血管内皮生长因子不同时间段经鼻给予新生大鼠缺氧缺血性脑损伤模型后对神经系统恢复效果的研究

发布时间：2018-09-10 11:21

【摘要】：目的：本研究建立7日龄SD大鼠缺氧缺血性脑损伤（hypoxic-ischemic braindamage，HIBD）模型，建模后将血管内皮生长因子（vascular endothelial growthfactor，VEGF）分两个不同时间段经鼻给予新生大鼠HIBD模型。通过观察新生大鼠运动行为学改变、改良神经功能缺失评分变化、脑组织形态学改变、脑组织病理改变以及脑细胞凋亡的变化，探讨VEGF在新生大鼠HIBD后发挥神经保护作用的最佳用药时间及安全有效的方法，为临床安全有效地应用外源性VEGF治疗新生儿缺氧缺血性脑病提供依据。方法：(1)7日龄SD大鼠64只，随机分为8组，每组8只：A组为假手术组，行颈部切口并分离组织后皮肤缝合不做HIBD处理，行假手术后当天处死；B H组均参照改良Rice法制备HIBD模型，B组HIBD模型建模后当天处死；C组建模后第6d处死；D组建模后第9d处死；E H组建模后将10mg/L的VEGF分两个时间段经鼻给予新生大鼠，其中E组、F组建模后第13d给药，每天1次，连续给药3d，E组建模后第6d处死；F组建模后第9d处死；G组、H组在46d给药，每天1次，连续给药3d，G组在建模后第6d处死；H组在建模后第9d处死。(2)各组新生大鼠处死前观察新生大鼠运动行为学改变、改良神经功能缺失评分变化，处死后观察脑组织病理改变、脑组织大体形态学改变、TUNEL法检测脑细胞凋亡的变化。结果：(1)新生大鼠运动行为学改变A组新生大鼠运动、感觉、反射和平衡能力正常。B H组HIBD模型建模后进行连续观察2h：所有新生大鼠相继出现间断性的抖动、皮肤青紫、躯体颤抖、尾部抽搐、翻身困难、尾部及臀部翘起并向左旋转、走路不稳等明显异常的运动行为学变化。HIBD后的新生大鼠右侧眼睛正常而左侧眼睛张开迟缓，眼裂小，上睑下垂。建模后第6d：C组、D组新生大鼠可以行走，，但行动迟缓，不能翻身，平衡能力差易向左侧倾倒；E组、F组可以行走，但不能顺利翻身，平衡能力较差向左侧偏斜；G组、H组可以行走，部分新生大鼠能较顺利翻身，平衡能力较差向左侧偏斜。建模后第9d：D组行走比较稳，大部分能翻身，但平衡能力较假手术组有明显差异；F组行走稳，可顺利翻身，平衡能力较HIBD组好；H组9天后行走稳，可顺利翻身，平衡能力较好。(2)新生大鼠改良神经功能缺失评分A组新生大鼠评分0，无神经功能缺失；B组14.63±1.41，严重神经功能损伤。建模后第6d：C组9.63±1.51，中度损伤；E组6.75±1.39，中度损伤；G组4.00±0.76，轻度损伤。建模后第9d：D组7.63±1.92，中度损伤；F组5.25±1.49，轻度损伤；H组2.75±0.89，轻度损伤。建模后4~6d给予VEGF治疗的G、H组评分分别低于1~3d给药的E、F组，给药治疗的E~H组均低于未给药的C、D组，且p0.05，差异有统计学意义。(3)脑组织大体形态学变化A组新生大鼠脑组织大体外观正常，两侧半球对称，无水肿、液化及萎缩；B H组HIBD模型建模后各时间点处死的新生大鼠脑组织左侧大脑出现水肿、苍白、液化等不同程度的异常改变。建模后第6d：C组新生大鼠脑组织左侧大脑大片坏死液化区，经VEGF治疗的E组、G组脑组织大体病变明显轻于未治疗各组，表现为中度的水肿，无大片液化坏死区。建模后第9d：D组新生大鼠脑组织左侧大脑萎缩明显，经VEGF治疗的F组、H组脑组织大体表现为轻中度的水肿。(4)脑组织病理改变（HE染色） A组新生大鼠脑组织结构层次清楚，细胞轮廓清晰，形态正常，细胞核居中。B~H组在建模后左侧大脑皮质区、海马区、丘脑出现神经细胞大片水肿、液化、坏死区。建模后第6d：C组新生大鼠脑组织切片左侧大脑大片坏死液化区，细胞数目明显减少；E组可见细胞坏死及水肿，但没有成大片坏死区域，细胞数目有所减少；G组可见明显水肿细胞，细胞数目减少，可见胶质细胞增生。建模后第9d：D组新生大鼠脑组织切片细胞数目减少更加明显，有胶质细胞增生；F细胞数目有所减少，细胞水肿减轻，胶质细胞增生；H组可见细胞数目减少，细胞间隙增宽，胶质细胞明显增生。(5) TUNEL法检测脑细胞凋亡A组切片染色后偶可见核棕染的凋亡细胞，凋亡细胞数3.50±0.93个。B组左侧缺血侧大脑切片可见大量核棕染的凋亡细胞，凋亡细胞数58.13±5.74个。建模后第6d：C组66.38±8.18个；E组为53.12±7.92个；G组为41.38±6.78个。建模后第9d：D组50.50±5.68个；F组为35.63±6.05个，H组为29.25±3.49个，凋亡细胞数明显低于未给药治疗的C、D组。建模后4~6d给予VEGF治疗的G、H组凋亡细胞数分别低于1~3d给药的E、F组，给药治疗的E~H组均低于未给药的C、D组，且p0.05，差异有统计学意义。结论：(1)经鼻将VEGF给予新生大鼠HIBD模型后可以有效改善大鼠神经行为功能，减轻脑水肿，改善神经细胞病理变化，减少神经细胞凋亡。提示VEGF可通过这种无创方法用于新生儿HIE的治疗。(2)本实验G组、H组效果优于E组、F组即在新生大鼠HIBD建模3d后应用效果优于起始应用。提示VEGF用于新生儿HIE的治疗可在脑水肿高峰过后应用。
[Abstract]:AIM: To establish a 7-day-old model of hypoxic-ischemic brain damage (HIBD) in SD rats. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) was injected nasally into the model of HIBD in neonatal rats in two different periods after the model was established. To explore the best time and safe and effective method for the use of vascular endothelial growth factor (VEGF) to exert neuroprotective effect after HIBD in neonatal rats, and to provide evidence for the safe and effective use of exogenous VEGF in the treatment of neonatal hypoxic-ischemic encephalopathy.
Methods: (1) Sixty-four seven-day-old SD rats were randomly divided into 8 groups, each group consisting of 8 rats: group A was sham-operated, the neck incision was made and the skin was sutured without HIBD treatment, and the rats were executed on the day after sham operation; group B was made HIBD model according to the modified Rice method, and group B was executed on the day after modeling; group C was executed on the 6th day after modeling; group D was executed on the day after modeling; The rats in group E were given 10 mg/L of vascular endothelial growth factor by nasal administration at the 9th day after the establishment of the model. The rats in group E were given 10 mg/L of vascular endothelial growth factor by nasal administration at the 13th day after the establishment of the model, once a day for 3 days, and the rats in group E were sacrificed at the 6th day after the establishment of the model. (2) The neonatal rats in each group were sacrificed on the 9th day to observe the changes of motor behavior, modified neurological deficit score, pathological changes of brain tissue, gross morphological changes of brain tissue, and apoptosis of brain cells by TUNEL.
