【作者】 诸葛小寅；

【导师】 曹云涛；

【作者基本信息】 遵义医学院 ， 儿科学， 2010， 硕士

【摘要】 目的：通过检测去铁胺(DFO)早期干预对新生大鼠缺氧缺血性脑损伤(HIBD)后海马齿状回(DG)巢蛋白(nestin)和5-溴-2-脱氧尿嘧啶核苷(BrdU)表达的影响,以了解内源性新生神经干细胞(NSCs)的变化,以及HIBD大鼠学习记忆功能改善情况,以探讨DFO对新生大鼠HIBD的保护作用。方法：选择7日龄新生Wistar大鼠制备新生鼠HIBD模型,设置假手术组、HIBD对照组、DFO干预组。观察大鼠体重变化和病侧大脑大体改变,并利用HE染色光镜下观察脑组织病理改变；通过石蜡切片间接免疫荧光组织化学方法和计算机图像分析技术检测各组大鼠术后4天海马DG区nestin、BrdU表达的变化；在大鼠32日龄时采用Morris水迷宫实验检测三组大鼠的学习记忆功能。结果：1.三组新生大鼠体重增加有统计学差异(P<0.05), HIBD对照组和DFO干预组大鼠术后体重的增加明显低于假手术组。2.病侧大脑大体改变：术后4天部分病侧大脑局部见苍白区,在大鼠40日龄时部分病侧大脑萎缩、局部有空洞。3.脑组织病理改变：HE染色显示假手术组大鼠脑组织无异常病理改变；HIBD对照组大鼠病侧海马CA1区锥体细胞层次减少、排列紊乱,出现大量神经细胞核固缩、碎裂；DFO干预组大鼠脑病理改变较HIBD对照组明显减轻。4. nestin表达：三组大鼠比较DG区nestin表达有统计学差异(P<0.05), HIBD对照组nestin阳性细胞荧光强度较假手术组增加(P＜0.05)；DFO干预组nestin阳性细胞荧光强度较HIBD对照组增加(P＜0.05)。5. BrdU的表达：三组大鼠比较DG区BrdU表达有统计学差异(P<0.05),HIBD对照组BrdU阳性细胞数较假手术组增加(P＜0.05)；DFO干预组BrdU阳性细胞数较HIBD对照组增加(P＜0.05)。6. Morris水迷宫学习记忆能力测定：三组连续5天的逃避潜伏期及穿环指数比较均有统计学差异(P＜0.05)；假手术组穿环指数高于HIBD对照组(P＜0.05),DFO干预组穿环指数高于HIBD对照组(P＜0.05),假手术组和DFO干预组比较穿环指数无统计学差异(P＞0.05)。结论：1.新生大鼠HIBD后存在大脑DG区内源性NSCs的代偿性再生；2.DFO早期干预可减轻新生大鼠HIBD后神经细胞的病理损伤；3.DFO早期干预后新生大鼠HIBD后海马DG区nestin、BrdU表达增加,提示DFO早期干预可促进内源性NSCs再生。4.HIBD使新生大鼠的空间学习记忆能力下降,而早期给予DFO干预较HIBD对照组有明显的改善作用。更多还原

【Abstract】 Objective:To explore the neuroprotective effects of early intervention of deferoxamine (DFO) on neonatal rats with hypoxic-ischemic brain damage (HIBD) by testing expression of bromodeoxyuridine (BrdU) and nestin in the dentate gyrus region which means the activation of endogenous neural stem cells (NSCs); and also the improvement of learning and memory capabilities as an evidence of the neuroprotective effects of DFO were examed in neonatal rats with HIBD. Methods:The HIBD model was made in 7 days old Wistar rats and the rats were divided into 3 groups randomly:sham operation group, control group of HIBD, DFO treated group. Histological examination of the CA1 hippocampal region of the damaged hemisphere was conducted by haematoxylin-eosin (HE) stain with optical microscope and the body weight of the rats were measured. Expressions of nestin and BrdU in the CA1 hippocampal region were tested by indirect immunofluorescence labelling of paraffin-embedded tissues and image quantitative analysis through computer at 4th days after operation and learning and memory capabilities of each group were evaluated by Morris water maze at the age of 32 days after birth. Results:1. There was significant difference in increase of body weight among the three groups (P<0.05). The sham operation group had a significantly higher increase in body weight than the other two groups.2. Anatomical pathology revealed the pale area in some right hemisphere of the rats at 4th day after hypoxia and cerebral atrophy and cavitas at the age of 32 days after birth.3. Histopathological changes (HE staining):There were no morphological anomalies in the sham operation group. The neuron number in the CA1 pyramidal cell layer of the right hippocampus was decreased and the cells arranged in disorder in the control group of HIBD. At the same time, lots of neurons were necrotic and had karyopycnosis in the CA1 region. Compared with the control group of HIBD, the necrotic neurons in the CA1 region were obviously reduced in the DFO treated group.4. Expression of nestin:There was significant difference in the expression of nestin in the DG hippocampal region among the three groups (P<0.05). Measurement of the fluorescence intensity of nestin-positive cells revealed that the DFO treated group was the highest compared with the control group of HIBD, while the sham operation group was the least (P<0.05).5. Expression of BrdU:There was significant difference in the expression of BrdU in the DG hippocampal region among the three groups (P<0.05). Measurement of the number of BrdU-positive cells revealed that the DFO treated group was the maximum compared with the control group of HIBD, while the sham operation group was the minimum (P<0.05).6. The use of Morris water maze in learning and memory:Both the latencies to find a hidden platform in the water maze on 5 consecutive days and the annulus-crossing index had significant differences among the three groups (P<0.05). The annulus-crossing index in the control group of HIBD was the minimum. But there was no significant difference between the sham operation group and the DFO treated group (P> 0.05). Conclusions:1. Expression of the endogenous NSCs increased in neonatal rats with HIBD, it means that the endogenous NSCs were activated after HIBD.2. Early intervention of DFO to the neonatal rats with HIBD can reduce the damage of the neurons in pathology.3. Early intervention of DFO to the neonatal rats with HIBD can increase the expression of BrdU and nestin, which means early intervention of DFO can improve the regeneration of the endogenous NSCs.4. Early intervention of DFO to the neonatal rats with HIBD can improve the capabilities of learning and memory which were waken due to HIBD.更多还原