【摘要】：目的：通過制作新生大鼠腦出血（intracranial hemorrhage，ICH）疾病模型及其產(chǎn)生的病理變化和對腦勻漿皮質(zhì)酮、海馬CA1區(qū)糖皮質(zhì)激素受體及神經(jīng)細胞的增殖的影響，以及進一步外源性糖皮質(zhì)激素干預治療后的變化，探討新生大鼠腦出血對下丘腦-垂體-腎上腺皮質(zhì)軸的影響及為外源性糖皮質(zhì)激素在新生兒腦出血的臨床應用提供理論依據(jù)。 方法：（1）將同窩新生10日齡SD大鼠，雌雄不限，隨機分為11組：每組8只，其中正常對照（control group，CON）3組：12h組、24h組和72h組；假手術(shù)（sham operated group，SHAM）3組：12h組、24h組和72h組；腦出血模型（ICH group）3組：12h組、24h組和72h組；腦出血模型+DEX組（DEX組）以及腦出血模型+RU486組（RU486組）。按自體血注入法制造ICH模型，假手術(shù)組只進針無注血，正常對照組大鼠不予特殊處理。DEX組在大鼠造模后立即給予地塞米松按1mg/kg腹腔內(nèi)注射；RU486組在大鼠造模前半小時予RU486注射液按20mg/kg腹腔內(nèi)注射，并于造模后腹腔內(nèi)注射地塞米松1mg/kg；其它組大鼠術(shù)后僅予生理鹽水（10ml/kg）腹腔內(nèi)注射。（2）行為測評采用：姿勢反射測試、轉(zhuǎn)圈或偏側(cè)行走和前肢放置實驗共同構(gòu)成的神經(jīng)損傷評分，在術(shù)前及術(shù)后72h完成。（3）大鼠腦勻漿皮質(zhì)酮水平測定采用放射免疫學方法。（4）應用尼氏染色、免疫熒光染色及BrdU標記檢測細胞增殖等方法分別觀察各組大鼠出血灶周圍神經(jīng)元形態(tài)學改變、海馬CA1區(qū)GR表達及腦室下區(qū)細胞增殖的情況。 結(jié)果：（1）尼氏染色：ICH組大鼠出血灶周邊細胞形態(tài)不規(guī)則，可見核固縮與核碎裂，尼氏體的數(shù)量明顯減少甚至消失，而CON組及SHAM組同區(qū)神經(jīng)元排列整齊，形態(tài)規(guī)則，尼氏體顆粒大而數(shù)量多，DEX干預后，，ICH后72h的出血灶周邊尼氏染色細胞表現(xiàn)出的壞死細胞數(shù)較少，雖然仍有部分神經(jīng)元胞體腫脹，而RU486組與單純鹽水干預的ICH組細胞形態(tài)無明顯差異。（2）NDS：實驗前所有新生大鼠行為能力正常。術(shù)后72h的提尾實驗中，ICH組大鼠持續(xù)屈曲損傷半球?qū)?cè)患肢，表現(xiàn)出各種不同姿勢，而CON與SHAM組均無上述表現(xiàn)，三組NDS對比差異有意義（P＜0.0001）。予DEX干預后，NDS總分、姿勢反射、轉(zhuǎn)圈或偏側(cè)行走以及前肢放置得分的減分率分別為：57.89%，54.55%，69.33%，54.84%，在72h末的NDS上，DEX組顯著低于鹽水干預的ICH組和RU486組。（3）腦勻漿皮質(zhì)酮水平：腦出血后12h，24h和72h，ICH組大鼠腦勻漿皮質(zhì)酮水平均較SHAM組及CON組大鼠比較明顯升高（P＜0.0001）。新生大鼠腦出血后，腦勻漿皮質(zhì)醇水平的變化趨勢為在腦出血后12h左右升高至峰值，后逐漸下降，在72h仍高于正常同齡大鼠腦勻漿皮質(zhì)酮水平。予DEX干預后，腦出血后72h的腦勻漿皮質(zhì)酮下降至正常水平（與正常大鼠比較P＞0.05），而RU486組與ICH組之間無差異。（4）免疫熒光染色：ICH組大鼠海馬CA1區(qū)神經(jīng)元GR平均光密度值（1.0125±0.0273）小于CON組（1.2824±0.0356）（P＜0.0001）；DEX組、RU486組及鹽水干預CON組之間無差異（P＞0.05）。（5）腦室下區(qū)細胞增殖情況：生后13d（即造模后72h）各干預組ICH大鼠BrdU+細胞數(shù)增加，顯著多于CON組同齡大鼠（P＜0.001），而各干預組內(nèi)差別不顯著（P＞0.05）。 結(jié)論：（1）新生大鼠ICH可導致HPA軸失調(diào)，表現(xiàn)為GC分泌增多，GR表達減少；（2）早期短期應用DEX可通過促進應激狀態(tài)下HPA軸功能穩(wěn)定，減少ICH所致內(nèi)源性皮質(zhì)酮的過度分泌，起到神經(jīng)保護作用；（3）DEX的短期應用對GR的表達及SVZ神經(jīng)細胞的增殖并無明顯影響。
[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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.34

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