HMGB1在幼年大鼠癫痫持续状态中的作用研究
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摘要
癫痫(epilepsy)是常见的神经系统慢性疾病之一,临床呈长期反复痫性发作的疾病过程,在儿童中发病率高,长期、频繁或严重的痫性发作会导致进一步的脑损伤甚至持久性神经精神障碍,给患者本人及家庭、社会造成很大的困扰。近年来,针对癫痫的病因、发病机制及治疗等的研究工作取得了很大进展,但是癫痫发生及发展的确切机制还未完全阐明。大量研究表明癫痫持续状态、脑外伤及缺血缺氧等脑损伤过程都伴有神经系统免疫炎症反应,而神经系统的免疫炎症反应可以增加神经元兴奋性,促进神经元损伤及癫痫发生。众多研究数据表明多种炎症介质参与了癫痫发生及发展过程,而抗炎治疗对部分类型的癫痫发作有效,可以减轻发作程度及神经病理学变化。
高迁移率族蛋白1(high-mobility group box1, HMGB1)是一种高度保守的非组DNA结合蛋白,具有稳定核酸结构、调节转录和基因表达等多种生物学功能。近年来发现在一定因素作用下可以释放到细胞外发挥促炎因子作用,HMGB1可以由坏死细胞被动释放或由活化的免疫细胞主动释放,通过与其受体如Toll样受体4(toll-like receptor-4, TLR4), Toll样受体2(TLR2)和晚期糖基化终末产物受体(receptor for advanced glycation end products, RAGE)等结合,激活核因子-κB (nuclear factor-κB,NF-κB)及其他信号传导通路,促进炎症因子及趋化因子的产生,从而发挥其促炎作用。TLR作为固有免疫受体,是近年来研究较多的一类受体,主要与感染细菌及内源性损伤相关分子结合发挥作用,其中TLR4是其中研究较热的受体之一,广泛分布于神经系统,包括神经元、小胶质细胞和星形胶质细胞,参与多种神经系统疾病过程。
研究证实HMGB1参与了败血症、关节炎、胰腺炎、呼吸障碍、脑缺血、脑外伤、脑炎等病理过程。而HMGB1的抑制剂如中和抗体,Box A等可以减轻多种疾病中HMGB1引起的炎症反应,发挥一定的保护作用,相反,给予重组HMGB1可以加重炎症反应及组织损伤。例如在败血症中给予HMGB1中和抗体可以减轻炎症反应,降低死亡率；在脑缺血和创伤性脑损伤中存在HMGB1的转位活化,给予HMGB1中和抗体可以抑制HMGB1活化及炎症反应,减轻脑损伤,而给予重组HMGB1可增加细胞因子合成,促进胶质细胞活化及加重神经元损伤。
近来有研究表明在成年小鼠癫痫发生中有HMGB1/TLR4信号通路的参与,在热性惊厥儿童中也观察到血浆HMGB1水平升高。但是在未成年机体癫痫发作中是否有HMGB1的参与以及HMGB1中和抗体在癫痫发作中是否有神经保护作用仍不清楚,研究表明癫痫发生的易感性、神经病理变化及预后都存在年龄依赖性,而HMGB1的表达也在一定程度上受到年龄影响,因此在本课题中,我们通过侧脑室注射海人酸(kainic acid, KA)建立生后21天(postnatal day21, P21)大鼠癫痫持续状态(status epilepticus, SE)模型,研究SE后早期阶段(3h-7d), HMGB1/TLR4通路在海马组织中的表达特点以及神经元损伤特点；并通过给予HMGB1中和抗体阻断HMGB1活性,研究HMGB1中和抗体对癫痫持续状态后海马炎症因子表达,胶质细胞活化状态及神经元损伤情况的影响,以明确HMGB1在幼鼠癫痫持续状态中的表达特点及作用。
第一部分：HMGB1/TLR4通路在幼年大鼠癫痫持续状态中表达变化的研究
目的：
研究海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马HMGB1/TLR4通路的表达变化。
材料与方法：
1.幼鼠癫痫持续状态模型的建立及分组
选用生后21天Wistar大鼠,水合氯醛(400mg/kg)腹腔注射麻醉后固定于立体定位仪。采用微量注射器缓慢将2nmol KA溶液(溶于1μl PBS)注入侧脑室(坐标为前囟后0.7mmm,中线外1.3mm,深度3.0mmm)。PBS对照组给予同等剂量PBS替代KA。痫性发作行为学评价根据Racine分级标准进行,持续全身发作或连续多次全身发作之间不能恢复正常状态超过30min者定义为癫痫持续状态(Racine分级Ⅳ-Ⅴ级),记录癫痫持续状态开始时间。在SE开始后2h给予水合氯醛(400mg/kg)腹腔注射终止发作。未达标准的大鼠予以剔除。
SE大鼠随机分为SE后3h、6h、12h、24h、3d和7d组,同时设立正常组和PBS对照组(根据预实验结果选取时间点为注射后24h)
2.采用尼氏染色和FJB染色观察神经元形态及损伤情况。
3.采用免疫组织化学技术检测各组大鼠海马HMGB1和TLR4蛋白表达变化。
4.采用Western blot方法检测各组大鼠海马HMGB1和TLR4蛋白表达变化。其中HMGB1蛋白检测又分为总蛋白和胞浆蛋白。
5.采用免疫荧光双重标记技术检测各组大鼠海马HMGB1在各类细胞(神经元、小胶质细胞和星形胶质细胞)中的分布变化。
结果：
1.海人酸侧脑室注射诱导P21大鼠癫痫持续状态
在海人酸注射后30min内,所有大鼠都出现呼吸急促、咀嚼、动须,点头、湿狗样抖动及前肢阵挛,之后逐渐出现旋转,站立,跌倒,强直阵挛发作。8%大鼠死亡,其中80%死亡出现于KA侧脑室注射后1h以内。观察大鼠癫痫持续状态发作2h后给予水合氯醛(400mg/kg)腹腔注射终止发作。而在PBS对照组无癫痫发作。
2.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马神经元损伤
尼氏染色和FJB染色显示正常组和PBS对照组大鼠海马无神经元损伤。SE后6h在CA3区和Hilus区开始出现神经元损伤,表现为尼氏染色阳性细胞数减少(P<0.01),并出现FJB阳性细胞,之后神经元损伤逐渐加重,SE后24h可见大量神经元损伤。而在CA1区明显的神经元损伤开始于SE后24h(P<0.05),3d明显(P<0.01),并且损伤较CA3区和Hilus区轻。
3.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马HMGB1蛋白的表达变化
Western blot分析显示海马HMGB1,总蛋白无明显变化,而胞浆HMGB1在SE后明显增加,开始于SE后3h(P<0.01),24h达高峰(P<0.01),之后逐渐降低,7d时仍高于正常组(P<0.05), PBS对照组与正常组无差异(P>0.05)。免疫组化显示在正常组大鼠和PBS对照组大鼠海马中,HMGB1主要分布于细胞核内,SE后3h开始出现于细胞浆中。在CA1区,SE后3h到12h,胞浆HMGB1主要出现于椎体细胞层中,在24h及以后,胞浆HMGB1在胶质形态细胞中增多。在CA3区和Hilus区,自SE后6h起可以观察到HMGB1染色缺失区域,该区域与尼氏染色和FJB染色显示的神经元损伤区域一致。