Results: (1) Neonatal rats had normal motor, sensory, reflex and balance abilities. The HIBD model of group B H was established and observed continuously for 2 hours. All the neonatal rats had intermittent shaking, blue skin, body trembling, tail twitching, difficulty in turning over, tail and buttock warping up and turning to the left, and no walking. After HIBD, the right eye of neonatal rats was normal, but the left eye was slow to open, the cleft was small, and the upper eyelid was drooping. Group G, group H can walk, part of the newborn rats can roll over smoothly, balance ability is poor to the left deviation. 9 days after modeling: group D walks more stable, most can roll over, but the balance ability is significantly different from the sham operation group; group F walks steadily, can roll over smoothly, balance ability is better than the HIBD group 9 days later; (2) Neonatal rats in group A scored 0, without neurological deficit; group B scored 14.63 + 1.41, with severe neurological impairment. Sixth day after modeling: group C 9.63 + 1.51, moderate injury; group E 6.75 + 1.39, moderate injury; group G 4.00 + 0.76, mild injury. Group D was 7.63 [1.92], moderate injury; Group F was 5.25 [1.49], mild injury; Group H was 2.75 [0.89], mild injury. After modeling, the scores of group G and group H were lower than those of group E and group F, which were given VEGF for 4-6 days, respectively. The scores of group E~H were lower than those of group C and group D, and the differences were statistically significant. (3) Gross morphological changes of brain tissue in group A were statistically significant. The brain tissue was normal in general appearance, bilateral hemisphere symmetry, no edema, liquefaction and atrophy; the left side of the brain tissue of neonatal rats in group B H was edema, paleness, liquefaction and other abnormal changes in varying degrees after the HIBD model was established. In group E, the gross lesion of brain tissue in group G was milder than that in untreated group, showing moderate edema without large areas of liquefied necrosis. In group B~H, there were large areas of edema, liquefaction and necrosis of neurons in the left cerebral cortex, hippocampus and thalamus. Necrosis and edema were observed, but there was no large area of necrosis, and the number of cells was decreased in group G. The number of edematous cells and glial cells were decreased in group G. The number of glial cells in brain slices of neonatal rats in group 9:D was decreased more obviously than that in group G. The number of F cells was decreased and the number of glial cells was edema. The number of apoptotic cells in group A was detected by TUNEL, and the number of apoptotic cells in group B was 3.50 (+ 0.93). A large number of apoptotic cells and apoptotic cells were found in the left ischemic side of the brain. On the 6th day after modeling, there were 66.38+8.18 cells in group C, 53.12+7.92 cells in group E, 41.38+6.78 cells in group G, 50.50+5.68 cells in group D, 35.63+6.05 cells in group F, 29.25+3.49 cells in group H, and the number of apoptotic cells in group H was significantly lower than that in group C and D. The number of apoptotic cells in group G treated with VEGF was lower than that in group 1 on the 4th to 6th day after modeling, respectively. The levels of E, F and E~H were lower than those of C and D, and the difference was statistically significant (p0.05).
Conclusion: (1) After intranasal administration of VEGF to HIBD model of neonatal rats, it can effectively improve neurobehavioral function, reduce brain edema, improve pathological changes of nerve cells and reduce apoptosis of nerve cells. The results showed that the application of VEGF in the treatment of neonatal HIE could be used after the peak of cerebral edema.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1

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