发育期脑出血后糖皮质激素及受体变化及其对神经细胞增殖的影响

发布时间：2018-09-19 07:40

【摘要】：目的：通过制作新生大鼠脑出血（intracranial hemorrhage，ICH）疾病模型及其产生的病理变化和对脑匀浆皮质酮、海马CA1区糖皮质激素受体及神经细胞的增殖的影响，以及进一步外源性糖皮质激素干预治疗后的变化，探讨新生大鼠脑出血对下丘脑-垂体-肾上腺皮质轴的影响及为外源性糖皮质激素在新生儿脑出血的临床应用提供理论依据。方法：（1）将同窝新生10日龄SD大鼠，雌雄不限，随机分为11组：每组8只，其中正常对照（control group，CON）3组：12h组、24h组和72h组；假手术（sham operated group，SHAM）3组：12h组、24h组和72h组；脑出血模型（ICH group）3组：12h组、24h组和72h组；脑出血模型+DEX组（DEX组）以及脑出血模型+RU486组（RU486组）。按自体血注入法制造ICH模型，假手术组只进针无注血，正常对照组大鼠不予特殊处理。DEX组在大鼠造模后立即给予地塞米松按1mg/kg腹腔内注射；RU486组在大鼠造模前半小时予RU486注射液按20mg/kg腹腔内注射，并于造模后腹腔内注射地塞米松1mg/kg；其它组大鼠术后仅予生理盐水（10ml/kg）腹腔内注射。（2）行为测评采用：姿势反射测试、转圈或偏侧行走和前肢放置实验共同构成的神经损伤评分，在术前及术后72h完成。（3）大鼠脑匀浆皮质酮水平测定采用放射免疫学方法。（4）应用尼氏染色、免疫荧光染色及BrdU标记检测细胞增殖等方法分别观察各组大鼠出血灶周围神经元形态学改变、海马CA1区GR表达及脑室下区细胞增殖的情况。结果：（1）尼氏染色：ICH组大鼠出血灶周边细胞形态不规则，可见核固缩与核碎裂，尼氏体的数量明显减少甚至消失，而CON组及SHAM组同区神经元排列整齐，形态规则，尼氏体颗粒大而数量多，DEX干预后，，ICH后72h的出血灶周边尼氏染色细胞表现出的坏死细胞数较少，虽然仍有部分神经元胞体肿胀，而RU486组与单纯盐水干预的ICH组细胞形态无明显差异。（2）NDS：实验前所有新生大鼠行为能力正常。术后72h的提尾实验中，ICH组大鼠持续屈曲损伤半球对侧患肢，表现出各种不同姿势，而CON与SHAM组均无上述表现，三组NDS对比差异有意义（P＜0.0001）。予DEX干预后，NDS总分、姿势反射、转圈或偏侧行走以及前肢放置得分的减分率分别为：57.89%，54.55%，69.33%，54.84%，在72h末的NDS上，DEX组显著低于盐水干预的ICH组和RU486组。（3）脑匀浆皮质酮水平：脑出血后12h，24h和72h，ICH组大鼠脑匀浆皮质酮水平均较SHAM组及CON组大鼠比较明显升高（P＜0.0001）。新生大鼠脑出血后，脑匀浆皮质醇水平的变化趋势为在脑出血后12h左右升高至峰值，后逐渐下降，在72h仍高于正常同龄大鼠脑匀浆皮质酮水平。予DEX干预后，脑出血后72h的脑匀浆皮质酮下降至正常水平（与正常大鼠比较P＞0.05），而RU486组与ICH组之间无差异。（4）免疫荧光染色：ICH组大鼠海马CA1区神经元GR平均光密度值（1.0125±0.0273）小于CON组（1.2824±0.0356）（P＜0.0001）；DEX组、RU486组及盐水干预CON组之间无差异（P＞0.05）。（5）脑室下区细胞增殖情况：生后13d（即造模后72h）各干预组ICH大鼠BrdU+细胞数增加，显著多于CON组同龄大鼠（P＜0.001），而各干预组内差别不显著（P＞0.05）。结论：（1）新生大鼠ICH可导致HPA轴失调，表现为GC分泌增多，GR表达减少；（2）早期短期应用DEX可通过促进应激状态下HPA轴功能稳定，减少ICH所致内源性皮质酮的过度分泌，起到神经保护作用；（3）DEX的短期应用对GR的表达及SVZ神经细胞的增殖并无明显影响。
[Abstract]:AIM: To investigate the effects of intracranial hemorrhage (ICH) on the proliferation of corticosterone, glucocorticoid receptor and neurons in hippocampal CA1 region and the pathological changes of intracranial hemorrhage (ICH) in neonatal rats. The effect of thalamus-pituitary-adrenal cortex axis and the clinical application of exogenous glucocorticoids in neonatal cerebral hemorrhage provide theoretical basis.
Methods: (1) SD rats aged 10 days were randomly divided into 11 groups: 8 rats in each group, including control group (CON) 3 groups: 12 hours group, 24 hours group and 72 hours group; sham operated group (SHAM) 3 groups: 12 hours group, 24 hours group and 72 hours group; ICH group 3 groups: 12 hours group, 24 hours group and 72 hours group; cerebral hemorrhage model + D group; EX group (DEX group) and RU486 group (RU486 group). ICH model was made by autologous blood injection. The sham operation group was only injected without blood injection, and the normal control group was not given special treatment. Intraperitoneal injection, and intraperitoneal injection of dexamethasone 1 mg / kg after modeling; other groups of rats were only given saline (10 ml / kg) intraperitoneal injection. (2) Behavior evaluation: postural reflex test, rotational or lateral walking and forelimb placement of the nerve injury score, before and after 72 hours to complete. (3) Rat brain homogenate skin. Radioimmunoassay was used to determine the level of plasma ketone. (4) Nissl staining, immunofluorescence staining and BrdU labeling were used to observe the morphological changes of neurons around the hemorrhagic focus, GR expression in hippocampus CA1 area and cell proliferation in subventricular area.
