NGF与癫痫后大鼠海马损伤修复及苔藓纤维发芽关系的研究

目的 探讨神经生长因子(NGF)与癫痫后大鼠海马损伤修复及与苔藓纤维发芽(MFS)的关系.方法 大鼠侧脑室内分别注入NGF.正、反义寡核苷酸和磷酸盐缓冲液,并使用红藻氨酸(KA)致痫后分别在48h、72h、1w和4w采用普通病理方法、免疫组化和Timm's染色法对鼠脑片进行研究.结果 KA致痫后48h大鼠海马NGF蛋白表达已可见显著升高,持续较高水平表达至少4w,并伴随大鼠海马明显MFS;给予NGF反义寡核苷酸能够减少NGF蛋白表达和MFS过程,但神经元损伤更为严重;大鼠行为学观察各癫痫组未见明显差异.结论 KA致痫后大鼠海马NGF蛋白表达升高并与MFS同时共存,但可被侧脑室内注入NGF反义寡核苷酸有效阻止,提示NGF可促进癫痫后大鼠海马损伤修复及MFS过程.

Abstract：

Objective To explore the correlation along nerve growth factor, the repairing of neuron damage and mossy fibers sprouting (MFS) in rat hippocampus after lcison. Methods Antisense or sense oligonucleotide to NGF and/ or PBS buffer was injected into the ventricles of rats of temporal lobe epilepsy model induced by KA and so as the control rats, then they were executed at 48h,72h, 1 w and 4w after lesion respectively. Immunohistochemical, pathobiology staining and Timm' s silver sulfide staining were used. Results The NGF-like protein expression increased at 48h after lesion, then the protein expression' s peak was at 72h and kept at a high level till about 4w, and with positive correlation with MFS.Timm' s staining showed that multiple aberrant Timm' s granules were found in the hippocampus of the KA model intracerebroventricularly injected with sense oligonucleotide to NGF and PBS. But antisense oligonucleotide to NGF alleviated these states. No obvious discrepancy of behavior between the epileptic rats injected antisense oligonucleotide and the sense oligonucleotide ones. Conclusions The NGF-like protein expression increased with time-dependence after lesion and coexisted with MFS, but antisense oligonucleotide to NGF could alleviate these states. It hinted that NGF can promote the repairing of neuron damage and mossy fibers sprouting (MFS) in rat hippocampus after leison.     

颅内不同起源肌阵挛致脑损伤的实验研究

目的 探讨颅内不同起源的肌阵挛与脑损伤的关系及其病理学特征.方法 选择GABAA受体拮抗剂SR95531在成年SD大鼠的初级运动皮层(PMC)、纹状体(CS)、丘脑网状核(NRT)以及L-5-HTP在幼年豚鼠桥脑背侧微量注射,建立皮层-丘脑轴起源和脑干起源的肌阵挛动物模型.分别在发作达峰后10 min、30 min对各组行股动脉取血以检测血清特异性神经元烯醇化酶(NSE),并留取诱导剂注射部位对侧额叶皮层及海马行HE染色及Nissl染色,TUNEL法检测细胞凋亡,免疫组化染色检测Bcl-2、Bax蛋白表达.结果 (1)PMC组、CS组和NRT组在发作达峰后30 min时血清NSE水平较对照组明显增高,差异有统计学意义(P＜0.05),其中以PMC组最明显.(2)HE染色见PMC组、CS组和NRT组额叶皮层及海马CA3区神经细胞明显变性及坏死,而桥脑背侧组未出现明显的病理学改变.Nissl染色见PMC组、CS组和NRT组额叶皮层神经细胞计数较对照组减少56.3%～66%,而桥脑背侧组无明显异常.(3)PMC组、CS组和NRT组额叶皮层和海马CA3区凋亡阳性细胞计数较对照组升高20.4～40.7倍,而桥脑背侧组凋亡阳性细胞计数无明显增加.(4)PMC组、CS组和NRT组额叶皮层和海马CA3区Bax蛋白表达均较对照组明显增高,Bcl-2蛋白表达明显降低,差异有统计学意义(P＜0.05),而桥脑背侧组与对照组比较差异无统计学意义.结论 PMC、CS和NRT等皮层-丘脑轴起源的肌阵挛发作可引起额叶皮层、海马神经细胞明显减少等组织学损伤,其损伤的发生与肌阵挛痫性放电激活神经细胞凋亡和坏死过程相关,因而属于惊厥性脑损伤.脑干起源的肌阵挛未见明显异常.