4.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马HMGB1的细胞定位
免疫荧光双标显示在正常组大鼠中,HMGB1位于神经元和星形胶质细胞细胞核内,无ED1阳性细胞。SE后6h,胞浆HMGB1主要出现于NeuN阳性的神经元细胞。SE后24h, HMGB1在胶质细胞中表达增多,并且星形胶质细胞表达HMGB1的比例及胞浆HMGB1阳性的星形胶质细胞明显增多(P<0.05),另外,出现ED1标记的小胶质细胞,主要表达于CA3区和Hilus区,分别有78.4±0.12%和81.2±0.08%的ED1阳性细胞与HMGB1共分布。
5.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马TLR4蛋白的表达变化
Western blot分析显示TLR4蛋白在正常组大鼠和PBS对照组大鼠海马中的表达量很少,而在SE后明显增加,开始于3h(P<0.01),24h达高峰(P<0.01),之后逐渐降低,7d时仍高于正常(P<0.01)。免疫组化结果显示在正常组大鼠和PBS对照组大鼠海马中,TLR4仅在部分大鼠有散在弱表达,SE后3h开始有表达,之后逐渐增多,在24h之前,TLR4阳性细胞主要分布于锥体细胞层,以神经元形态为主,在24h之后,出现在胶质细胞形态细胞中。
结论：
1.在海人酸侧脑室注射诱导P21大鼠癫痫持续状态后早期阶段,海马HMGB1/TLR4通路活化,并与神经元损伤相关。
2. HMGB1的活化即核浆转位及胞外释放首先发生在神经元,随后出现于胶质细胞。
第二部分：HMGB1中和抗体在幼年大鼠癫痫持续状态中的作用研究
目的：
研究HMGB1中和抗体对P21大鼠癫痫持续状态后海马炎症反应及神经元损伤的作用。
方法：
1.幼鼠癫痫持续状态模型的建立
癫痫持续状态模型建立方法同第一部分。
2.分组及药物干预
SE大鼠随机分为模型组(KA+IgY组)和HMGB1中和抗体干预低、中、高剂量组(KA+anti-HMGB1组)。模型组通过侧脑室注射给予对照IgY (4μg),干预组分别给予HMGB1中和抗体1、2、4μg。给药时间为SE终止即刻及SE后12h。同时设PBS对照组,在相应时间给予对照IgY (4μg)。
3.实时定量RT-PCR
SE后6h取海马组织提取RNA,采用实时定量RT-PCR方法检测海马炎症因子白介素1β(IL-1β)和肿瘤坏死因子α(TNF-a) mRNA的表达变化。
4.免疫组织化学
SE后3d取脑组织制作石蜡切片,通过免疫组织化学方法检测海马Ibal、ED1和GFAP的表达变化。
5.尼氏染色和FJB染色
SE后3d,采用尼氏染色和FJB染色方法检测海马神经元形态及损伤情况。结果：
1. HMGB1中和抗体对海马炎症因子的影响
实时定量RT-PCR结果显示SE后6h,与对照组相比,KA+IgY组大鼠海马IL-1β和TNF-a mRNA表达明显增高(P<0.01)。而侧脑室注射中、高剂量中和抗体(2和4μg)可以明显抑制海马IL-1p和TNF-a mRNA表达(P<0.05),低剂量中和抗体(1μg)则无明显抑制作用(P>0.05)。
2. HMGB1中和抗体对海马胶质细胞的影响
免疫组织化学结果显示SE后3d,KA+IgY组大鼠海马小胶质细胞(Ibal和ED1)和星形胶质细胞(GFAP)免疫染色较对照组明显增强(P<0.01)。而侧脑室注射中、高剂量中和抗体(2和4μg)可以明显抑制海马Iba1、ED1和GFAP免疫染色强度(P<0.01),低剂量中和抗体(1μg)则无明显抑制作用(P>0.05)。
3. HMGB1中和抗体对海马神经元损伤的影响
尼氏染色和FJB染色显示SE后3d,对照组海马神经元形态正常,无损伤,KA+IgY组大鼠海马神经元损伤明显,表现为尼氏染色阳性神经元明显减少,而FJB染色阳性神经元显著增多(P<0.01),且CA3区、Hilus区较CA1区神经元损伤明显。而侧脑室注射中、高剂量中和抗体(2和4μg)可以明显减轻神经元损伤(P<0.05),低剂量中和抗体(1μg)则无明显作用(P>0.05)。
结论：
1.海人酸侧脑室注射诱导的P21大鼠癫痫持续状态可以促进海马炎症因子合成,胶质细胞活化及神经元损伤。
2.侧脑室注射HMGB1中和抗体可以剂量依赖性的抑制海马炎症因子合成及胶质细胞活化,减轻神经元损伤,发挥神经保护作用。
Epilepsy is one of the common neurological chronic disorders, with long-tern and repeated features. It has high morbidity in children. Frequently and seriously epileptic seizures may lead to further brain injury and persistent neurologic and psychogenic disorders, bringing troubles to the patients and family and society. Recent years, alought great evolution has been made in the etiology, pathology and treatment of epilepsy, the exact mechanism of epileptogenesis still remains unclear. Increasing evidence indicates that brain injury such as status epilepticus (SE), trauma, and stroke was accompanied by neuroinflammation, which may contribute to promoting neuronal excitability, neuronal death, and epileptogenesis. Accumulating data shows that many inflammatory factors participated in the development of epilepsy, and anti-inflammatory therapies could reduce seizures and neuropathological changes in some forms of epilepsy.
