粒细胞集落刺激因子对脑小血管病大鼠神经元活性、血管超微结构及神经元核抗原阳性表达的影响

Effects of G-CSF on neuronal activity,vascular ultrastructure and positive expression of NeuN in rats with cerebral small vessel disease

目的探究粒细胞集落刺激因子(G-CSF)对脑小血管病(CSVD)大鼠神经元活性、血管超微结构及神经元核抗原(NeuN)阳性表达的影响。方法2020年1月至2021年1月,将40只SD雄性大鼠按随机数字表法分为假手术组10只和模型组大鼠30只;采用同种系微栓子体外注入法制备CSVD大鼠模型,最终20只造模成功,再平均分为CSVD模型组(CSVD组)和CSVD模型大鼠给予G-CSF干预治疗组(G-CSF组),每组10只。造模成功后,将G-CSF组大鼠经皮下注射G-CSF 50μg/kg,1天1次;假手术组和CSVD组大鼠均经皮下注射等量的生理盐水干预,1天1次。三组大鼠均连续注射7 d。采用苏木精-伊红染色(HE染色)观察海马组织形态学变化,TUNEL检测神经元细胞凋亡情况,透射电镜观察微血管结构和内皮形态,免疫组织化学检测NeuN阳性表达,逆转录聚合酶链反应(RT-PCR)检测NeuN和血管内皮生长因子(VEGF)mRNA表达。结果假手术组大鼠脑组织结构没有出现异常;CSVD组大鼠脑组织结构异常,海马区病灶严重,海马组织结构无规则;神经组织大量坏死,神经元缩小;干预后的G-CSF组脑组织结构得到明显改善(P<0.05)。TUNEL检测结果显示,与假手术组神经元细胞凋亡率(21.34±4.43)%相比,CSVD组神经元细胞凋亡率(50.31±2.92)%增多(P<0.05),G-CSF组神经元细胞凋亡率(30.90±8.10)%较CSVD组明显减少(P<0.05)。假手术组微血管结构正常,CSVD组微血管结构破坏及内皮细胞损伤明显,而G-CSF组较CSVD组微血管结构和内皮细胞损伤得到明显改善(P<0.05)。免疫组织化学结果发现CSVD组大鼠NeuN阳性表达(0.375±0.020)%明显低于假手术组(0.572±0.015)%(P<0.05);G-CSF组中NeuN阳性表达(0.551±0.012)%较CSVD组明显升高(P<0.05)。RT-PCR检测结果显示,CSVD组大鼠NeuN(0.11±0.04)mRNA和VEGFmRNA(0.76±0.31)表达均低于假手术组NeuN(0.48±0.13)mRNA和VEGFmRNA(1.45±0.21)表达(P<0.05);G-CSF组NeuN(0.32±0.11)和VEGFmRNA(1.31±0.26)表达明显高于CSVD组(P<0.05)。结论G-CSF可改善CSVD大鼠微血管内皮细胞形态、促进神经元存活而减少凋亡发生,同时有效促进NeuN的阳性表达,进而发挥脑保护作用。

Objective To explore the effect of granulocyte colony-stimulating factor(G-CSF)on neuronal activity,vascular ultrastructure and positive expression of neuronal nuclear antigen(NeuN)in rats with cerebral small vessel disease(CSVD).Methods From January 2020 to January 2021,40 SD male rats were divided into 10 rats in the sham operation group and 30 rats in the model group according to the random number table method.Finally,20 rats were successfully modeled,and then they were equally divided into the CSVD model group(CSVD group)and CSVD model rats given the G-CSF intervention treatment group(G-CSF group),with 10 rats in each group.After successful modeling,the rats in the G-CSF group were subcutaneously injected with G-CSF 50μg/kg,once a day;the rats in the sham-operated group and the CSVD group were treated by subcutaneous injection of the same amount of normal saline once per day.All three groups of rats were continuously injected for 7 days.Hematoxylin-eosin staining(HE staining)was used to observe the morphological changes of hippocampus,TUNEL to detect neuronal apoptosis,transmission electron microscopy to observe the micro vascular structure and endothelial morphology,immunohistochemistry to detect the positive expression of NeuN,reverse transcription-polymerase chain reaction(RT-PCR)detection of NeuN and human vascular endothelial growth factor(VEGF)mRNA expression.Results The brain tissue structure of the rats in the sham operation group was not abnormal;the brain tissue structure of the rats in the CSVD group was abnormal,with severe lesions and irregular tissue structure in the hippocampus.The nerve tissue was massively necrotic,and the neurons were shrunken;the brain tissue structure was significantly improved in the G-CSF group after intervention(P<0.05).The results of TUNEL showed that compared with the sham operation group(21.34±4.43)%,the neuron apoptosis rate in the CSVD group(50.31±2.92)%increased(P<0.05),and the neuron apoptosis rate in the G-CSF group(30.90±8.10)%was significantly lower than that in the CSVD group(P<0.05).The micro vascular structure was normal in the sham operation group,and the micro vascular structure and endothelial cell damage in the CSVD group were significantly improved,while those in the G-CSF group were significantly improved compared with those in the CSVD group(P<0.05).Immunohistochemical results showed that the positive expression of NeuN in the CSVD group was(0.375±0.020)%significantly lower than that in the sham operation group(0.572±0.015)%(P<0.05),while the positive expression of NeuN in the G-CSF group(0.551±0.012)%was significantly higher than that in the CSVD group(P<0.05).The RT-PCR results showed that the expression levels of NeuN(0.11±0.04)mRNA and VEGF(0.76±0.31)mRNA in the CSVD group were lower than those in the sham operation group(0.48±0.13)mRNA and VEGF(1.45±0.21)mRNA(P<0.05);the expression levels of NeuN(0.32±0.11)and VEGF mRNA(1.31±0.26)in the G-CSF group were significantly higher than those in the CSVD group(P<0.05).Conclusion G-CSF can improve the morphology of microvascular endothelial cells in CSVD rats,promote the survival of neurons,reduce the occurrence of apoptosis,and effectively promote the positive expression of NeuN,thereby exerting a protective effect on the brain.