Abstract：

Objective To explore the possibility of brain damage caused by myoclonic seizures of different origins and its pathologic characteristics. Methods Fifty-six adult SD rats were randomly divided into pontine micturition center (PMC) microinjection group (n=16), corpus striatum (CS) microinjection group (n=16), reticular thalamic nucleus (RTN) microinjection group (n=16), and normal control group (n=8);24 infant Guinea Pigs were randomized into dorsal pons microinjection group (n=16) and normal control group (n=8);Models of myoclonic seizures of different origins were established by microinjecting SR95531 into the PMC, CS and NRT of adult SD rats, and microinjecting L-5-HTP into the dorsal pons of Guinea Pigs, respectively. Blood from femoral artery was drawn 10 and 30 minutes after the peak time of myoclonic seizures from PMC, CS and NRT areas of rats and from dorsal pons of Guinea Pigs to detect the level of neuron-specific enolase (NSE). All animals were sacrificed 10 and 30 minutes after the peak time of myoclonic seizures and the contralateral brain at the microinjecting sites were isolated for following study: morphology in the frontal cortex and the CA3 region of hippocampus was observed by HE staining and Nissl staining;apoptosis cells in the frontal cortex and the CA3 region of hippocampus were detected by TUNEL;protein expressions of apoptosis-related Bcl-2 and Bax were detected by immunohistochemistry. Results Significant neurodegeneration and neuronal necrosis were found by HE staining in the frontal cortex and the CA3 region of hippocampus of the myoclonus originating from PMC, CS and NRT;however, no obvious histopathologic changes were observed in the cortex of Guinea Pigs with myoclonus arising from the dorsal pons. Cell count at the frontal cortex by Nissl staining was less by 56.3%-66% (30 minutes of peak time) than that in the normal control group;however, no obvious loss of cell was observed in the cortex of the Guinea pigs with myoclonus originating from dorsal pons. The serum level of NSE was increased in all rats with myoclonus originating from PMC, CS and NRT, especially in those from PMC. TUNEL-positive cells in the frontal cortex and hippocampus caused by myoclonic seizures originating from PMC, CS and NRT were increased by 20.4-40.7 times remarkably as compared with those in the controls, but no significant increase of apoptosis cells in the Guinea pigs with myoclonus originating from dorsal pons was noted. As compared with those in the controls, significant increase of Bax protein expression and obvious decrease of Bcl-2 protein expression in the frontal cortex and hippocampus of those animals with myoclonus arising from PMC, CS and NRT were found (P<0.05);no obvious differences in those of the animals with myoclonus arising from dorsal pons were noted as compared with those of its control group (P>0.05).Conclusion The myoclonus seizure arising from the axis of cortical-thalamus but not the dorsal pons can induce the decrease of neurocytes in the susceptive area of cortex and CA3 region. The brain damage in the cortex with the myoclonic seizures originating from the axis of cortical-thalamus is caused by the epileptic discharges of myoclonus which activates the process of necrosis and apoptosis of the neurocytes in brain, which belongs to the brain injury induced by seizures. However, there is no obvious damage in cortex of the models with the myoclonus originating from the dorsal pons of Guinea Pigs.     

柴胡疏肝汤对戊四氮致痫大鼠海马及额叶皮质c-fos表达的影响

目的 观察柴胡疏肝汤对戊四氮致痫大鼠海马及额叶皮质c-fos表达的影响.方法 选取经戊四氮诱导制作的癫痫模型大鼠48只,按随机数字表法分成6组:癫痫模型组、德巴金组、定痫丸组、柴胡疏肝汤低剂量组、柴胡疏肝汤中剂量组和柴胡疏肝汤高剂量组,每组各8只;另设正常对照组8只.正常对照组和癫痫模型组常规饲养,其他各组给予药物德巴金,定痫丸,低、中、高剂量柴胡疏肝汤处理,均连续灌胃治疗5周.免疫组化染色检测各组大鼠海马及额叶皮质c-fos的表达情况.结果 戊四氮致痫后大鼠海马及额叶皮质c-fos表达明显增强,而应用中、高剂量的柴胡疏肝汤,德巴金和定痫丸治疗后,大鼠海马及额叶皮质c-fos表达均明显减弱,与癫痫模型组比较差异均有统计学意义(P<0.05),而柴胡疏肝汤低剂量组大鼠海马及额叶皮质c-fos表达无明显减弱.结论 柴胡疏肝汤的抗癫痫作用机制可能与其含有柴胡皂甙及其他多种有效的抗癫痫成份多靶点干预海马及额叶皮质c-fos表达水平有关.

Abstract：

Objective To observe the effect of chaihushugantang on the expression of c-fos in the hippocampus and frontal lobe cortex of pentylenetetrazole (PTZ)-induced epileptic rats. Methods Forty-eight PTZ-induced epileptic rats were randomly divided into 6 groups: epileptic model group,Valproate treatment group, Dingxianwan treatment group, and lower-dose, medium-dose and high-dose chaihushugantang treatment groups (n=8). Normal control group was also employed (n=8). The epileptic rats in the normal control group and epileptic model group were fed normally. Rats of the treatment groups were performed intragastric administration of medicines (Valproate, Dingxianwan and chaihushugantang) for 5 weeks in succession respectively. The expression of c-fos in the hippocampus and frontal lobe cortex of all the rats was observed. Results The expression of c-fos in the hippocampus and frontal lobe cortex of rats in the epileptic model group was significantly increased, while the c-fos expression in the hippocampus and the frontal lobe cortex of rats in the medium-dose and high-dose chaihushugantang treatment groups and Valproate treatment group and Dingxianwan treatment group was significantly decreased as compared with that in epileptic model group (P<0.05). But the c-fos expression in the hippocampus and the frontal lobe cortex of rats in the low-dose chaihushugantang treatment group showed no obvious decrease. Conclusion Chaihushugantang has good antiepileptic effect, might through affecting the c-fos expression in the epileptic rats. The antiepileptic mechanism of chaihushugantang can be related to saikosaponins and other antiepileptic constituents in it.     