High-mobility group box1(HMGB1) is a highly conserved, ubiquitous non-histone DNA-binding protein that participates in stabilization of nucleosome formation and regulation of gene transcription. HMGB1has recently been defined as a key cytokine. It is released into the extracellular milieu actively by immune cells or passively by necrotic or damaged cells and triggers inflammatory responses and tissue injury. HMGB1can bind to multiple receptors such as the receptor for advanced glycation end products (RAGE), toll-like receptor-4(TLR4) and TLR2. The pro-inflammatory effect of HMGB1is exerted by activating nuclear factor-κB (NF-κB) or other pathways which promotes the chemotaxis and the production of cytokines. Toll-like receptors are innate immune receptors and respond to microbial structures and endogenous danger factors. Among the TLRs, TLR4is one of receptors which are the focus of concern. TLR4was expressed widely in the nervous system, including in microglia, astrocytes and neurons, and participated in many neurological diseases.
It has been reported that HMGB1contributes to the pathophysiology of sepsis, arthritis, respiratory disorders, pancreatitis, cerebral ischemia, traumatic brain injury, encephalitis and so on. The inhibitors of HMGB1such as anti-HMGB1antibody and Box A could alleviate inflammatory reaction and exert protective effects. On the contrary, injection recombinant HMGB1may promote inflammatory response and aggravate neuronal damage. For example, anti-HMGB1antibody could decrease the lethality of sepsis. HMGB1translocates from the nucleus into the cytoplasm and increases in cerebrospinal fluid and serum in cerebral ischemia and traumatic brain injury models. Anti-HMGB1antibody may inhibit the HMGB1translocation and inflammatory reaction and ameliorate brain infarction. However, recombinant HMGB1could prompt the induction of pro-inflammatory mediators and increase the neuronal death in vivo and in vitro.
Recently, it has been reported that the HMGB1-TLR4axis is activated in adult mice models of acute and chronic seizure, and elevated serum levels of HMGB1are observed in febrile seizure patients. However, so far, the role of HMGB1in epilepsy of immature brain and the role of anti-HMGB1antibody in the neuronal damage and inflammation following SE have not been explored. In addition, experimental evidence indicated that the susceptivity and the neuropathological changes of epilepsy are age-dependently, moreover, maturational stage could also affect the expression pattern of HMGB1. Therefore, in order to investigate the effect of HMGB1after SE in immature brain, we used postnatal day21(P21) wistar rats to induce SE models by an intracerebroventricular kainic acid (KA). We firstly investigated the expression of HMGB1/TLR4signal pathway and neuronal damage in hippocampus during the early phase of KA-induced SE (from3h to7d). Further, we studied the effects of anti-HMGB1antibody on the hippocampal damage and inflammatory reaction after KA-induced SE by an intracerebroventricular injection of anti-HMGBl antibody.
Chapter Ⅰ
The expression of HMGB1/TLR4signal pathway in immature hippocampus after status epilepticus
Objective
To investigate the expression changes of HMGB1/TLR4in P21rat hippocampus after KA-induced status epilepticus.
Materials and methods
1. KA-induced SE model
P21male Wistar rats were used. Rats were anesthetized using chloral hydrate (400mg/kg, intraperitoneally) and injected stereotaxically with KA (2nmol in1ml0.01M PBS) into the lateral ventricle at the following coordinates from the bregma:0.7mm posterior,1.3mm lateral, and3.0mm deep. The control rats received equal volumes of PBS. Seizures were behaviorally recorded in five grades using Racine's scale. The onset of SE was defined as the first grade4or greater seizure that progressed to similar repeated or prolonged behavioral seizures. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after the onset of SE. Rats that did not reach grade4were removed from further experiments.
The rats subjected to SE were further assigned randomly into six time point groups:3h,6h,12h,24h,3d and7d after the onset of SE. In addition to the above groups, normal-control group and PBS-control group (24h after injection) were also included.
2. Nissl and Fluoro-Jade B stainings were used to observe the neuronal morphology and neuronal damage in the hippocampus of P21rats after SE.
3. Immunohistochemistry was performed to examine the dynamic expression of HMGB1and TLR4in different subfields of hippocampus of P21rats after SE. The detection of HMGB1included the total HMGB1and cytoplasmic HMGB1.
4. Western blot was used to detect the dynamic expression of HMGB1and TLR4proteins in the hippocampus of P21rats after SE.
5. Double-labeling immunofluorescence staining was used to identify the cellular distribution (neuron, microglia and astrocytes) of HMGB1in the hippocampus of P21rats after SE.
Results
1.KA-induced SE
With in30min, all the rats showed polypnea, head nodding, facial automatisms, wet-dog shakes and forelimb clonus. Then, rats appeared rearing, generalized tonic-clonic seizures and transient loss of postural control.8%of rats died, and80%of the rats were died within the1hour after the intracerebroventricular injection of KA. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after the onset of SE. No seizure was observed in normal rats and PBS-injected rats.
2. Neuronal damage in the hippocampus of P21rats after KA-induced SE
Nissl and Fluoro-Jade B staining showed that there was no neuronal damage in normal rats and PBS-injected rats. Neuronal damage was occurred early in the CA3and Hilus at6h after SE, it was showed that the Nissl-positive cell counts were decreased (P<0.01), and FJB positive cells appeared. The neuron cell counts analysis showed a time-dependent decrease of Nissl-positive neurons in the hippocampus after KA-induced SE. At24h after SE, there was a substantial neuronal damage in the CA3and Hilus. However, delayed neuronal damage of CA1pyramidal cell layer was appeared lightly at24h after SE (P<0.05), and the degree of damage was light in CA1than that in CA3and Hilus.
