吡非尼酮对大鼠蛛网膜下腔出血后柔脑膜纤维化的影响

发布时间：2018-08-06 16:37

【摘要】：目的：蛛网膜下腔出血(subarachnoid hemorrhage, SAH)是由于各种原因造成颅内出血，血液流入蛛网膜下腔形成的。按其发生机制不同可分为：外伤性SAH、自发性SAH、继发性SAH。外伤性SAH是颅脑外伤导致的；自发性SAH是指无明显诱因SAH，主要颅内动静脉畸形或动脉瘤破裂出血造成；继发性SAH是由于脑室内或脑实质内出血弥散到蛛网膜下腔形成的。SAH后急性期神经系统并发症主要有脑血管痉挛造成的缺血性脑损伤、出血部位发生再出血、急性脑积水等。慢性脑积水是其远期主要并发症之一，严重影响患者的预后、生活质量。SAH作为神经内外科常见危重急症，目前对急性期并发症研究较多，对脑积水的研究相对较少，对脑积水发生的具体机制尚未明确。多数学者认为：SAH后急性脑积水的发生是由于血凝块堵塞脑脊液循环通路造成的梗阻性脑积水，起病比较急,主要表现为神情淡漠、反应迟钝、原有症状加重、重症病人出现昏迷、意识障碍。SAH后慢性脑积水的发生主要原因在于出血造成的应激、炎症反应刺激蛛网膜、软脑膜、蛛网膜小梁等柔脑膜结构发生纤维化病变，使局部结构发生纤维化粘连，造成脑脊液的吸收减少，导致交通性脑积水；堵塞脑脊液循环通路造成脑脊液循环受阻，形成梗阻性脑积水。吡非尼酮(Pirfenidone, PFD)是一种小分子化合物，化学式：C12Hl1NO，PFD分子能够通过细胞膜，无需受体。口服给药后经胃肠道吸收，迅速在体内各组织中广泛分布，可透过血脑屏障。大量实验证实PFD在抗纤维化治疗方面的疗效显著且不良反应少。主要应用于特异性肺纤维化、肾脏、肝脏、心肌组织、多发硬化症和神经纤维瘤等抗纤维化疾病的治疗研究。利用PFD预防及治疗SAH后的柔脑膜纤维化、减低SAH后慢性脑积水的发生率的研究尚未见报道。本研究拟观察PFD与大鼠SAH后脑脊液中强致纤维化因子之一转化生长因子-β1（transforming growth factor beta1，TGF-β1）的关系，以及其对柔脑膜纤维化的影响。方法: 采用清洁级SD大鼠80只，雌雄不限，体重250±10g，随机分为正常对照组、假手术组和模型组；模型组又分为干预组和对照组。采用血管内穿刺法建立SAH动物模型，干预组给予PFD100mg/kg灌胃给药，对照组给予安慰剂灌胃，连续14天，分别在3、6、10、14、21天自枕大池采集脑脊液标本，检测各组脑脊液中TGF-β1的浓度；第21天将试验动物灌注取脑，经MASSON染色处理后，用病理图文分析系统图像处理软件Image-Pro Plus6.0分析图像，测量柔脑膜的厚度和灰度值，以100×厚度/灰度值作为胶原纤维含量的指标，所得数据均采用SPSS13.0软件进行统计学分析。观察其对柔脑膜纤维化的程度的影响。结果: 1各组大鼠一般情况的观察比较空白组大鼠进食好，精神好，被毛有光泽，活动灵活，未见明显异常状态，随着饲养时间的延长，体型渐长，体重不断增加。假手术组大鼠麻醉清醒后表现为竖毛、精神萎靡、饮食摄水减少、活动减少，于术后第1日恢复正常，进食增多，精神状态佳，被毛光泽，活动灵活，无异常状态出现，随着饲养时间的延长，体型渐长，体重不断增加。模型组大鼠麻醉清醒后表现为竖毛、精神萎靡、嗜睡，定向力差、于术后1周内均精神萎靡、饮食摄水减少，活动减少，于术后1周后渐恢复，精神较前好转，饮食摄水增加，但与空白组及假手术组相比活动减少，反应较迟钝。随着饲养时间延长，体型有所增长。 2模型制作观察及神经行为评分模型制作24小时后，空白组和假手术组灌注取脑后，蛛网膜下腔未见血液成分，颅底未见血凝块。模型组灌注取脑后大脑腹侧面可见血凝块存在，主要位于前颅窝及颅底动脉环周围，包绕脑底部主要血管，，并且穿刺侧出血量相对较多，颅底相对应位置可见血凝块。模型制作24小时候，参照Bederson方法进行神经行为功能缺损评分。将评分2分以上为纳入模型成功标准。结果提示:空白组和假手术组没有功能缺损，模型组神经功能缺损评分平均为2.50±0.56分，与空白对照组进行比较，评分有统计学意义。 3转化因子-β1及柔脑膜纤维化影响对照组大鼠脑脊液中TGF-β1的浓度出现2次升高，与空白组比较差异有显著性（P0.05）。干预组脑脊液中TGF-β1的浓度也出现2次升高，但最高值明显低于对照组（P0.05）。空白组、假手术组无明显变化。MASSON染色显示对照组与干预组较空白组、假手术组柔脑膜增厚且灰度值明显减低（P0.05），其中干预组较对照组柔脑膜纤维化明显减低（P0.05）。空白组与假手术组柔脑膜无明显纤维化。结论: 1SAH发生后脑脊液中TGF-β1浓度明显升高，并呈双时相性。 2SAH21天后可出现柔脑膜纤维化现象。 3SAH发生后应用PFD干预可降低脑脊液中的TGF-β1的浓度，特别是能够明显减低第二时相的升高程度，能够明显减低柔脑膜的纤维化程度。
[Abstract]:Objective:
Subarachnoid hemorrhage (subarachnoid hemorrhage, SAH) is due to various causes causing intracranial hemorrhage and blood flow into the subarachnoid cavity. According to its mechanism, it can be divided into traumatic SAH, spontaneous SAH, and secondary SAH. traumatic SAH, which is caused by craniocerebral trauma; spontaneous SAH refers to no obvious inducement SAH and major intracranial