大鼠大脑皮层星形胶质细胞体外培养方法改良

目的建立大鼠大脑皮层星形胶质细胞(As)的培养方法,以进一步对大脑皮层As进行体外实验研究。方法在M cCarthy方法基础上改用出生后5 d的SD大鼠的大脑皮层,制备单细胞悬液后,接种于培养瓶,培养箱中1 h后换瓶,3 d后换液,细胞融合成单层后传代,传3代后采用间接免疫荧光法监测As特异性标志胶质纤维酸性蛋白(GFAP)。阳性细胞确认为As。结果体外培养的大脑皮层As经历了贴壁、去除成纤维细胞、纯化和传3代,GFAP阳性细胞达95%以上。结论大脑皮层As体外培养成功,并有较高的纯度,为进一步进行As的研究创造了条件。

正常灌注压突破综合征

正常灌注压突破综合征是脑血管疾病术中或术后的严重并发症。本文就近年来正常灌注压突破综合征的研究进展进行综述,重点探讨其发病机制、预测和预防。

丹参与电针对脑缺血再灌注损伤大鼠GFAP和iNOS表达的影响

目的:观察脑缺血再灌注损伤后大鼠脑胶质原纤维酸性蛋白(GFAP)及诱导型一氧化氮合酶(iNOS)的表达,以及丹参与电针对其表达的影响。方法:将60只SD大鼠随机分为假手术组、模型组、丹参组、电针组、丹参+电针组,采用大脑中动脉线栓法建立脑缺血再灌注模型。造模后,丹参组给予丹参注射液5g/kg腹腔注射,电针组刺激"百会"、"大椎"穴,疏密波,频率2~15Hz,持续30min,丹参+电针组给予丹参注射液腹腔注射和电针,各组治疗均每天1次,连续4d。进行神经功能缺损评分、干湿重法测脑组织含水量,采用HE染色观察脑组织的病理形态学变化,免疫组织化学染色法检测各组大鼠纹状体GFAP、iNOS的表达。结果:模型组神经功能缺损评分和脑组织含水量均明显高于假手术组(P<0.01);与模型组比较,丹参组、电针组、丹参+电针组神经功能缺损评分和脑组织含水量均显著降低(P<0.05或P<0.01),并且丹参+电针组的评分和含水量显著低于丹参组、电针组(P<0.05或P<0.01);HE染色显示的病理形态学改变与上述一致。模型组GFAP、iNOS的表达均明显高于假手术组(P<0.01);与模型组比较,丹参组、电针组、丹参+电针组GFAP、iNOS的表达均明显降低(P<0.05或P<0.01),并且丹参+电针组的表达明显低于丹参组、电针组(P<0.05或P<0.01)。结论:丹参和电针对脑缺血再灌注损伤有神经保护作用,可能与其抑制星型胶质细胞的过度活化,降低iNOS的表达有关,且二者合用效果更佳。

Effects of P2Y_1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways

Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion （tMCAO） and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blotting, enzyme linked immunosorbent assay （ELISA） were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium （MRS2179） reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein （CREB）, and reduction of phosphorylated Janus kinase2 （JAK2） and signal transducer and activator of transcription3 （STAT3, Ser727）. After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 （inhibitor of JAK2） reduced phosphorylation of STAT3 （Ser727） as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase （PI3-K）, decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 （MEK 1/2） U0126, an important molecule of Ras/extracellular signal- regulated kinase （ERK） signaling pathway, decreased the phosphorylation of JAK2, STAT3 （Ser727）, Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Neurogenesis by Activation of Inherent Neural Stem Cells in the Rat Hippocampus after Cerebral Infarction