Results: (1) Nissl staining: In ICH group, the cells around the hemorrhagic focus were irregular, nucleus pyknosis and nucleus fragmentation were observed, and the number of Nissl bodies was significantly reduced or even disappeared. In CON group and SHAM group, the neurons in the same area were arranged regularly, and the Nissl granules were large and large. After DEX intervention, the Nissl staining cell surface around the hemorrhagic focus 72 hours after ICH. The number of necrotic cells was small, although some neurons were still swollen, but there was no significant difference in cell morphology between RU486 group and ICH group. (2) NDS: The behavior of all neonatal rats was normal before the experiment. After DEX intervention, the total scores of NDS, postural reflex, circumflex or lateral walking and forelimb placement were 57.89%, 54.55%, 69.33%, 54.84% respectively. At the end of 72 hours, the scores of NDS in DEX group were significantly lower than those in ICH group and RU486 group. Corticosterone level: At 12h, 24h and 72h after cerebral hemorrhage, corticosterone level in ICH group was significantly higher than that in SHAM group and CON group (P The level of corticosterone in homogenate decreased to normal level 72 hours after intracerebral hemorrhage (P > 0.05 compared with normal rats), but there was no difference between RU486 group and ICH group. (4) Immunofluorescence staining: The average GR optical density of hippocampal CA1 neurons in ICH group (1.0125.0273) was lower than that in CON group (1.2824.0356) (P < 0.0001). There was no significant difference between DEX group, RU486 group and CON group (P > 0.05). (5) Proliferation of cells in subventricular area: The number of BrdU + cells in ICH rats in each intervention group increased significantly on the 13th day after birth (72 hours after modeling), but there was no significant difference among the intervention groups (P > 0.05).
CONCLUSIONS: (1) ICH can induce HPA axis dysfunction in neonatal rats, which is manifested by increased GC secretion and decreased GR expression; (2) Short-term application of DEX in early stage can promote the stability of HPA axis and reduce the excessive secretion of endogenous corticosterone induced by ICH, thus playing a neuroprotective role; (3) Short-term application of DEX can increase the expression of GR and the expression of SVZ neurons. Reproduction has no obvious effect.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34

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