3. Time-course expression of HMGB1in the hippocampus of P21rats after KA-induced SE
Western blot analysis showed that the total HMGB1protein level did not changed after SE, but the cytoplasmic HMGB1protein significantly upregulated after SE. The elevation of HMGB1was observed at3h after the onset of SE (P<0.01), peaked at24h (P<0.01), and decreased thereafter. At7days, a subtle elevation was still detected in the hippocampus (P<0.05). There was no significant difference between the normal rats and PBS-injected rats (P>0.05). Immunohistochemistry showed that in the normal and the PBS-injected rat hippocampus, HMGB1immunoreactivity was detected mostly in the nuclei. When rats were subjected to SE, HMGB1cytoplasmic translocation was observed in the hippocampus as early as3h after SE. From3h to12h after SE, the cytoplasmic translocation of HMGB1was obvioused mainly in the pyramidal neurons of CA1region, and after12h, the cytoplasmic HMGB1staining was occurred in the cell with glia morphology. In the pyramidal cell layer of CA3and hilus, HMGB1staining loss was observed from6h after SE, the staining loss region was in line with the region which undergoing degenerative changes at these time points.
4. The colocalization of HMGB1with different cellular markers in the hippocampus of P21rats after KA-induced SE
Double-labeling immunofluorescence showed that in the normal rat hippocampus, HMGB1immunoreactivity was observed in the nuclei of both neurons and astrocytes, and there were no ED1-positive cells in normal hippocampus. At6h after SE, cytoplasmic HMGB1staining was present mainly in the neurons of pyramidal layer. At24h post SE, the percentages of HMGB1-positive astrocytes and the cytoplasmic HMGB1-positive astrocytes were substantially increased (P<0.05). Moreover, the immunoreactivity of ED1, a marker of activated microglia/macrophages, was appeared mainly in the CA3and hilus. And double-labeling assay detected that78.4±0.12%and81.2±0.08%of ED1-positive cells were double stained by HMGB1in the CA3and hilus, respectively.
5. Time-course expression of TLR4protein in the hippocampus of P21rats after KA-induced SE
Western blotting analysis demonstrated that the expression of TLR4protein was very low in the normal and PBS control group. After SE, the expression of TLR4protein was progressively increased from3h (P<0.01) in the hippocampus, peaked at24h (P<0.01), and decreased thereafter.7days after SE, the elevation of TLR4was still detected in the hippocampus (P<0.01). Immunohistochemistry showed that in some of the normal rat hippocampus, faint immunoreactivity was observed in only a few scattered cells present in pyramidal layer. After SE, the immunostaining of TLR4protein in the hippocampus was significantly increased from3h after SE. before24h after SE, the TLR4-positive cells were mainly distributed in the pyramidal layer, and after24h, the TLR4immunoreactivity was also observed in the cells with glia morphology. In the subfields of hippocampus, the TLR4immunostaining was distributed mainly in the dentate hilus and CA3.
Conclusion
1. The expression of HMGB1/TLR4axis was transiently activated during the early phase of KA-induced SE in P21rats, and the elevation of HMGB1/TLR4was related with the neuronal damage.
2. The translocation of HMGB1from nuclei to cytoplasm and the extracellular release was first occurred in neurons, and then occurred in the glias.
Chapter Ⅱ
The effects of HMGB1antibody in juvenile rat hippocampus after kainic acid-induced status epilepticus
Objective To investigate the effects of anti-HMGB1antibody on hippocampal damage and
inflammatory reaction after KA-induced SE in postnatal day21rats.
Materials and methods
1. KA-induced SE model
The model was induced according to the method in chapter I.
2. Experimental design
Rats were selected randomly to receive anti-HMGB1antibody (1,2, and4μg, IgY subclass) or nonimmune control IgY (4μg) by an intracerebroventricular injection immediately after the termination of SE and12h after the onset of SE. The PBS-injected rats received control IgY (4μg) at the same time point. The dose schedule was selected on the basis of previous report and preliminary experiments. Rats were killed6h (n=6/group) after SE to quantify the cytokine production. To evaluate the microglial activation and neuronal damage, rats were killed3d after SE (n=6/group)
3. Real-time quantitative PCR
Rats were decapitated at6h after SE, and the hippocampi were dissected and stored at-80℃. The hippocampal mRNA expression of IL-1β and TNF-a was investigated by real-time PCR.
4. Immunohistochemistry
Rats were decapitated at3d after SE, and the brain was dissected and made into paraffin sections. The immunoreactivities of Ibal、ED1and GFAP were investigated by immunohistochemistry.
5. Nissl and Fluoro-Jade B stainings
Rats were decapitated at3d after SE, and the brain was dissected and made into paraffin sections. The neuronal damage was detected by Nissl and Fluoro-Jade B staining.
Results
1. Effects of anti-HMGB1antibody on the gene expression of inflammatory factors in the hippocampus of P21rats at6h after SE.
The levels of IL-1β and TNF-a mRNA were significantly increased in the KA group when compared with the PBS group (P<0.01). An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably suppressed the mRNA expression of IL-1β and TNF-a (P<0.05). However, no significant decreases were observed in the group treated with1μg of antibody (P>0.05).
2. Effects of anti-HMGB1antibody on glial activation in the hippocampus of P21rats at3d after status epilepticus
Immunohistochemistry showed that the immunoreactivities of Ibal, ED1and GFAP were increased when compared with the PBS group (P<0.01). An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably suppressed the immunoreactivities of Ibal, ED1and GFAP in the hippocampus (P<0.01), but no significant inhibition were observed in the group treated with1μg of antibody (P>0.05).
3. Effects of anti-HMGB1antibody on the neuronal damage in the hippocampus of P21rats at3d after status epilepticus
Nissl and Fluoro-Jade B staining showed that there was no neuronal damage was observed in the PBS group. However, the neuronal damage was significant in the KA+IgY group, and the damage was obvious in CA3and DG. An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably attenuated the neuronal damage (P<0.05), but no significant inhibition were observed in the group treated with1μg of antibody (P>0.05).
