基于组织病理学的大鼠脑干原发性功能损伤程度研究

目的:利用自制改进的Marmarou模型建立不同损伤高度的弥漫性轴索损伤(Diffuse axonal injury,DAI)大鼠模型,探讨通过苏木精-伊红(Hematoxylin eosin staining,HE)染色和β-淀粉样前体蛋白(β-Amyloid precursor protein,β-APP)免疫组化互相验证结合的方式对大鼠脑干原发性功能损伤程度进行定量研究。方法:健康成年雄性(Sprague-dawley,SD)大鼠24只,随机分为0 m正常组、1.25 m致伤组和2.25 m致伤组,采用改进的经典Marmarou模型制作大鼠脑干部位的DAI模型,正常组不进行打击。在致伤后进行过量麻醉取材制作HE染色切片和β-APP免疫组化染色切片,其中对HE染色切片计算脑干感兴趣区域内出血占比、脑水肿个数和锥体束出血占比;对β-APP免疫组化切片测定脑干感兴趣区域内的阳性细胞面积和累计光密度值(Integral optical density,IOD)。将不同损伤程度脑组织切片的HE染色分析与β-APP免疫组化染色结合做相关性分析。结果:不同致伤高度组的HE染色切片在光镜下可见神经束疏散、紊乱,血管淤滞,出现不同程度的散点状或长条状出血,细胞核显示不清,突触结构模糊不清等经典DAI病理特征;β-APP免疫组化出现轻度轴索损伤变化。测定脑干感兴趣区域内β-APP的阳性细胞面积和IOD值与HE染色的出血面积占比和锥体束出血面积占比呈正相关。结论:采用HE染色计算的大鼠脑干位置出血面积占比和锥体束区域出血面积占比和β-APP免疫组化计算的阳性细胞面积和IOD值可以定量区分大鼠脑干损伤程度,为判断大鼠脑干原发性功能损伤提供新角度,有助于为交通事故和法医检测等脑损伤机制的研究提供实验参考。

通过脑干听觉诱发电位观察电项针治疗脑干缺血大鼠即时效应

目的采用电凝椎动脉的方法建立脑干缺血大鼠模型,通过转棒实验、Bederson评分、脑干听觉诱发电位检测观察电项针对脑干缺血大鼠模型的即时疗效。分析通过针灸及电项针对脑干缺血大鼠进行即刻治疗时脑干听觉诱发电位(BAEP)的变化情况。方法选取清洁级健康雄性SD成年大鼠60只,经常规适应性喂养及转棒实验适应性训练1周后开始正式实验。将大鼠随机分为4组,分别为假手术组、模型组、电项针组、针刺组。记录各组大鼠治疗前后于转棒上停留时间及Bederson评分情况。在造模结束24 h后(治疗前)进行第1次BAEP检测,首次治疗结束后立即进行第2次BAEP检测,比较4组大鼠Ⅰ、Ⅲ、V波振幅及Ⅰ、Ⅲ、Ⅴ波的波峰潜伏期。结果相较于假手术组,模型组、针刺组、电项针组3组大鼠造模24 h后于转棒上停留时间明显减少(P<0.01),Bederson评分明显增高;即刻治疗后,电项针组相较模型组及针刺组于转棒上停留时间明显增加(P<0.05),Bederson评分明显增高;模型组、针刺组、电项针组大鼠造模24 h后进行BAEP检测,Ⅲ、Ⅴ波振幅明显压低,Ⅲ、Ⅴ波峰潜伏期波峰明显延长(P<0.01),假手术组不出现变化,Ⅰ波无明显变化(P>0.05),即刻治疗后电项针组较针刺组及模型组大鼠Ⅲ、Ⅴ波振幅抬高(P<0.05),Ⅲ、Ⅴ波峰潜伏期缩短(P<0.05),Ⅰ波无明显变化(P>0.05)。结论电项针即刻治疗可明显改善脑干大鼠的脑干血液循环,其效果优于即刻针刺治疗。

Cognitive improvement following transvenous adipose-derived mesenchymal stem cell transplantation in a rat model of traumatic brain injury

The effects of adipose-derived mesenchymal stem cell (ADMSC) transplantation for the repair of traumatic brain injury remain poorly understood. The present study observed neurological functional changes in a rat model of traumatic brain injury following ADMSC transplantation via the tail vein. Cell transplants were observed in injured cerebral cortex, and expression of brain-derived nerve growth factor was significantly increased in the injured hippocampus following transplantation. Results demonstrated that transvenous ADMSC transplants migrated to the injured cerebral cortex and significantly improved cognitive function.