movement. The secondary SAH is the ischemic brain injury caused by cerebral vasospasm, bleeding sites and acute hydrocephalus. Chronic hydrocephalus is the main complication of the acute phase of.SAH in the acute phase of the acute phase after the hemorrhage of the intraventricular or intraventricular hemorrhage to the subarachnoid cavity. One of the disease, which seriously affects the prognosis of the patients, the quality of life.SAH is a common critical emergency in the internal and external department. At present, there are many studies on the acute complications, the research on hydrocephalus is relatively few, the specific mechanism of hydrocephalus is not clear. Most scholars believe that the occurrence of acute hydrocephalus after SAH is due to the clogging of the brain ridge with blood clot. The obstructive hydrocephalus caused by the fluid circulation pathway is very urgent, mainly manifested in the indifference, the slow reaction, the aggravation of the original symptoms and the coma in the severe patients. The main cause of the chronic hydrocephalus after.SAH is the stress caused by bleeding, and the inflammatory reaction stimulates the arachnoid, the pia meninges, the arachnoid trabecula and so on. The occurrence of fibrosis, the local structure of fibrosis adhesion, resulting in the absorption of cerebrospinal fluid absorption, causing traffic hydrocephalus; blocking the cerebrospinal fluid circulation pathway caused by the cerebrospinal fluid circulation obstruction, the formation of obstructive hydrocephalus. Pirfenidone (PFD) is a small molecular compound, chemical formula: C12Hl1NO, PFD molecules can pass Cell membrane, without the need for receptor. After oral administration, it is absorbed by the gastrointestinal tract and is widely distributed in various tissues in the body and through the blood brain barrier. A large number of experiments have proved that PFD has significant effects on anti fibrosis and less adverse reactions. It is mainly used in specific pulmonary fibrosis, kidney, liver, myocardial tissue, multiple sclerosis and neurofibroma. Research on the treatment of anti fibrosis diseases. The study on the use of PFD to prevent and treat SAH after SAH has not been reported. This study is to observe the relationship between PFD and rat SAH, one of the strong fibrotic factors transforming growth factor - beta 1 (transforming growth factor beta1, TGF- beta 1). Line, and its effect on the fibrosis of the soft meninges.
