β, from Biosource Europe SA, Belgium). The cytokine contents in the brain tissue were expressed as picogram per milligram protein.

Total/nuclear protein extraction 4.11.

To extract cortex total protein, proper size of tissues were mechanically lysed in 20 mM Tris (pH 7.6, 0.2% SDS, 1% Triton X-100, 1% deoxycholate, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.11 IU/ml aprotinin) (all from Sigma, Shanghai, China). Homogenates were centrifuged at 14,000g for 15 min at 4 1C. The supernatant was collected and stored at  80 1C until analysis. Cortex nuclear protein was extracted following the previous studies (Zhou et al., 2007). Briefly, 50 mg of fresh cortex was homogenized in 0.4 ml of ice-cold buffer A, which contain 10 mM HEPES (pH 7.9), 2 mM MgCl2,10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT) and 0.5 mM PMSF (all from Sigma, Shanghai, China). Then 15 μl Nonidet P-40 was added to the system. After centrifuged the mixture, we discarded the supernatant (cytoplasmic fraction) and resuspended the nuclear pellet with 100 μl buffer B, which consist of 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 25% (v/v) glycerol. The mixture was centrifuged at 14,000g at 4 1C for 15 min. The supernatant containing nuclear proteins was collected and stored at 80 1C until analysis.

4.9.

253

Western blot analysis

Equal amounts (40 μg) of total protein or nuclear protein were separated in 10% SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane. The membrane was blocked with 5% defatted milk for 2 h at room temperature, then incubated overnight at 4 1C with primary antibodies. For primary antibodies we used rabbit anti-TLR4 (1:200; Santa Cruz Biotechnology, Santa

TUNEL staining and cell counting

The TUNEL staining performed according to our previous study (Zhuang et al., 2012) was used to detected the apoptotic cells. In briefly, an In situ cell death detection Kit POD (ISCDD, Boehringer Mannheim) was used. The procedures ran according to protocol of the kit and the other references. Briefly, sections at 5 μm thickness with a microtome were deparaffinized, rehydrated, and washed with distilled water. The tissues were digested with 20 g/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) at room temperature for 15 min. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide/methanol in PBS at 37 1C for 30 min. Then the sections were incubated with terminal deoxynucleotidyl transferase at 37 1C for 60 min to add the dioxigenin-conjugatd dUTP to the 30 –OH ends of fragmented DNA. Anti-digoxigenin antibody peroxidase was applied to the sections to detect the labeled nucleotides. The sections were stained with DAB and counterstained slightly with hematoxylin. The positive cells were identified, counted and analyzed under the light microscope by an investigator blinded to the grouping. And apoptotic neuron counting was also restricted to the external granular lamina layer of the occipital cortex. Every third coronary section starting from 3.0 mm posterior to the optic chiasma was collected and a total of 10 sections from each animal were used for quantification. Six random high power fields (  400) in each coronary section were chosen bilaterally, and the mean number of apoptotic neurons in the six views was regarded as the data of each section. The final average proportion of apoptotic neuronal cells of the sections was regarded as the data for each sample and the severity of brain damage was

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evaluated by apoptotic index, defined as the average percentage of TUNEL positive neuronal cells.

4.12.

Statistical analysis

Results were expressed as means7SEM and SPSS 17.0 and GrahPad Prism 5.0 software was used for the statistical analysis. Mortality Rate between groups was analyzed by Fischer exact test. All other data were statistically analyzed using a one-way analysis of variance followed by Tukey post hoc test. A difference with Po0.05 was considered statistically significant.

Acknowledgments This work is supported by Grants from National Natural Science Foundation of China (nos. 81070921 and 81271297 for J.-X. Shi, no. 81000503 for M.-L. Zhou).

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