丙戊酸在大鼠蛛网膜下腔出血后早期脑损伤中的保护作用及机制研究

发布时间：2018-05-04 18:27

本文选题：蛛网膜下腔出血 + 早期脑损伤　；参考：《浙江大学》2014年博士论文

【摘要】：研究背景蛛网膜下腔出血约占全部脑卒中的5%-10%,其中大部分的出血原因为颅内动脉瘤破裂。过去数十年来,脑血管痉挛和动脉瘤破裂再出血一直被认为是导致高死亡和高致残的一个主要因素。但多中心的临床试验研究表明迟发性血管痉挛的减轻并没有改善蛛网膜下腔出血患者的预后。因此有学者提出了早期脑损伤是蛛网膜下腔出血致死致残的主要原因。早期脑损伤这一概念指从蛛网膜下腔出血开始至72小时这一时间窗之内的脑损伤。目前对蛛网膜下腔出血后早期脑损伤的病理生理学机制已经有了初步的认识,主要包括颅内压升高、脑血流量降低、脑灌注压下降、有氧呼吸的抑制、血脑屏障破坏、脑水肿及神经元的死亡。然而,具体的损伤机制及信号通路还需要进一步的深入研究。丙戊酸是临床上治疗癫痫和双向情感障碍的一线药物,近年来的研究表明其有神经保护作用。丙戊酸在MCAO脑缺血模型、颅脑外伤模型、脊髓损伤模型中均能减轻神经损伤,改善神经功能。其可能的神经保护机制：保护血脑屏障,抑制炎症细胞的浸润、炎症因子的表达,减少细胞凋亡等。本研究中我们将在蛛网膜下腔出血模型中探讨丙戊酸的神经保护作用,并通过检测血脑屏障和细胞凋亡两方面来明确丙戊酸的神经保护机制。实验方法 1.采用线栓穿刺法制作蛛网膜下腔出血模型,成年雄性SD大鼠被随机分为假手术组(Sham组),蛛网膜下腔出血+生理盐水组(SAH+Vehicle组)和蛛网膜下腔出血+丙戊酸组(SAH+VPA组),术后24小时通过对死亡率,神经功能缺陷评分,脑水肿含量进行测定来检测脑损伤程度。 2.成年雄性SD大鼠被随机分为假手术组,蛛网膜下腔出血+生理盐水组和蛛网膜下腔出血+丙戊酸组,术后24小时测量脑组织Evans blue含量,免疫组织化学检测IgG染色和MMP9细胞定位,巨噬细胞计数,采用RT-PCR测量炎症因子(MMP9, IL-1β, IL-6, TNF-α)的基因表达,Western blot检测蛋白(pERKl/2, MMP9, Occludin, Claudin-5)的表达,明胶酶谱检测MMP9的活性。 3.成年雄性大鼠被随机分为假手术组,蛛网膜下腔出血+生理盐水组和蛛网膜下腔出血+丙戊酸组,术后24小时采用Western blot检测凋亡相关蛋白(Bcl-2,cleaved caspase3)以及HSP70, pAkt蛋白的表达,TUNEL法测定细胞凋亡程度。结果 1、术后24小时SAH+VPA治疗组神经功能较SAH+Vehicle组明显改善(P0.05),SAH+VPA治疗组的脑水含量较SAH+Vehicle组明显减少(P0.05),但两组间死亡率没有明显差别。 2、术后24小时脑组织中Evans blue含量SAH+VPA组明显少于SAH+Vehicle组(P0.05), IgG染色和脑实质内的巨噬细胞数SAH+VPA组明显少于SAH+Vehicle组,MMP9主要定位于神经元和血管内皮细胞,炎症因子(MMP9, IL-1β, IL-6, TNF-α)的基因表达在SAH+VPA组也明显减少。明胶酶谱实验表明SAH+VPA组的MMP9活性明显低于SAH+Vehicle组(P0.05),western blot提示紧密连接蛋白(occluding、claudin-5) SAH+VPA组明显高于SAH+Vehicle组(P0.05),而蛋白pERK1/2, MMP9的水平则相反。 3、术后24小时SAH+VPA组的HSP70, pAkt, Bcl-2蛋白表达量明显高于SAH+Vehicle组(P0.05), cleaved caspase3的蛋白表达量SAH+VPA组明显少于SAH+Vehicle组(P0.05), SAH组和VPA组均出现细胞凋亡,但SAH+VPA组凋亡指数明显低于SAH+Vehicle组(P0.05)。结论 1.丙戊酸在蛛网膜下腔出血后急性期减轻了脑组织水肿,并改善大鼠神经功能。 2.丙戊酸减轻了蛛网膜下腔出血后血脑屏障的破坏,抑制炎症细胞的侵入。 3.丙戊酸激活Akt通路,增强HSP70蛋白的表达来减少细胞凋亡。
[Abstract]:Research background
Subarachnoid hemorrhage accounts for about 5%-10% of all stroke, most of which are caused by ruptured intracranial aneurysms. Over the past few decades, cerebral vasospasm and aneurysm rupture and rebleeding have been considered as a major cause of high death and high disability. However, a multicenter clinical trial indicates delayed vasospasm. Reduction did not improve the prognosis of patients with subarachnoid hemorrhage. Therefore, some scholars suggested that early brain injury is the main cause of sublethal hemorrhage.
The concept of early brain injury refers to brain damage from the beginning of subarachnoid hemorrhage to 72 hours. The pathophysiological mechanism of early brain injury after subarachnoid hemorrhage has been preliminarily recognized, including intracranial pressure, cerebral blood flow, cerebral perfusion pressure, inhibition of aerobic respiration, and blood brain. Barrier destruction, brain edema and neuronal death. However, specific injury mechanisms and signaling pathways need further in-depth study.
Valproic acid is a first-line drug in the clinical treatment of epilepsy and bipolar disorder. Recent studies have shown that it has neuroprotective effects. Valproic acid can reduce nerve damage and improve nerve function in the MCAO cerebral ischemia model, brain injury model, and spinal cord injury model. The possible neuroprotective mechanism: protecting the blood brain barrier and inhibiting inflammation Infiltration of cells, expression of inflammatory factors, and reduction of apoptosis.