Method:
80 SD rats were divided into the normal control group, the sham operation group and the model group, and the model group was divided into the intervention group and the control group. The model group was divided into the intervention group and the control group. The SAH animal model was established by intravascular puncture, the intervention group was given the medicine of PFD100mg/kg and the control group was given the placebo for 14 days, respectively, at 3,6,10,14, The concentration of TGF- beta 1 in cerebrospinal fluid was collected at 21 days from the big cistern, and the concentration of the cerebrospinal fluid in the cerebrospinal fluid was detected. After twenty-first days, the experimental animals were perfused to take the brain. After MASSON staining, the image processing software Image-Pro Plus6.0 was analyzed by the pathological graphic analysis system. The thickness and gray value of the soft meninges were measured, and the 100 x thickness / gray value was used as the collagen fiber. The data were analyzed by SPSS13.0 software. The effect of SPSS13.0 on the degree of leptomeningeal fibrosis was observed.
Result:
1 observation and comparison of the general situation of rats in each group
The rats in blank group had good eating, good spirit, glossy coat, flexible movement and no obvious abnormality. With the prolongation of feeding time, the body shape and weight increased gradually.
The rats in the sham operation group showed the erect hair after sober anesthesia, depressed spirit, reduced food intake, reduced activity, resumed normal in first days after the operation, increased eating and had better mental state, was flexible, and had no abnormal state. As the feeding time extended, the body type grew gradually, and the weight was constantly increasing.
The rats in the model group were shown to be erect, depressed, sleepy, and poor Orienteering after 1 weeks of anesthesia. They were depressed in the spirit, reduced food intake and decreased activity after 1 weeks of operation. The spirits were gradually recovered after 1 weeks, the spirit was better and the food intake was increased, but the activity was reduced and the reaction was slow compared with the blank group and the sham operation group. With the feeding time prolonged, There is a growth in shape.
2 model making observation and neurobehavioral score
After 24 hours of making the model, there was no blood component in the subarachnoid space and no blood clot in the subarachnoid space. The blood clot was found on the ventral side of the brain of the model group, mainly located around the anterior cranial fossa and the ring of the skull base artery. The blood vessels were wrapped around the base of the brain, and the amount of bleeding on the puncture side was relatively large, and the skull base was relatively large. The blood clot was seen in the relative position. When the model was made 24, the Bederson method was used to score the neurobehavioral function defect score. The score of the score was more than 2 points. The results showed that there was no functional defect in the blank group and the sham operation group, and the average score of the neural function defect was 2.50 + 0.56 points in the model group, compared with the blank control group. Compared, the score was statistically significant.
3 transformation factor - beta 1 and the influence of the soft meningeal fibrosis
The concentration of TGF- beta 1 in the cerebrospinal fluid of the control group increased 2 times, compared with the blank group (P0.05). The concentration of TGF- beta 1 in the cerebrospinal fluid of the intervention group was also increased 2 times, but the highest value was significantly lower than that of the control group (P0.05). There was no obvious change in the sham group and the.MASSON staining showed that the control group and the intervention group were more than the blank group and the false hand. In the operation group, the soft meninges were thickened and the gray value decreased significantly (P0.05), and the fibrosis in the intervention group was significantly lower than that in the control group (P0.05). There was no obvious fibrosis in the blank group and the sham group.
Conclusion:
The concentration of TGF- beta 1 in CSF increased significantly after 1SAH.
The phenomenon of soft meningeal fibrosis appears after 2SAH21 days.
The use of PFD intervention after 3SAH can reduce the concentration of TGF- beta 1 in cerebrospinal fluid, especially to reduce the level of second phase obviously, and reduce the degree of fibrosis of the meningomeninges.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.35

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