养血清脑颗粒和丙戊酸联用对戊四氮点燃癫痫大鼠的影响

目的 探讨养血清脑颗粒(YXQNG)联用丙戊酸(VPA)对戊四氮(PTZ)慢性点燃模型大鼠癫痫发作、EEG、认知功能及颞叶、海马T型Ca2+通道蛋白(Cav3.2)表达的影响. 方法 成年雄性SD大鼠40只按随机数字表法分为PTZ组、VPA组、VPA+YXQNG组、NS组,每组10只.前3组大鼠腹腔注射PTZ溶液制作慢性点燃模型,VPA组大鼠在注射PTZ前1 h给予VPA灌胃;VPA+YXQNG组除给予VPA外,注射PTZ前1.5 h给予YXQNG灌胃;NS组腹腔注射生理盐水,每天一次.8周后观察各组大鼠的行为学变化、Y型电迷宫检查大鼠的正确反应率、捕记EEG并应用免疫组化染色检测颞叶和海马Cav3.2的表达. 结果 给药8周后PTZ组大鼠全部达到完全点燃(连续3 d出现Ⅳ级发作或达到Ⅴ级发作),VPA组和VPA+YXQNG组大鼠仅出现0～Ⅱ级发作;Y型电迷宫检查结果显示VPA+YXQNG组大鼠正确反应率高于PTZ组,差异有统计学意义(P＜0.05);EEG结果显示PTZ组大鼠癫痫发作时EEG有明显异常放电,总功率高于用药前,差异有统计学意义(P＜0.05).VPA组、VPA+YXQNG组大鼠用药前后EEG总功率的差值均高于PTZ组,差异有统计学意义(P＜0.05);免疫组化染色结果显示VPA组、VPA+YXQNG组大鼠颞叶和海马Cav3.2表达低于PTZ组,VPA+YXQNG组大鼠颞叶和海马Cav3.2表达低于VPA组,差异均有统计学意义(P＜0.05). 结论 YXQNG和VPA联用能降低癫痫大鼠发作级别、改善认知功能、减少脑部异常放电并降低脑组织Cav3.2水平,有抗癫痫和脑保护作用.

Abstract：

Objective To explore the effet of Yangxue Qingnao granule (YXQNG) on seizures and cognition function of pentylenetetrazole (PTZ)-kindled chronic epileptic rats models, expression of Cav3.2 in the hippocampus and the temporal lobe of these rats, and EEG features of the rats. Methods Forty healthy adult male SD rats were equally divided into 4 groups at random: PTZ group, VPA treatment group, VPA+YXQNG treatment group, normal saline (NS)-control group (n=10). PTZ solution was intraperitoneally injected for 8 weeks to induce the kindling model in the above 3 groups except the NS-control group. VPA by intragastric administration was given to the rats in the VPA treatment group 1 h before PTZ injection; YXQNG and VPA by intragastric administration were given to the rats in the VPA+YXQNG treatment group 1.5 h before PTZ injection. Behavioral changes of the rats were observed 8 weeks after PTZ injection; accuracy rate of response of the rats were examined by electric maze test;EEG was performed; and the expression ofT-type Ca2+ channel protein (Cav3.2) in the temporal lobe and hippocampus was detected by immunohistochemical staining. Results Rats in the PTZ group appeared grade Ⅳ or Ⅴ seizures for 3 consecutive d, and rats in the VPA treatment group, VPA+YXQNG treatment group appeared grade 0-Ⅱ seizures. The accuracy rate of response of the rats in the VPA+YXQNG treatment group was significantly higher than that in the PTZ group (P＜0.05). EEG indicated that paradoxical discharge was noted in rats of PTZ group when seizures appeared, and the total power at the time was obviously higher than that before PTZ injection (P＜0.05). The D-value of total power of EEG in rats of the VPA treatment group and VPA+YXQNG treatment group before and after treatment was significantly higher than that in the PTZ group (P＜0.05). And the level of Cav3.2 in the temporal and hippocampus in rats of the VPA treatment group and VPA+YXQNG treatment group was significantly lower than that in the PTZ group (P＜0.05); as compared with that in the VPA treatment group, the expression of Cav3.2 in the temporal and hippocampus in rats of the VPA+YXQNG treatment group was significantly reduced (P＜0.05). Conclusion The combination use of YXQNG and VPA can decrease the seizure stage, the paradoxical discharge of the brain and the level of Cav3.2 in brain tissue,and improve the cognitive function of the PTZ-kindled rats, indicating that using VAP and YXQNG simultaneously can treat epileptic seizure and protect the neurons.     