Conclusions
1. KA-induced SE markedly increased the mRNA expression of IL-1β and TNF-a, microglial activation, and neuronal damage in the hippocampus.
2. An intracerebroventricular injection of anti-HMGB1antibody dose dependently inhibited the synthesis of cytokines, glial activation, and neuronal losses in the hippocampus after SE.
高迁移率族蛋白1(high-mobility group box1, HMGB1)是一种高度保守的非组DNA结合蛋白,具有稳定核酸结构、调节转录和基因表达等多种生物学功能。近年来发现在一定因素作用下可以释放到细胞外发挥促炎因子作用,HMGB1可以由坏死细胞被动释放或由活化的免疫细胞主动释放,通过与其受体如Toll样受体4(toll-like receptor-4, TLR4), Toll样受体2(TLR2)和晚期糖基化终末产物受体(receptor for advanced glycation end products, RAGE)等结合,激活核因子-κB (nuclear factor-κB,NF-κB)及其他信号传导通路,促进炎症因子及趋化因子的产生,从而发挥其促炎作用。TLR作为固有免疫受体,是近年来研究较多的一类受体,主要与感染细菌及内源性损伤相关分子结合发挥作用,其中TLR4是其中研究较热的受体之一,广泛分布于神经系统,包括神经元、小胶质细胞和星形胶质细胞,参与多种神经系统疾病过程。
研究证实HMGB1参与了败血症、关节炎、胰腺炎、呼吸障碍、脑缺血、脑外伤、脑炎等病理过程。而HMGB1的抑制剂如中和抗体,Box A等可以减轻多种疾病中HMGB1引起的炎症反应,发挥一定的保护作用,相反,给予重组HMGB1可以加重炎症反应及组织损伤。例如在败血症中给予HMGB1中和抗体可以减轻炎症反应,降低死亡率；在脑缺血和创伤性脑损伤中存在HMGB1的转位活化,给予HMGB1中和抗体可以抑制HMGB1活化及炎症反应,减轻脑损伤,而给予重组HMGB1可增加细胞因子合成,促进胶质细胞活化及加重神经元损伤。
近来有研究表明在成年小鼠癫痫发生中有HMGB1/TLR4信号通路的参与,在热性惊厥儿童中也观察到血浆HMGB1水平升高。但是在未成年机体癫痫发作中是否有HMGB1的参与以及HMGB1中和抗体在癫痫发作中是否有神经保护作用仍不清楚,研究表明癫痫发生的易感性、神经病理变化及预后都存在年龄依赖性,而HMGB1的表达也在一定程度上受到年龄影响,因此在本课题中,我们通过侧脑室注射海人酸(kainic acid, KA)建立生后21天(postnatal day21, P21)大鼠癫痫持续状态(status epilepticus, SE)模型,研究SE后早期阶段(3h-7d), HMGB1/TLR4通路在海马组织中的表达特点以及神经元损伤特点；并通过给予HMGB1中和抗体阻断HMGB1活性,研究HMGB1中和抗体对癫痫持续状态后海马炎症因子表达,胶质细胞活化状态及神经元损伤情况的影响,以明确HMGB1在幼鼠癫痫持续状态中的表达特点及作用。
第一部分：HMGB1/TLR4通路在幼年大鼠癫痫持续状态中表达变化的研究
目的：
研究海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马HMGB1/TLR4通路的表达变化。
材料与方法：
1.幼鼠癫痫持续状态模型的建立及分组
选用生后21天Wistar大鼠,水合氯醛(400mg/kg)腹腔注射麻醉后固定于立体定位仪。采用微量注射器缓慢将2nmol KA溶液(溶于1μl PBS)注入侧脑室(坐标为前囟后0.7mmm,中线外1.3mm,深度3.0mmm)。PBS对照组给予同等剂量PBS替代KA。痫性发作行为学评价根据Racine分级标准进行,持续全身发作或连续多次全身发作之间不能恢复正常状态超过30min者定义为癫痫持续状态(Racine分级Ⅳ-Ⅴ级),记录癫痫持续状态开始时间。在SE开始后2h给予水合氯醛(400mg/kg)腹腔注射终止发作。未达标准的大鼠予以剔除。
SE大鼠随机分为SE后3h、6h、12h、24h、3d和7d组,同时设立正常组和PBS对照组(根据预实验结果选取时间点为注射后24h)
2.采用尼氏染色和FJB染色观察神经元形态及损伤情况。
3.采用免疫组织化学技术检测各组大鼠海马HMGB1和TLR4蛋白表达变化。
4.采用Western blot方法检测各组大鼠海马HMGB1和TLR4蛋白表达变化。其中HMGB1蛋白检测又分为总蛋白和胞浆蛋白。
5.采用免疫荧光双重标记技术检测各组大鼠海马HMGB1在各类细胞(神经元、小胶质细胞和星形胶质细胞)中的分布变化。
结果：
1.海人酸侧脑室注射诱导P21大鼠癫痫持续状态
在海人酸注射后30min内,所有大鼠都出现呼吸急促、咀嚼、动须,点头、湿狗样抖动及前肢阵挛,之后逐渐出现旋转,站立,跌倒,强直阵挛发作。8%大鼠死亡,其中80%死亡出现于KA侧脑室注射后1h以内。观察大鼠癫痫持续状态发作2h后给予水合氯醛(400mg/kg)腹腔注射终止发作。而在PBS对照组无癫痫发作。
2.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马神经元损伤
尼氏染色和FJB染色显示正常组和PBS对照组大鼠海马无神经元损伤。SE后6h在CA3区和Hilus区开始出现神经元损伤,表现为尼氏染色阳性细胞数减少(P<0.01),并出现FJB阳性细胞,之后神经元损伤逐渐加重,SE后24h可见大量神经元损伤。而在CA1区明显的神经元损伤开始于SE后24h(P<0.05),3d明显(P<0.01),并且损伤较CA3区和Hilus区轻。
3.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马HMGB1蛋白的表达变化
Western blot分析显示海马HMGB1,总蛋白无明显变化,而胞浆HMGB1在SE后明显增加,开始于SE后3h(P<0.01),24h达高峰(P<0.01),之后逐渐降低,7d时仍高于正常组(P<0.05), PBS对照组与正常组无差异(P>0.05)。免疫组化显示在正常组大鼠和PBS对照组大鼠海马中,HMGB1主要分布于细胞核内,SE后3h开始出现于细胞浆中。在CA1区,SE后3h到12h,胞浆HMGB1主要出现于椎体细胞层中,在24h及以后,胞浆HMGB1在胶质形态细胞中增多。在CA3区和Hilus区,自SE后6h起可以观察到HMGB1染色缺失区域,该区域与尼氏染色和FJB染色显示的神经元损伤区域一致。