Objective To investigate the changes of neural stem cells (NSCs) in the rat hippocampus after cerebral infarction (CI) and to evaluate the neurogenesis caused by the activation of NSCs. Methods CI models of rats were made and rats were assigned to 6 groups: sham-operated, 1 day, 3 days, 7 days, 14 days, and 28 days after CI. The dynamic expression of bromodeoxyuridine (BrdU), polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), and neuronal nuclear antigen (NeuN) were determined by immunohistochemistry and immunofluorescence staining. BrdU was used to mark the proliferated NSCs. PSA-NCAM was used to mark the plasticity of activated NSCs. GFAP and NeuN were used to mark the differentiated NSCs. Results Compared with the controls, the number of BrdU+ cells in the hippocampus increased significantly at 1 day after CI (P<0.05), reached peak at 7 days after CI (P<0.05), decreased but still elevated compared with the controls at 14 days after CI (P<0.05), and nearly unchanged at 28 days after CI. The number of BrdU+/PSA-NCAM+ cells increased significantly at 7 days after CI (P<0.05), reached peak at 14 days after CI (P<0.05), and decreased but still elevated compared with the controls at 28 days after CI (P<0.05). The number of BrdU+/PSA-NCAM+ cells was equal to 60% of the number of BrdU+ cells in all the same period. The number of BrdU+/NeuN+ cells in the hippocampus increased significantly at 14 days after CI (P<0.05) and reached peak at 28 day after CI (P<0.05). The number of BrdU+/GFAP+cells in the hippocampus nearly unchanged after CI. Conclusion CI can stimulate the proliferation of inherent NSCs, and most proliferated NSCs may differentiate into neurons and represent neural plasticity.

Neural stem cell activation and glial proliferation in the hippocampal CA3 region of posttraumatic epileptic rats

The present study observed the dynamic expression of CD133, nuclear factor-κB and glial fibrUlary acidic protein in the hippocampal CA3 area of the experimental posttraumatic epilepsy rats to investigate whether gliosis occurs after posttraumatic epilepsy. CD133 and nuclear factor-κB expression was increased at 1 day after posttraumatic epilepsy, peaked at 7 days, and gradually decreased up to 14 days, as seen by double-irnmunohistochemical staining. Glial fibrillary acidic protein/nuclear factor-EB double-labeled cells increased with time and peaked at 14 days after posttraumatic epilepsy. Results show that activation of hippocampal neural stem cells and glial proliferation after posttraumatic epilepsy-induced oxidative stress increases hippocampal glial cell density.

Clinicopathological and immunohistochemical features of pilomyxoid astrocytoma: a report of six cases

Objective： The aim of this study was to study the clinicopathological and immunohistochemical features of pilo- myxoid astrocytoma （PMA）. Methods： The clinical and pathologic features in six cases of PMA were analyzed. Immunohisto- chemical staining for glial fibrillary acidic protein （GFAP）, synaptophysin （Syn）, Chromogranin A （CgA）, cytokeratin （AEI/AE3）, epithelial membrane antigen （EMA） and Ki67 was performed on paraffin-embedded sections. Results： Among the six cases, five occurred in female patients, one was male, the age at diagnosis ranged from 2 to 15 years. Four cases were located in the hypothalamic area and optic pathway, one case in the third ventricle, and one case in left parietal lobe. On imaging, PMAs often appears as well-circumscribed mass. Microscopically, the tumor was composed of monomorphous bipolar （piloid） cells setting in a prominent myxoid background with an angiocentric radiating growth pattern in some areas. PMA lacked biphasic pattern, Rosenthal fibers and eosinophilic granular bodies which were usually typical in a classic pilocytic astrocytoma （PA）. Immunohistochemcal study showed that the tumor cells were diffusely positive for GFAP. Syn positive staining was observed in one case. The Ki67 labeling index measured less than 5%. Conclusion： PMA is a distinct aggressive variant of pilocytic astrocytoma with special histological and immunohistochemical features. It is typically a rare tumor of early childhood. Im- munohistochemical staining for GFAP and Syn is helpful in differential diagnosis.

All-trans Retinoic Acid Induced the Differentiation of Human Glioma Cells

OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.