心肌营养素1修饰的神经干细胞移植对癫(癎)持续状态大鼠海马损伤及苔藓纤维发芽的影响

Objective To observe the survival,migration and differentiation of grafted neural stem cells(NSCs)transfected with cardiotrophin-1(CT1)in hippocampus in status epilepticus(SE)rats,and investigate its effect on neuron loss and mossy fiber sprouting(MFS)in hippocampus of SE rats.Methods (1)Lithium-pilocarpine induced SE model rats were divided into 3 groups randomly:CT1-NSCs transplantation group(n=18);NScs transplantation group(n=18)and SE model group(n=18).Another 18 rats served as normal control group.Each group was further divided into 3 time points testing groups(n=6 at each point)corresponding to 1,4 and 8 weeks after transplantation respectively.(2)Under the confocal microscopy,the survival,migration and differentiation of the grafted cells were observed by immunofluorescenee.(3)Morphological changes and neuron loss in the hippocampal CA1 region were examined by Nissl staining.(4)MFS in hippocampal dentate gyrus in rats was obserred by Timm histochemistry.Results(1)At 4 and 8 weeks post-tmusplantation,the numbers of double-labeled NF-200 and EGFP pesitive cells in the CT1-NSCs group were significantly hisher than those in NSCs group.In the former group most of the grafted NSCs migrated away from the needle tract,but in the latter group,grafted ceHs remained at the transplantation site.(2)The numbers of neuron in the hippocampal CA1 region reduced gradually after SE.The numbers of neuron in the CA1 region in CT1-NSCs transplantation rats (68.85±11.49,60.89±12.17 and 51.51±13.34 in 1,4,8 weeks after transplantation respectivelv)were greater than that in NSCs transplantation rats(67.92±10.78,42.56±11.47 and 30.49±10.12).tvalue were 4.650 and 5.334 in 4 and 8 weeks after transplantation(P<0.05).(3)Aberrant MFS in the inner molecular layer of dentate gyrus was observed,and the scores of MFS gradually increased with timelapse.The scores of MFS in CT1-NSCs transplantation rats(0.77±0.04,2.48±0.89 and 2.39±0.82 in 1,4,8 weeks after transplantation respectively)were significant lower than that in NSCs transplantation rats (1.12±0.62,3.17±0.64 and 3.88±0.51,t=6.059,9.511 and 9.728,P<0.05).Conclusions CT1 could promote the survival,migration and differentiation of engrafted NSCs in hippocampud in SE rats.Engrafted NSCs transfected with CT1 have effect on repair of the injured hippocampus,and could inhibit hippocampus MFS in SE rats.     

心肌营养素1修饰的神经干细胞移植对癫(癎)持续状态大鼠海马损伤及苔藓纤维发芽的影响

Objective To observe the survival,migration and differentiation of grafted neural stem cells(NSCs)transfected with cardiotrophin-1(CT1)in hippocampus in status epilepticus(SE)rats,and investigate its effect on neuron loss and mossy fiber sprouting(MFS)in hippocampus of SE rats.Methods (1)Lithium-pilocarpine induced SE model rats were divided into 3 groups randomly:CT1-NSCs transplantation group(n=18);NScs transplantation group(n=18)and SE model group(n=18).Another 18 rats served as normal control group.Each group was further divided into 3 time points testing groups(n=6 at each point)corresponding to 1,4 and 8 weeks after transplantation respectively.(2)Under the confocal microscopy,the survival,migration and differentiation of the grafted cells were observed by immunofluorescenee.(3)Morphological changes and neuron loss in the hippocampal CA1 region were examined by Nissl staining.(4)MFS in hippocampal dentate gyrus in rats was obserred by Timm histochemistry.Results(1)At 4 and 8 weeks post-tmusplantation,the numbers of double-labeled NF-200 and EGFP pesitive cells in the CT1-NSCs group were significantly hisher than those in NSCs group.In the former group most of the grafted NSCs migrated away from the needle tract,but in the latter group,grafted ceHs remained at the transplantation site.(2)The numbers of neuron in the hippocampal CA1 region reduced gradually after SE.The numbers of neuron in the CA1 region in CT1-NSCs transplantation rats (68.85±11.49,60.89±12.17 and 51.51±13.34 in 1,4,8 weeks after transplantation respectivelv)were greater than that in NSCs transplantation rats(67.92±10.78,42.56±11.47 and 30.49±10.12).tvalue were 4.650 and 5.334 in 4 and 8 weeks after transplantation(P<0.05).(3)Aberrant MFS in the inner molecular layer of dentate gyrus was observed,and the scores of MFS gradually increased with timelapse.The scores of MFS in CT1-NSCs transplantation rats(0.77±0.04,2.48±0.89 and 2.39±0.82 in 1,4,8 weeks after transplantation respectively)were significant lower than that in NSCs transplantation rats (1.12±0.62,3.17±0.64 and 3.88±0.51,t=6.059,9.511 and 9.728,P<0.05).Conclusions CT1 could promote the survival,migration and differentiation of engrafted NSCs in hippocampud in SE rats.Engrafted NSCs transfected with CT1 have effect on repair of the injured hippocampus,and could inhibit hippocampus MFS in SE rats.     