4.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马HMGB1的细胞定位
免疫荧光双标显示在正常组大鼠中,HMGB1位于神经元和星形胶质细胞细胞核内,无ED1阳性细胞。SE后6h,胞浆HMGB1主要出现于NeuN阳性的神经元细胞。SE后24h, HMGB1在胶质细胞中表达增多,并且星形胶质细胞表达HMGB1的比例及胞浆HMGB1阳性的星形胶质细胞明显增多(P<0.05),另外,出现ED1标记的小胶质细胞,主要表达于CA3区和Hilus区,分别有78.4±0.12%和81.2±0.08%的ED1阳性细胞与HMGB1共分布。
5.海人酸侧脑室注射诱导P21大鼠癫痫持续状态后海马TLR4蛋白的表达变化
Western blot分析显示TLR4蛋白在正常组大鼠和PBS对照组大鼠海马中的表达量很少,而在SE后明显增加,开始于3h(P<0.01),24h达高峰(P<0.01),之后逐渐降低,7d时仍高于正常(P<0.01)。免疫组化结果显示在正常组大鼠和PBS对照组大鼠海马中,TLR4仅在部分大鼠有散在弱表达,SE后3h开始有表达,之后逐渐增多,在24h之前,TLR4阳性细胞主要分布于锥体细胞层,以神经元形态为主,在24h之后,出现在胶质细胞形态细胞中。
结论：
1.在海人酸侧脑室注射诱导P21大鼠癫痫持续状态后早期阶段,海马HMGB1/TLR4通路活化,并与神经元损伤相关。
2. HMGB1的活化即核浆转位及胞外释放首先发生在神经元,随后出现于胶质细胞。
第二部分：HMGB1中和抗体在幼年大鼠癫痫持续状态中的作用研究
目的：
研究HMGB1中和抗体对P21大鼠癫痫持续状态后海马炎症反应及神经元损伤的作用。
方法：
1.幼鼠癫痫持续状态模型的建立
癫痫持续状态模型建立方法同第一部分。
2.分组及药物干预
SE大鼠随机分为模型组(KA+IgY组)和HMGB1中和抗体干预低、中、高剂量组(KA+anti-HMGB1组)。模型组通过侧脑室注射给予对照IgY (4μg),干预组分别给予HMGB1中和抗体1、2、4μg。给药时间为SE终止即刻及SE后12h。同时设PBS对照组,在相应时间给予对照IgY (4μg)。
3.实时定量RT-PCR
SE后6h取海马组织提取RNA,采用实时定量RT-PCR方法检测海马炎症因子白介素1β(IL-1β)和肿瘤坏死因子α(TNF-a) mRNA的表达变化。
4.免疫组织化学
SE后3d取脑组织制作石蜡切片,通过免疫组织化学方法检测海马Ibal、ED1和GFAP的表达变化。
5.尼氏染色和FJB染色
SE后3d,采用尼氏染色和FJB染色方法检测海马神经元形态及损伤情况。结果：
1. HMGB1中和抗体对海马炎症因子的影响
实时定量RT-PCR结果显示SE后6h,与对照组相比,KA+IgY组大鼠海马IL-1β和TNF-a mRNA表达明显增高(P<0.01)。而侧脑室注射中、高剂量中和抗体(2和4μg)可以明显抑制海马IL-1p和TNF-a mRNA表达(P<0.05),低剂量中和抗体(1μg)则无明显抑制作用(P>0.05)。
2. HMGB1中和抗体对海马胶质细胞的影响
免疫组织化学结果显示SE后3d,KA+IgY组大鼠海马小胶质细胞(Ibal和ED1)和星形胶质细胞(GFAP)免疫染色较对照组明显增强(P<0.01)。而侧脑室注射中、高剂量中和抗体(2和4μg)可以明显抑制海马Iba1、ED1和GFAP免疫染色强度(P<0.01),低剂量中和抗体(1μg)则无明显抑制作用(P>0.05)。
3. HMGB1中和抗体对海马神经元损伤的影响
尼氏染色和FJB染色显示SE后3d,对照组海马神经元形态正常,无损伤,KA+IgY组大鼠海马神经元损伤明显,表现为尼氏染色阳性神经元明显减少,而FJB染色阳性神经元显著增多(P<0.01),且CA3区、Hilus区较CA1区神经元损伤明显。而侧脑室注射中、高剂量中和抗体(2和4μg)可以明显减轻神经元损伤(P<0.05),低剂量中和抗体(1μg)则无明显作用(P>0.05)。
结论：
1.海人酸侧脑室注射诱导的P21大鼠癫痫持续状态可以促进海马炎症因子合成,胶质细胞活化及神经元损伤。
2.侧脑室注射HMGB1中和抗体可以剂量依赖性的抑制海马炎症因子合成及胶质细胞活化,减轻神经元损伤,发挥神经保护作用。
Epilepsy is one of the common neurological chronic disorders, with long-tern and repeated features. It has high morbidity in children. Frequently and seriously epileptic seizures may lead to further brain injury and persistent neurologic and psychogenic disorders, bringing troubles to the patients and family and society. Recent years, alought great evolution has been made in the etiology, pathology and treatment of epilepsy, the exact mechanism of epileptogenesis still remains unclear. Increasing evidence indicates that brain injury such as status epilepticus (SE), trauma, and stroke was accompanied by neuroinflammation, which may contribute to promoting neuronal excitability, neuronal death, and epileptogenesis. Accumulating data shows that many inflammatory factors participated in the development of epilepsy, and anti-inflammatory therapies could reduce seizures and neuropathological changes in some forms of epilepsy.