In this study, we will explore the neuroprotective effect of valproic acid in the subarachnoid hemorrhage model and identify the neuroprotective mechanism of valproic acid by detecting the blood brain barrier and apoptosis in two aspects.
Experimental method
1. the adult male SD rats were randomly divided into sham operation group (group Sham), subarachnoid hemorrhage + physiological saline group (group SAH+Vehicle) and subarachnoid hemorrhage + valproic acid group (group SAH+VPA). The mortality, neurological deficit score and brain edema were measured at 24 hours after operation. The degree of brain damage was determined.
2. adult male SD rats were randomly divided into sham operation group, subarachnoid hemorrhage + physiological saline group and subarachnoid hemorrhage + valproic acid group. The content of Evans blue in brain tissue was measured at 24 hours after operation. Immunohistochemistry was used to detect IgG staining and MMP9 cell location, macrophage count, and RT-PCR measurement of inflammatory factors (MMP9, IL-1 beta, IL-6, TNF-). Gene expression, Western blot detection of pERKl/2 (MMP9, Occludin, Claudin-5) expression, gelatinase spectrum detection MMP9 activity.
3. adult male rats were randomly divided into sham operation group, subarachnoid hemorrhage + physiological saline group and subarachnoid hemorrhage + valproic acid group. 24 hours after operation, Western blot was used to detect apoptosis related protein (Bcl-2, cleaved Caspase3), HSP70, pAkt protein expression, and TUNEL method was used to determine the degree of apoptosis.
Result
1, 24 hours after the operation, the nerve function in the SAH+VPA group was significantly better than that in the SAH+Vehicle group (P0.05). The brain water content in the SAH+VPA group was significantly lower than that in the SAH+Vehicle group (P0.05), but there was no significant difference in the mortality rate between the two groups.
2, the content of Evans blue in group SAH+VPA was significantly less than that in group SAH+Vehicle (P0.05) after 24 hours of operation, and the number of SAH+VPA in IgG staining and the number of macrophages in brain parenchyma was significantly less than that in group SAH+Vehicle, MMP9 mainly located in neurons and vascular endothelial cells, and the gene expression of inflammatory factors (MMP9, IL-1 beta, IL-6, and alpha) was also significantly reduced. The MMP9 activity of the SAH+VPA group was significantly lower than that of the SAH+Vehicle group (P0.05), and Western blot suggested that the SAH+VPA group of the tightly connexin (occluding, claudin-5) was obviously higher than the SAH+Vehicle group (P0.05), but the level of protein pERK1/2 was the opposite.
3, the expression of HSP70, pAkt, Bcl-2 protein in group SAH+VPA was significantly higher than that in group SAH+Vehicle (P0.05), and the expression of cleaved Caspase3 in SAH+VPA group was significantly less than that in SAH+Vehicle group (P0.05).
conclusion
1. valproic acid alleviated brain edema and improved neurological function in acute phase after subarachnoid hemorrhage.
2. valproic acid alleviated the destruction of blood-brain barrier after subarachnoid hemorrhage and inhibited the invasion of inflammatory cells.
3. valproic acid activates the Akt pathway and enhances the expression of HSP70 protein to reduce apoptosis.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.35

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