心肌营养素1修饰的神经干细胞移植对癫(癎)持续状态大鼠海马损伤及苔藓纤维发芽的影响

Objective To observe the survival,migration and differentiation of grafted neural stem cells(NSCs)transfected with cardiotrophin-1(CT1)in hippocampus in status epilepticus(SE)rats,and investigate its effect on neuron loss and mossy fiber sprouting(MFS)in hippocampus of SE rats.Methods (1)Lithium-pilocarpine induced SE model rats were divided into 3 groups randomly:CT1-NSCs transplantation group(n=18);NScs transplantation group(n=18)and SE model group(n=18).Another 18 rats served as normal control group.Each group was further divided into 3 time points testing groups(n=6 at each point)corresponding to 1,4 and 8 weeks after transplantation respectively.(2)Under the confocal microscopy,the survival,migration and differentiation of the grafted cells were observed by immunofluorescenee.(3)Morphological changes and neuron loss in the hippocampal CA1 region were examined by Nissl staining.(4)MFS in hippocampal dentate gyrus in rats was obserred by Timm histochemistry.Results(1)At 4 and 8 weeks post-tmusplantation,the numbers of double-labeled NF-200 and EGFP pesitive cells in the CT1-NSCs group were significantly hisher than those in NSCs group.In the former group most of the grafted NSCs migrated away from the needle tract,but in the latter group,grafted ceHs remained at the transplantation site.(2)The numbers of neuron in the hippocampal CA1 region reduced gradually after SE.The numbers of neuron in the CA1 region in CT1-NSCs transplantation rats (68.85±11.49,60.89±12.17 and 51.51±13.34 in 1,4,8 weeks after transplantation respectivelv)were greater than that in NSCs transplantation rats(67.92±10.78,42.56±11.47 and 30.49±10.12).tvalue were 4.650 and 5.334 in 4 and 8 weeks after transplantation(P<0.05).(3)Aberrant MFS in the inner molecular layer of dentate gyrus was observed,and the scores of MFS gradually increased with timelapse.The scores of MFS in CT1-NSCs transplantation rats(0.77±0.04,2.48±0.89 and 2.39±0.82 in 1,4,8 weeks after transplantation respectively)were significant lower than that in NSCs transplantation rats (1.12±0.62,3.17±0.64 and 3.88±0.51,t=6.059,9.511 and 9.728,P<0.05).Conclusions CT1 could promote the survival,migration and differentiation of engrafted NSCs in hippocampud in SE rats.Engrafted NSCs transfected with CT1 have effect on repair of the injured hippocampus,and could inhibit hippocampus MFS in SE rats.     

心肌营养素1修饰的神经干细胞移植对癫(癎)持续状态大鼠海马损伤及苔藓纤维发芽的影响

Objective To observe the survival,migration and differentiation of grafted neural stem cells(NSCs)transfected with cardiotrophin-1(CT1)in hippocampus in status epilepticus(SE)rats,and investigate its effect on neuron loss and mossy fiber sprouting(MFS)in hippocampus of SE rats.Methods (1)Lithium-pilocarpine induced SE model rats were divided into 3 groups randomly:CT1-NSCs transplantation group(n=18);NScs transplantation group(n=18)and SE model group(n=18).Another 18 rats served as normal control group.Each group was further divided into 3 time points testing groups(n=6 at each point)corresponding to 1,4 and 8 weeks after transplantation respectively.(2)Under the confocal microscopy,the survival,migration and differentiation of the grafted cells were observed by immunofluorescenee.(3)Morphological changes and neuron loss in the hippocampal CA1 region were examined by Nissl staining.(4)MFS in hippocampal dentate gyrus in rats was obserred by Timm histochemistry.Results(1)At 4 and 8 weeks post-tmusplantation,the numbers of double-labeled NF-200 and EGFP pesitive cells in the CT1-NSCs group were significantly hisher than those in NSCs group.In the former group most of the grafted NSCs migrated away from the needle tract,but in the latter group,grafted ceHs remained at the transplantation site.(2)The numbers of neuron in the hippocampal CA1 region reduced gradually after SE.The numbers of neuron in the CA1 region in CT1-NSCs transplantation rats (68.85±11.49,60.89±12.17 and 51.51±13.34 in 1,4,8 weeks after transplantation respectivelv)were greater than that in NSCs transplantation rats(67.92±10.78,42.56±11.47 and 30.49±10.12).tvalue were 4.650 and 5.334 in 4 and 8 weeks after transplantation(P<0.05).(3)Aberrant MFS in the inner molecular layer of dentate gyrus was observed,and the scores of MFS gradually increased with timelapse.The scores of MFS in CT1-NSCs transplantation rats(0.77±0.04,2.48±0.89 and 2.39±0.82 in 1,4,8 weeks after transplantation respectively)were significant lower than that in NSCs transplantation rats (1.12±0.62,3.17±0.64 and 3.88±0.51,t=6.059,9.511 and 9.728,P<0.05).Conclusions CT1 could promote the survival,migration and differentiation of engrafted NSCs in hippocampud in SE rats.Engrafted NSCs transfected with CT1 have effect on repair of the injured hippocampus,and could inhibit hippocampus MFS in SE rats.     

心肌营养素1修饰的神经干细胞移植对癫(癎)持续状态大鼠海马损伤及苔藓纤维发芽的影响

Objective To observe the survival,migration and differentiation of grafted neural stem cells(NSCs)transfected with cardiotrophin-1(CT1)in hippocampus in status epilepticus(SE)rats,and investigate its effect on neuron loss and mossy fiber sprouting(MFS)in hippocampus of SE rats.Methods (1)Lithium-pilocarpine induced SE model rats were divided into 3 groups randomly:CT1-NSCs transplantation group(n=18);NScs transplantation group(n=18)and SE model group(n=18).Another 18 rats served as normal control group.Each group was further divided into 3 time points testing groups(n=6 at each point)corresponding to 1,4 and 8 weeks after transplantation respectively.(2)Under the confocal microscopy,the survival,migration and differentiation of the grafted cells were observed by immunofluorescenee.(3)Morphological changes and neuron loss in the hippocampal CA1 region were examined by Nissl staining.(4)MFS in hippocampal dentate gyrus in rats was obserred by Timm histochemistry.Results(1)At 4 and 8 weeks post-tmusplantation,the numbers of double-labeled NF-200 and EGFP pesitive cells in the CT1-NSCs group were significantly hisher than those in NSCs group.In the former group most of the grafted NSCs migrated away from the needle tract,but in the latter group,grafted ceHs remained at the transplantation site.(2)The numbers of neuron in the hippocampal CA1 region reduced gradually after SE.The numbers of neuron in the CA1 region in CT1-NSCs transplantation rats (68.85±11.49,60.89±12.17 and 51.51±13.34 in 1,4,8 weeks after transplantation respectivelv)were greater than that in NSCs transplantation rats(67.92±10.78,42.56±11.47 and 30.49±10.12).tvalue were 4.650 and 5.334 in 4 and 8 weeks after transplantation(P<0.05).(3)Aberrant MFS in the inner molecular layer of dentate gyrus was observed,and the scores of MFS gradually increased with timelapse.The scores of MFS in CT1-NSCs transplantation rats(0.77±0.04,2.48±0.89 and 2.39±0.82 in 1,4,8 weeks after transplantation respectively)were significant lower than that in NSCs transplantation rats (1.12±0.62,3.17±0.64 and 3.88±0.51,t=6.059,9.511 and 9.728,P<0.05).Conclusions CT1 could promote the survival,migration and differentiation of engrafted NSCs in hippocampud in SE rats.Engrafted NSCs transfected with CT1 have effect on repair of the injured hippocampus,and could inhibit hippocampus MFS in SE rats.     