High-mobility group box1(HMGB1) is a highly conserved, ubiquitous non-histone DNA-binding protein that participates in stabilization of nucleosome formation and regulation of gene transcription. HMGB1has recently been defined as a key cytokine. It is released into the extracellular milieu actively by immune cells or passively by necrotic or damaged cells and triggers inflammatory responses and tissue injury. HMGB1can bind to multiple receptors such as the receptor for advanced glycation end products (RAGE), toll-like receptor-4(TLR4) and TLR2. The pro-inflammatory effect of HMGB1is exerted by activating nuclear factor-κB (NF-κB) or other pathways which promotes the chemotaxis and the production of cytokines. Toll-like receptors are innate immune receptors and respond to microbial structures and endogenous danger factors. Among the TLRs, TLR4is one of receptors which are the focus of concern. TLR4was expressed widely in the nervous system, including in microglia, astrocytes and neurons, and participated in many neurological diseases.
It has been reported that HMGB1contributes to the pathophysiology of sepsis, arthritis, respiratory disorders, pancreatitis, cerebral ischemia, traumatic brain injury, encephalitis and so on. The inhibitors of HMGB1such as anti-HMGB1antibody and Box A could alleviate inflammatory reaction and exert protective effects. On the contrary, injection recombinant HMGB1may promote inflammatory response and aggravate neuronal damage. For example, anti-HMGB1antibody could decrease the lethality of sepsis. HMGB1translocates from the nucleus into the cytoplasm and increases in cerebrospinal fluid and serum in cerebral ischemia and traumatic brain injury models. Anti-HMGB1antibody may inhibit the HMGB1translocation and inflammatory reaction and ameliorate brain infarction. However, recombinant HMGB1could prompt the induction of pro-inflammatory mediators and increase the neuronal death in vivo and in vitro.
Recently, it has been reported that the HMGB1-TLR4axis is activated in adult mice models of acute and chronic seizure, and elevated serum levels of HMGB1are observed in febrile seizure patients. However, so far, the role of HMGB1in epilepsy of immature brain and the role of anti-HMGB1antibody in the neuronal damage and inflammation following SE have not been explored. In addition, experimental evidence indicated that the susceptivity and the neuropathological changes of epilepsy are age-dependently, moreover, maturational stage could also affect the expression pattern of HMGB1. Therefore, in order to investigate the effect of HMGB1after SE in immature brain, we used postnatal day21(P21) wistar rats to induce SE models by an intracerebroventricular kainic acid (KA). We firstly investigated the expression of HMGB1/TLR4signal pathway and neuronal damage in hippocampus during the early phase of KA-induced SE (from3h to7d). Further, we studied the effects of anti-HMGB1antibody on the hippocampal damage and inflammatory reaction after KA-induced SE by an intracerebroventricular injection of anti-HMGBl antibody.
Chapter Ⅰ
The expression of HMGB1/TLR4signal pathway in immature hippocampus after status epilepticus
Objective
To investigate the expression changes of HMGB1/TLR4in P21rat hippocampus after KA-induced status epilepticus.
Materials and methods
1. KA-induced SE model
P21male Wistar rats were used. Rats were anesthetized using chloral hydrate (400mg/kg, intraperitoneally) and injected stereotaxically with KA (2nmol in1ml0.01M PBS) into the lateral ventricle at the following coordinates from the bregma:0.7mm posterior,1.3mm lateral, and3.0mm deep. The control rats received equal volumes of PBS. Seizures were behaviorally recorded in five grades using Racine's scale. The onset of SE was defined as the first grade4or greater seizure that progressed to similar repeated or prolonged behavioral seizures. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after the onset of SE. Rats that did not reach grade4were removed from further experiments.
The rats subjected to SE were further assigned randomly into six time point groups:3h,6h,12h,24h,3d and7d after the onset of SE. In addition to the above groups, normal-control group and PBS-control group (24h after injection) were also included.
2. Nissl and Fluoro-Jade B stainings were used to observe the neuronal morphology and neuronal damage in the hippocampus of P21rats after SE.
3. Immunohistochemistry was performed to examine the dynamic expression of HMGB1and TLR4in different subfields of hippocampus of P21rats after SE. The detection of HMGB1included the total HMGB1and cytoplasmic HMGB1.
4. Western blot was used to detect the dynamic expression of HMGB1and TLR4proteins in the hippocampus of P21rats after SE.
5. Double-labeling immunofluorescence staining was used to identify the cellular distribution (neuron, microglia and astrocytes) of HMGB1in the hippocampus of P21rats after SE.
Results
1.KA-induced SE
With in30min, all the rats showed polypnea, head nodding, facial automatisms, wet-dog shakes and forelimb clonus. Then, rats appeared rearing, generalized tonic-clonic seizures and transient loss of postural control.8%of rats died, and80%of the rats were died within the1hour after the intracerebroventricular injection of KA. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after the onset of SE. No seizure was observed in normal rats and PBS-injected rats.
2. Neuronal damage in the hippocampus of P21rats after KA-induced SE
Nissl and Fluoro-Jade B staining showed that there was no neuronal damage in normal rats and PBS-injected rats. Neuronal damage was occurred early in the CA3and Hilus at6h after SE, it was showed that the Nissl-positive cell counts were decreased (P<0.01), and FJB positive cells appeared. The neuron cell counts analysis showed a time-dependent decrease of Nissl-positive neurons in the hippocampus after KA-induced SE. At24h after SE, there was a substantial neuronal damage in the CA3and Hilus. However, delayed neuronal damage of CA1pyramidal cell layer was appeared lightly at24h after SE (P<0.05), and the degree of damage was light in CA1than that in CA3and Hilus.