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.

Effect of catgut implantation at acupoints on GABA_B and mGluR1 expressions in brain stem of rats with spasticity after stroke

OBJECTIVE: To investigate the effect of catgut implantation at acupoints on the expressions of γ-amino butyric acid B receptor(GABAB ) and metabotropic glutamate receptor 1(mGluR1) in the brain stem of rats with spasticity after stroke.METHODS: In total, 60 male Sprague-Dawley rats were randomly divided into three groups: a sham group(n=10), a model group(n=25) and a treatment group(n=25). The rats in both the model group and the treatment group were subjected to middle cerebral artery occlusion to establish a model of focal cerebral ischemia. Rats with limb-spasm met the inclusion criteria. Only the left carotid artery was isolated in sham group rats. Three days after modeling, the treatment group was subjected to catgut implantation at Dazhui(GV 14), Guanyuan(CV 4), and Zhongwan(CV 12). Neurological deficit symptoms were assessed with the Zea-Longa neurological deficit score. The Modified Ashworth Scale(MAS), and isolated muscle tone were used to evaluate spasticity before and after treatment. Immunohistochemistry was applied to determine the expression of GABAB and mGluR1 in the rat brain stem after treatment.RESULTS: After treatment, neural impairment symptoms had significantly improved in the treatment group when compared to the model group(P<0.05). Both MAS and isolated muscle tone in the treatment group were significantly decreased when compared with the model group(P<0.05),and were also lower than before treatment. GABAB expression was significantly higher and mGluR1 was lower in the treatment group when compared with the model group(P<0.01 and P<0.05, respectively).CONCLUSION: Catgut implantation at Dazhui(GV 14), Guanyuan(CV 4), and Zhongwan(CV 12), can relieve limb spasticity by increasing the expression of GABAB and reducing the expression of mGluR1 in the brain stem of rats after stroke.

In vivo tracing of superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells transplanted for traumatic brain injury by susceptibility weighted imaging in a rat model

Objective：To label rat bone marrow mesenchymal stem cells （BMSCs） with superparamagnetic iron oxide （SPIO） in vitro, and to monitor the survival and location of these labeled BMSCs in a rat model of traumatic brain injury （TBI） by susceptibility weighted imaging （SWI）sequence.Methods：BMSCs were cultured in vitro and then labeled with SPIO. Totally 24 male Sprague Dawley （SD） rats weighing 200-250 g were randomly divided into 4 groups： Groups A-D （n=6 for each group）. Moderate TBI models of all the rats were developed in the left hemisphere following Feeney＇s method. Group A was the experimental group and stereotaxic transplantation of BMSCs labeled with SPIO into the region nearby the contusion was conducted in this group 24 hours after TBI modeling. The other three groups were control groups with transplantation of SPIO, unlabeled BMSCs and injection of nutrient solution respectively conducted in Groups B, C and D at the same time. Monitoring of these SPIO-labeled BMSCs by SWI was performed one day,one week and three weeks after implantation.Results： Numerous BMSCs were successfully labeled with SPIO. They were positive for Prussian blue staining and intracytoplasm positive blue stained particles were found under a microscope （×200）. Scattered little iron particles were observed in the vesicles by electron microscopy （×5000）. MRI of the transplantation sites of the left hemisphere demonstrated a low signal intensity on magnitude images,phase images and SWI images for all the test rats in Group A, and the lesion in the left parietal cortex demonstrated a semicircular low intensity on SWI images, which clearly showed the distribution and migration of BMSCs in the first and third weeks. For Group B, a low signal intensity by MRI was only observed on the first day but undetected during the following examination. No signals were observed in Groups C and D at any time points.Conclusion：SWI sequence in vivo can consecutively and noninvasively trace and demonstrate the status and distribution of BMSCs labeled with SPIO in the brain of TBI model rats.