心肌营养素1修饰的神经干细胞移植对癫(癎)持续状态大鼠海马损伤及苔藓纤维发芽的影响

Objective To observe the survival,migration and differentiation of grafted neural stem cells(NSCs)transfected with cardiotrophin-1(CT1)in hippocampus in status epilepticus(SE)rats,and investigate its effect on neuron loss and mossy fiber sprouting(MFS)in hippocampus of SE rats.Methods (1)Lithium-pilocarpine induced SE model rats were divided into 3 groups randomly:CT1-NSCs transplantation group(n=18);NScs transplantation group(n=18)and SE model group(n=18).Another 18 rats served as normal control group.Each group was further divided into 3 time points testing groups(n=6 at each point)corresponding to 1,4 and 8 weeks after transplantation respectively.(2)Under the confocal microscopy,the survival,migration and differentiation of the grafted cells were observed by immunofluorescenee.(3)Morphological changes and neuron loss in the hippocampal CA1 region were examined by Nissl staining.(4)MFS in hippocampal dentate gyrus in rats was obserred by Timm histochemistry.Results(1)At 4 and 8 weeks post-tmusplantation,the numbers of double-labeled NF-200 and EGFP pesitive cells in the CT1-NSCs group were significantly hisher than those in NSCs group.In the former group most of the grafted NSCs migrated away from the needle tract,but in the latter group,grafted ceHs remained at the transplantation site.(2)The numbers of neuron in the hippocampal CA1 region reduced gradually after SE.The numbers of neuron in the CA1 region in CT1-NSCs transplantation rats (68.85±11.49,60.89±12.17 and 51.51±13.34 in 1,4,8 weeks after transplantation respectivelv)were greater than that in NSCs transplantation rats(67.92±10.78,42.56±11.47 and 30.49±10.12).tvalue were 4.650 and 5.334 in 4 and 8 weeks after transplantation(P<0.05).(3)Aberrant MFS in the inner molecular layer of dentate gyrus was observed,and the scores of MFS gradually increased with timelapse.The scores of MFS in CT1-NSCs transplantation rats(0.77±0.04,2.48±0.89 and 2.39±0.82 in 1,4,8 weeks after transplantation respectively)were significant lower than that in NSCs transplantation rats (1.12±0.62,3.17±0.64 and 3.88±0.51,t=6.059,9.511 and 9.728,P<0.05).Conclusions CT1 could promote the survival,migration and differentiation of engrafted NSCs in hippocampud in SE rats.Engrafted NSCs transfected with CT1 have effect on repair of the injured hippocampus,and could inhibit hippocampus MFS in SE rats.     

红藻氨酸诱导性癫痫发作大鼠海马cNOS及iNOS的变化及作用

目的研究组成型一氧化氮合酶（ｃＮＯＳ）及诱导型一氧化氮合酶（ｉＮＯＳ）在红藻氨酸（ＫＡ）诱导癫痫发作中的变化及作用。方法采用免疫组织化学方法显示ｃＮＯＳ及ｉＮＯＳ的变化;Ｎｉｓｓｌ染色显示神经元的损害。结果ＫＡ３０分钟ｃＮＯＳ较对照组明显增加（Ｐ＜０．０５）,随后下降至正常水平;ＫＡ诱导２小时ｉＮＯＳ明显升高,以ＣＡ１区为著,至ＫＡ６小时达高峰,然后在高水平缓慢下降;Ｎｉｓｓｌ染色神经元变性坏死ＣＡ３＝齿状回＞ＣＡ１。结论ＫＡ诱导癫痫发作导致海马ｃＮＯＳ及ｉＮＯＳ表达增多,神经元的坏死与ＮＯＳ表达增多无明显关系。     

雌激素影响海人酸杏仁核点燃大鼠癫(癎)发作机制的初步探讨

目的:探讨雌激素(E)和姜黄素(C)影响癫发作的机制。方法:用E和C单独及联用连续处理去势雌性大鼠5d,第6天以海人酸(KA)杏仁核点燃法制备癫大鼠模型,观察大鼠癫发作的行为学表现,用免疫组化方法检测海马组织c-Jun蛋白的表达。结果:E加C组(EC KA组)大鼠癫重度发作的严重程度较E组(E KA组)明显减轻(P<0.05)。E KA组海马中c-Jun蛋白表达最多,C组(C KA组)及对照组(KA组)均表达较少且没有任何差异;EC KA组海马的CA1区c-Jun蛋白表达较E KA组明显减少(P<0.05)。结论:C能一定程度上减轻E引起的癫发作加重,它可能通过抑制c-Jun/核转录因子激活蛋白-1(activate-protein1,AP-1)活性,使E作用的AP-1通路受阻,从而减轻了E的促神经元兴奋作用。     