3. Time-course expression of HMGB1in the hippocampus of P21rats after KA-induced SE
Western blot analysis showed that the total HMGB1protein level did not changed after SE, but the cytoplasmic HMGB1protein significantly upregulated after SE. The elevation of HMGB1was observed at3h after the onset of SE (P<0.01), peaked at24h (P<0.01), and decreased thereafter. At7days, a subtle elevation was still detected in the hippocampus (P<0.05). There was no significant difference between the normal rats and PBS-injected rats (P>0.05). Immunohistochemistry showed that in the normal and the PBS-injected rat hippocampus, HMGB1immunoreactivity was detected mostly in the nuclei. When rats were subjected to SE, HMGB1cytoplasmic translocation was observed in the hippocampus as early as3h after SE. From3h to12h after SE, the cytoplasmic translocation of HMGB1was obvioused mainly in the pyramidal neurons of CA1region, and after12h, the cytoplasmic HMGB1staining was occurred in the cell with glia morphology. In the pyramidal cell layer of CA3and hilus, HMGB1staining loss was observed from6h after SE, the staining loss region was in line with the region which undergoing degenerative changes at these time points.
4. The colocalization of HMGB1with different cellular markers in the hippocampus of P21rats after KA-induced SE
Double-labeling immunofluorescence showed that in the normal rat hippocampus, HMGB1immunoreactivity was observed in the nuclei of both neurons and astrocytes, and there were no ED1-positive cells in normal hippocampus. At6h after SE, cytoplasmic HMGB1staining was present mainly in the neurons of pyramidal layer. At24h post SE, the percentages of HMGB1-positive astrocytes and the cytoplasmic HMGB1-positive astrocytes were substantially increased (P<0.05). Moreover, the immunoreactivity of ED1, a marker of activated microglia/macrophages, was appeared mainly in the CA3and hilus. And double-labeling assay detected that78.4±0.12%and81.2±0.08%of ED1-positive cells were double stained by HMGB1in the CA3and hilus, respectively.
5. Time-course expression of TLR4protein in the hippocampus of P21rats after KA-induced SE
Western blotting analysis demonstrated that the expression of TLR4protein was very low in the normal and PBS control group. After SE, the expression of TLR4protein was progressively increased from3h (P<0.01) in the hippocampus, peaked at24h (P<0.01), and decreased thereafter.7days after SE, the elevation of TLR4was still detected in the hippocampus (P<0.01). Immunohistochemistry showed that in some of the normal rat hippocampus, faint immunoreactivity was observed in only a few scattered cells present in pyramidal layer. After SE, the immunostaining of TLR4protein in the hippocampus was significantly increased from3h after SE. before24h after SE, the TLR4-positive cells were mainly distributed in the pyramidal layer, and after24h, the TLR4immunoreactivity was also observed in the cells with glia morphology. In the subfields of hippocampus, the TLR4immunostaining was distributed mainly in the dentate hilus and CA3.
Conclusion
1. The expression of HMGB1/TLR4axis was transiently activated during the early phase of KA-induced SE in P21rats, and the elevation of HMGB1/TLR4was related with the neuronal damage.
2. The translocation of HMGB1from nuclei to cytoplasm and the extracellular release was first occurred in neurons, and then occurred in the glias.
Chapter Ⅱ
The effects of HMGB1antibody in juvenile rat hippocampus after kainic acid-induced status epilepticus
Objective To investigate the effects of anti-HMGB1antibody on hippocampal damage and
inflammatory reaction after KA-induced SE in postnatal day21rats.
Materials and methods
1. KA-induced SE model
The model was induced according to the method in chapter I.
2. Experimental design
Rats were selected randomly to receive anti-HMGB1antibody (1,2, and4μg, IgY subclass) or nonimmune control IgY (4μg) by an intracerebroventricular injection immediately after the termination of SE and12h after the onset of SE. The PBS-injected rats received control IgY (4μg) at the same time point. The dose schedule was selected on the basis of previous report and preliminary experiments. Rats were killed6h (n=6/group) after SE to quantify the cytokine production. To evaluate the microglial activation and neuronal damage, rats were killed3d after SE (n=6/group)
3. Real-time quantitative PCR
Rats were decapitated at6h after SE, and the hippocampi were dissected and stored at-80℃. The hippocampal mRNA expression of IL-1β and TNF-a was investigated by real-time PCR.
4. Immunohistochemistry
Rats were decapitated at3d after SE, and the brain was dissected and made into paraffin sections. The immunoreactivities of Ibal、ED1and GFAP were investigated by immunohistochemistry.
5. Nissl and Fluoro-Jade B stainings
Rats were decapitated at3d after SE, and the brain was dissected and made into paraffin sections. The neuronal damage was detected by Nissl and Fluoro-Jade B staining.
Results
1. Effects of anti-HMGB1antibody on the gene expression of inflammatory factors in the hippocampus of P21rats at6h after SE.
The levels of IL-1β and TNF-a mRNA were significantly increased in the KA group when compared with the PBS group (P<0.01). An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably suppressed the mRNA expression of IL-1β and TNF-a (P<0.05). However, no significant decreases were observed in the group treated with1μg of antibody (P>0.05).
2. Effects of anti-HMGB1antibody on glial activation in the hippocampus of P21rats at3d after status epilepticus
Immunohistochemistry showed that the immunoreactivities of Ibal, ED1and GFAP were increased when compared with the PBS group (P<0.01). An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably suppressed the immunoreactivities of Ibal, ED1and GFAP in the hippocampus (P<0.01), but no significant inhibition were observed in the group treated with1μg of antibody (P>0.05).
3. Effects of anti-HMGB1antibody on the neuronal damage in the hippocampus of P21rats at3d after status epilepticus
Nissl and Fluoro-Jade B staining showed that there was no neuronal damage was observed in the PBS group. However, the neuronal damage was significant in the KA+IgY group, and the damage was obvious in CA3and DG. An intracerebroventricular injection of anti-HMGB1antibody (2and4μg) notably attenuated the neuronal damage (P<0.05), but no significant inhibition were observed in the group treated with1μg of antibody (P>0.05).
Conclusions
1. KA-induced SE markedly increased the mRNA expression of IL-1β and TNF-a, microglial activation, and neuronal damage in the hippocampus.
2. An intracerebroventricular injection of anti-HMGB1antibody dose dependently inhibited the synthesis of cytokines, glial activation, and neuronal losses in the hippocampus after SE.
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