Effects of electroacupuncture at Fengchi(GB20)on motor function and GFAP/NeuN expression around the ischemic tissue of the motor cortex in MCAO rats

Objective:To investigate the potential mechanism of electroacupuncture(EA)at bilateral Fengchi(GB20)in treating cerebral ischemia-reperfusion injury and to provide a scientific basis for future experimental research and clinical applications.Methods:Forty male specific-pathogen-free Sprague-Dawley rats were randomly divided into four groups:a normal group,a normal with EA group,a model group,and a model with EA group,with 10 rats in each group.The normal group received no intervention.The normal with EA group received EA at bilateral Fengchi(GB20).The model group underwent middle cerebral artery occlusion(MCAO)using the suture.The model with EA group underwent MCAO and received EA at bilateral Fengchi(GB20).Cerebral blood flow was monitored using a laser Doppler cerebral blood flow meter.Neurologic damage was assessed using the neurologic deficit score,and motor ability was observed using the CatWalk gait system.The expression of glial fibrillary acidic protein(GFAP)and neuronal nuclei(NeuN)protein,the neuron markers,was detected by Western blotting.The protein expression levels of GFAP and NeuN,as well as the number of positive cells in the motor cortex,were detected using immunofluorescence.Results:Compared to the normal group,the cerebral blood flow values in the model group and the model with EA group decreased by more than 50%during the modeling process(P<0.01)and returned to pre-modeling levels after reperfusion(P>0.05).The neurologic deficit score increased(P<0.05),the average motor velocity decreased(P<0.05),GFAP protein expression and the number of positive cells in the motor cortex increased(P<0.05),and the NeuN protein expression and the number of positive cells decreased(P<0.05)in the model group.Compared to the model group,the neurologic deficit score decreased(P<0.05),the average motor velocity accelerated(P<0.05),GFAP and NeuN protein expression and the number of positive cells in the motor cortex increased(P<0.01)in the model with EA group.Conclusion:EA at bilateral Fengchi(GB20)can reduce neuronal loss and increase GFAP and NeuN protein expression in the motor cortex of rats after ischemia-reperfusion,improve the motor function after ischemic stroke,and accelerate the recovery of balance and stability of the affected limbs.

重组人粒细胞集落刺激因子对大鼠脑缺血再灌注损伤后AQP4和GFAP表达的影响

目的探讨重组人粒细胞集落刺激因子(rh G-CSF,瑞白,150μg,山东齐鲁药业)对SD大鼠脑缺血再灌注后血脑屏障的保护机制。方法将雄性SD大鼠48只随机分为:假手术组、模型组和治疗组,每组16只,利用改良Longa线栓法制作大鼠中动脉缺血模型,治疗组于缺血2 h后实施再灌注并给予rh G-CSF(50μg/kg)腹部皮下注射,假手术组及模型组给予同等剂量生理盐水。采用Longa 5分制评分标准进行神经功能评分,利用分光光度仪计算缺血大脑半球脑组织内中伊文思蓝含量,免疫组化法检测各组大鼠脑组织中水通道蛋白-4(AQP4)、胶质纤维酸性蛋白(GFAP)含量变化和电镜下观察血脑屏障的超微结构。结果治疗组神经功能评分(1.36±0.63)低于模型组(2.50±0.65)(U=16,P<0.01);与假手术组右侧脑组织内伊文思蓝含量(7.38±2.71)相比较,模型组右侧伊文思蓝的含量(37.15±2.30)明显增多(t=30.60,P<0.01),治疗组伊文思蓝的含量(22.75±4.61)较模型组降低(t=-16.73,P<0.05);治疗组AQP4、GFAP表达的灰度值(180.67±7.72、160.64±5.07)均较模型组增高(t=24.16,P<0.01;t=17.98,P<0.01);假手术组大鼠脑组织AQP4、GFAP表达的灰度值(202.08±5.80、173.73±4.40)高于模型组(t=46.07,P<0.01;t=31.07,P<0.01),电镜下观察到假手术组内皮细胞紧密连接完整,血脑屏障周围组织完好,而模型组和治疗组脑血管内皮细胞连接间隙增加,且连接内皮细胞的基质和基膜完整性缺失。结论 rh G-CSF对脑缺血再灌注损伤具有保护作用,推测其可能机制为rh G-CSF通过抑制星形胶质细胞过度活化,下调GFAP、AQP4的表达,减轻脑水肿,从而保护血脑屏障完整性。

Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

OBJECTIVE: Inactivation of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells(NSCs).Qingnaoyizhi decoction(QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid(CSF) containing QNYZD(CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway.METHODS: The protein expression levels of glial fibrillary acidic protein(GFAP), tubulin, drosophila mothers against decapentaplegic protein(SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor(AG490),CSF-QNYZD, and CSF-XDZ(CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay.RESULTS: Compared with the control group,CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3.CONCLUSION: Enhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferationin treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

Changes of serum Tau, GFAP, TNF-α and malonaldehyde after blast-related traumatic brain injury

Objective： To determine the changes of serum Tau protein, glial fibrillary acidic protein （GFAP）, tumor necrosis factor alpha （TNF-α）, and malonaldehyde （MDA） in rats after blast-related traumatic brain injury （BTBI） and to provide relative information for further studies on BTBI mechanism and seek specific biomarkers for BTBI. Methods： Ninety male Sprague-Dawley rats were randomly assigned into three groups： control group, moderate blast injury group, and severe blast injury group （n=30 for each）. Rats in the moderate and severe blast injury groups were respectively exposed to corresponding levels of BTBI. After explosion, serum levels of Tau, GFAP, TNF-α, and MDA in each group were determined by Elisa assay at different time points after injury （8 h, 24 h, 3 d, and 6 d）. The extent of brain damage was detected by Nissl staining and TUNEL assay. Results： Serum levels of Tau and GFAP rapidly increased and reached the peak at 24 h after either moderate or severe blast injury. All the values were significantly higher than control group at all time points （P〈0.05）. Serum TNF-α level of both injury groups peaked at 8 h after BTBI and stayed significantly higher than control group at all time points （P〈0.05）. Serum MDA of two injury groups began to significantly increase at 3 d and the level stayed significantly higher than control group until 6 d （P〈0.05）. Moreover, unlike the other biomarkers, serum MDA of severe blast injury group was significantly higher than moderate blast injury group at 6 d （P〈0.05）. Conclusion： The changes of serum Tau, GFAP, and TNF-α showed a good sensitivity at the acute phase after BTBI （within 24 h）. However, their specificity and correlation with the extent of injury were limited in this experiment. Moreover, although the change of serum MDA showed a poor sensitivity and specificity to the diagnosis of BTBI during the first few days, it can reflect the injury degree at 6 d after injury. Therefore, further studies are needed to improve the methods of detecting more serum markers and investigate the significance of multiple markers in diagnosing BTBI.

Stem cells modified by brain-derived neurotrophic factor to promote stem cells differentiation into neurons and enhance neuromotor function after brain injury

Objective： To promote stem cells differentiation into neurons and enhance neuromotor function after brain injury through brain-derived neurotrophic factor （BDNF） induction. Methods： Recombinant adenovirus vector was applied to the transfection of BDNF into human-derived umbilical cord mesenchymal stem cells （UCMSCs）. Enzyme linked immunosorbent assay （ELISA） was used to determine the secretion phase of BDNF. The brain injury model of athymic mice induced by hydraulic pressure percussion was established for transplantation of stem cells into the edge of injury site. Nerve function scores were obtained, and the expression level of transfected and non-transfected BDNF, proportion of neuron specific enolase （NSE） and glial fibrillary acidic protein （GFAP）, and the number of apoptosis cells were compared respectively. Results： The BDNF expression achieved its stabilization at a high level 72 hours after gene transfection. The mouse obtained a better score of nerve function, and the proportion of the NSE-positive cells increased significantly （P〈0.05）, but GFAP-positive cells decreased in BDNF- UCMSCs group compared with the other two groups （P〈0.05）. At the site of high expression of BDNF, the number of apoptosis cells decreased markedly. Conclusion： BDNF gene can promote the differentiation of the stem cells into neurons rather than glial cells, and enhance neuromotor function after brain injury.