Temporal lobe epilepsy animal model established by stereotaxic microinjection of kainic acid***★

BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high doses of drug are required and the success rate of model induction is low. It is necessary to develop an improved method to establish a temporal lobe epilepsy （TLE） animal model. OBJECTIVE： To explore an economic, stable and efficient method of establishing a TLE animal model. DESIGN, TIME AND SETTING： A completely randomized, controlled study. The experiments were performed in the Cellular Function Laboratory of the Physiology Department, Anhui Medical University from March to July 2007. MATERIALS： Twenty adult male Wistar rats, weighing 230-260 g, were provided by the Experimental Animal Centre of Nanjing Medical University. Kainic acid was purchased from Sigma in USA. Type SN-2 stereotaxic apparatus was made by Narishge in Japan. METHODS： Wistar rats were randomly divided into a kainic acid （KA） group （n = 12） and a normal saline （NS） group （n = 8）. For intrahippocampal microinjection, a burr hole was drilled in the skull at the following stereotaxic coordinates： anteroposterior （AP） 4.1 mm caudal to bregma; lateral （ML） 4.2 mm right lateral to the midline. Rats in the KA group were injected with 2.5 μL KA （0.4 g/L） into the center of the CA3 region, while in the NS group the same volume of NS was injected into the same site. MAIN OUTCOME MEASURES： Both groups were monitored under a video capture system for 12 weeks to record spontaneous seizures. Intracranial eletroencepholograph （IEEG） recordings in vivo were performed after the behavioral observations. After the IEEG recordings, hippocampi were processed into coronal sections. Nissl and Timm stainings were then performed to observe and confirm pathology. RESULTS： Twenty rats were involved in the final analysis. Behavioral observations： the eadiest spontaneous onset of epilepsy appeared 2 weeks a     

下丘脑内基因转染对大鼠癫痫行为的影响

目的探讨下丘脑过度兴奋对于颞叶癫痫行为学变化的影响，从而进一步阐明下丘脑与颢叶癫痫的关系以及谷氨酸受体2亚基Q型(glutamate receptor 2Q，GluR2Q)在癫痫发病中的作用机制。方法20只Wistar大鼠随机分为海人酸(kainic acid or kainite，KA)组(KA对照组)与KA GluR2Q组，分别观察两组大鼠的癫痫行为。结果KA对照组大鼠癫痫发作程度较轻，主要以部分性发作为主且发作次数少，持续时间短，较少出现全面性发作。KA GluR2Q组大鼠癫痫发作程度剧烈，部分性癫痫发作较KA对照组更早、更频繁且由部分性发作转化为全面性发作的比率高于KA对照组。结论通过HVJ-脂质体基因转染技术将GluR2Q基因转染到下丘脑乳头体可以提高其兴奋性，并使该兴奋性冲动通过下丘脑与海马之间的联络纤维传导至海马齿状回及CA3、CA1区，使海马区原有的兴奋性加强，表现为癫痫行为的加重，从而促进了癫痫的发展及传播。     

托吡酯对颞叶癫痫大鼠海马神经细胞粘附分子表达的影响

目的观察托吡酯对红藻氨酸(KA)诱导颞叶癫痫大鼠海马突触重建标记物神经细胞粘附分子(NCAM)表达的影响,进一步探讨托吡酯的抗痫作用机制。方法采用免疫组织化学染色观察KA诱导癫痫大鼠及托吡酯给药大鼠海马神经细胞粘附分子表达水平,并对两组NCAM表达进行比较。结果KA组NCAM表达水平各时间点平均NCAM阳性密度OD率均明显高于N S组和KA TPM组(P<0.01)。结论本研究提示托吡酯可能通过抑制突触重建的形成,减少痫性放电,从而控制癫痫发作。     

Dentate granule cells are essential for kainic acid-induced wet dog shakes but not for seizures

The purpose of this study was to determine the role that dentate granule cells play in wet dog shakes (WDS), behavioral seizures, and hippocampal cell loss caused by systemic administration of kainic acid (KA). Rats were given bilateral injections of colchicine (COL) into the hippocampal formation to selectively lesion dentate granule cells. Two weeks later, they were injected subcutaneously with KA and were observed for WDS and seizures. Seizures were terminated with pentobarbital 2.5 hr after KA injection, and the rats were killed 48 hr later. The integrity of hippocampal cell populations and projections to the hippocampal formation from entorhinal cortex was assessed with radioimmunoassay and immunostaining for methionine-enkephalin (ME) and dynorphin (DYN) A, as well as with Timm and Nissl staining. Results indicate that COL injections eliminated KA-induced WDS, did not affect the latency to onset of seizures, and potentiated KA-induced cell loss in the CA3 region of hippocampus. COL lesions eliminated ME and DYN immunostaining of granule cells, but not ME immunostaining of entorhinal afferents to the dentate gyrus or Ammon's horn. These findings indicate that granule cells are an essential neuronal link in the expression of KA-induced WDS, but that seizures propagate along other pathways in the limbic system.     

A guinea pig model of mesial temporal lobe epilepsy following nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid

Purpose: Models of temporal lobe epilepsy are commonly utilized to study focal epileptogenesis and ictogenesis. The criteria that define animal models representative of human mesial temporal lobe may vary in different laboratories. We describe herein a focal epilepsy model of mesial temporal (hippocampal) origin that relies on the analysis of interictal and ictal electroencephalography (EEG) patterns and on their correlation with seizure symptoms and neuropathologic findings. The study is based on guinea pigs, a species seldom utilized to develop chronic epilepsy models. Methods: Young adult guinea pigs were bilaterally implanted under isoflurane anesthesia with epidural electrodes over somatosensory cortex and depth electrodes in CA1 hippocampal region. A stainless steel guide cannula was positioned unilaterally in the right dorsal hippocampus to inject 1 μl of 0.9% NaCl solution containing 1 μg kainic acid (KA). One week after surgery, continuous 24 h/day video‐EEG monitoring was performed 48 h before and every other week after KA injection, for no <1 month. EEG data were recorded wide‐band at 2 kHz. After video‐EEG monitoring, brains were analyzed for thionine and Timm staining and glial fibrillary acid protein (GFAP) immunostaining. Key Findings: Unilateral injection of KA in dorsal hippocampus of guinea pigs induces an acute nonconvulsive status epilepticus (SE) that terminates within 24 h (n = 22). Chronic seizures with very mild motor signs (undetectable without EEG monitoring) and highly variable recurrence patterns appear in 45.5% (10 of 22) KA‐treated animals, with variable delays from the initial SE. In these animals interictal events, CA1 cell loss, gliosis, and altered Timm staining pattern were observed. The induction of a chronic condition did not correlate with the duration of the nonconvulsive acute SE, but correlated with the extension and quality of neuropathologic damage. Significance: We demonstrate that a model of hippocampal (mesial temporal lobe) epilepsy can be developed in the guinea pig by intrahippocampal injection of KA. Seizure events in this model show little behavioral signs and may be overlooked without extensive video‐EEG monitoring. The establishment of a chronic epileptic condition correlates with the extension of the hippocampal damage (mainly cell loss and gliosis) and not with the intensity of the initial SE.     

白藜芦醇对戊四氮致痫大鼠脑脊液、血清S100B蛋白的影响

目的研究白藜芦醇（Res）对戊四氮致痫大鼠脑脊液、血清S100B蛋白的影响。方法采用戊四氮（PTZ）腹腔注射建立慢性癫痫模型,造模成功后予以Res（15mg／kg·d）灌胃干预10d;采用酶联免疫吸附法测定脑脊液、血清S100B蛋白含量,海马标本行Nissl染色。结果经28d连续给药,18只大鼠符合Racine点燃标准,Res干预组大鼠痫性发作潜伏期明显延长,且发作时间明显低于癫痫模型组、二甲基亚砜组（P〈0．05）。海马Nissl染色提示Res干预对大鼠海马CAl、CA3区神经元有保护作用（P〈0．05）,而对齿状回保护作用不明显（P〉0．05）。Res干预组大鼠脑脊液、血清S100B蛋白含量低于癫痫模型组、二甲基亚砜组（P〈0．05）。结论Res降低PTZ致痫大鼠脑脊液、血清SIOOB蛋白含量,或许减缓癫痫发作脑损伤发挥神经保护作用。     

Consequences of pilocarpine-induced recurrent seizures in neonatal rats

Accumulated evidence have shown that a series of morphological alternations occur in patients with epilepsy and in different epileptic animal models. Given most of animal model studies have been focused on adulthood stage, the effect of recurrent seizures to immature brain in neonatal period has not been well established. This study was designed to observe the certain morphological changes following recurrent seizures occurred in the neonatal rats. For seizure induction, neonatal Wistar rats were intraperitoneally injected with pilocarpine on postnatal day 1 (P1), P4 and P7. Rat pups were grouped and sacrificed at 1d, 7d, 14d and 42d after the last pilocarpine injection respectively. Bromodeoxyuridine (BrdU) was intraperitoneally administered 36h before the rats were sacrificed. BrdU single and double labeling with neuronal markers were used to analyze cell proliferation and differentiation. Nissl and Timm staining were performed to evaluate cell loss and mossy fiber sprouting. Rats with neonatal seizures had a significant reduction in the number of Bromodeoxyuridine-(BrdU) labeled cells in the dentate gyrus compared with the control groups when the animals were killed either 1 or 7 days after the third seizure (P<0.05) but there was no difference between two groups on P21. On the contrary, BrdU-labeled cells significantly increased in the experimental group compared with control group on P49 (P<0.05). The majority of the BrdU-labeled cells colocalized with neuronal marker-NF200 (Neurofilament-200). Nissl staining showed that there was no obvious neuronal loss after seizure induction over all different time points. Rats with the survival time of 42 days after neonatal seizures developed to increased mossy fiber sprouting in both the CA3 region and supragranular zone of the dentate gyrus compared with the control groups (P<0.05). Taken together, the present findings suggest that synaptic reorganization only occurs at the later time point following recurrent seizures in neonatal rats, and neonatal recurrent seizures can modulate neurogenesis oppositely over different time window with a down-regulation at early time and up-regulation afterwards.