Influence of mild hypothermia on vascular endothelial growth factor and infarct volume in brain tissues after cerebral ischemia in rats

BACKGROUND: It has been demonstrated that mild hypothermia has obvious protective effect on both whole and local cerebral ischemia. However, the definite mechanism is still unclear for the brain protection of mild hypothermia on cerebral edema, inhibiting inflammatory reaction, stabilizing blood brain barrier, etc. OBJECTIVE: To investigate the effect of mild hypothermia on the expression of vascular endothelial growth factor and the infarct volume after cerebral ischemia in rats, and analyze the brain protective mechanism of mild hypothermia. DESIGN: A randomized grouping and controlled animal trial. SETTING: Department of Neurology, People's Hospital of Yunyang Medical College. MATERIALS: Twenty adult male SD rats of clean degree, weighing (250±30) g, were provided by the animal experimental center, School of Medicine, Wuhan University. The kits for SP immunohistochemistry were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. METHODS: The experiments were carried out in the laboratory of Department of Neurology, Renmen Hospital of Wuhan University from May to July 2005. ① The 20 rats were divided randomly into normal temperature group (n =10) and mild hypothermia group (n =10). Models of permanent middle cerebral artery occlusion were established with modified nylon suture embolization. The rats were assessed with the Longa standards: 0 point for without nerve dysfunction; 1 for mild neurological deficit (fore claws could no extend completely); 2 for moderate neurological deficit (circling towards the affected side); 3 for severe neurological deficit (tilting towards the affected side); 4 for coma and unconscious; 1-3 points represented that models were successfully established. The rats of the normal temperature group were fed at room temperature, and those in the mild hypothermia group were induced by hypothermia from 2 hours postoperatively, and the rectal temperature was kept at 34-35 ℃ for 72 hours. ② Measurement of infarct volume: All the rats were anesthetized by intraperitoneal injection overdose sodium pentobarbital 7 days postoperatively, and then the heads were cut down to harvest brain. The brain tissues were placed into -20 ℃ refrigerator for 20 minutes, coronal sections of 2 mm were prepared. The infarct sites were not stained, whereas normal brain tissues were stained as red. The infarct volumes were calculated by using MPLAS-500 multimedia color pathological image&&word analytical system. ③ Counting positive cells of vascular endothelial growth factor protein: The brains were harvested by cutting heads, then coronal sections of 2 mm were prepared. Routine dehydration, hyalinization, wax immersion and embedding were performed, then the detected with SP immunohistochemistry, the kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. The cells whose cytoplasm was yellow-brown were positive ones, a single sample as a unit, peri-ischemic site and ischemic core were selected, and the corresponding sites in controlateral hemisphere were taken as controls. Five visual fields were selected from each site to be observed under microscope, the cells were counted, and the average number of positive cells was calculated in each group. The numbers of positive cells were determined with the image analytical apparatus. MAIN OUTCOME MEASURES: Number of the positive cells of vascular endothelial growth factor protein; Infarct volume of rat brain tissue. RESULTS: All the 20 rats were involved in the analysis of results. ① Number of positive cells of vascular endothelial growth factor protein in brain tissue: It was obviously lower in the mild hypothermia group than in the normal temperature group [(24.02±5.05), (36.07±2.69) cells/high power visual field, P < 0.01]. ② Comparison of infarct volume of brain tissue: After MCAO, it was obviously smaller in the mild hypothermia group than in the normal temperature group [(153.25±23.14), (253.45±36.21) mm3, P < 0.01]. CONCLUSION: Mild hypothermia can inhibit the expression of vascular endothelial growth factor and decrease the volume of cerebral infarction. The inhibition of mild hypothermia on the expression of vascular endothelial growth factor may be one of the brain protective mechanisms.     

微创手术治疗高血压脑出血

目的 探讨微创手术治疗高血压脑出血的临床疗效。方法 132例高血压脑出血分成微创手术组(68例)和传统开颅手术组(64例)，分析两组手术的特点和手术时机，比较两组手术治疗的疗效。结果 微创组术后GOS良好23例、中残24例、重残9例、植物生存3例、死亡9例；传统组术GOS良好16例、中残15例、重残12例、植物生存6例、死亡15例。两组超早期或早期手术均有良好的预后，而微创组效果更佳。结论 微创手术治疗高血压脑出血能明显提高临床疗效，降低病死率。     

In vitro differentiation of human adipose-derived adult stromal cells into neuron-like cells in hippocampal astrocyte conditioned medium

BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal astrocyte conditioned medium (HCAM) can induce in vitro differentiation from hADASC to neuron-like cells is still unclear. OBJECTIVE: To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells. DESIGN: Randomized control study. SETTING: Department of Neurology, Taixing People's Hospital; Central Laboratory, North China Coal Medical College. MATERIALS: Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20－35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation. In addition, 8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences. Rabbit-anti-human Nestin polyclonal antibody, rabbit-anti-human glial fibriliary acidic protein (GFAP) polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2 (MAP-2) polyclonal antibody were provided by Wuhan Boster Company. METHODS: The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope. Immunocytochemistry, immunofluorescence and Western blotting were used to evaluate the expressions of Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. MAIN OUTCOME MEASURES: Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. RESULTS: On the 3rd day of culture, partial hADASC started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. Five days later, immunohistochemical detection suggested that expression of Nestin (10.5±0.037) was found out in cells; meanwhile, expressions of GFAP (38.4±0.052) and neuro-specific enolase (NSE) (15.7±0.023) were also found out in cells; however, expression of MAP-2 was not observed. Western blot indicated that, 5 days after effect of HCAM, Nestin was found out in hADASC; meanwhile, expressions of GFAP and neuro-specific enolase were also found out; however, expression of MAP-2 was not observed. CONCLUSION: HCAM can induce the differentiation from hADASC to neuron-like cells in vitro.     

Effects of human amniotic fluid on cartilage regeneration from free perichondrial grafts in rabbits.

After the chondrogenic potential of free grafts of perichondrium was shown in several experimental studies, perichondrium has been used to reconstruct cartilage tissue in various clinical situations. This study investigates the effects of human amniotic fluid on neochondrogenesis from free perichondrial grafts in a rabbit model. Since this fluid contains high concentrations of hyaluronic acid, hyaluronic acid-stimulating activator, growth factors, and extracellular matrix precursors during the second trimester, it may have a stimulating effect on neochondrogenesis. Perichondrial grafts, measuring 20 x 20 mm2 were obtained from the ears of 144 New Zealand young rabbits and were sutured over the paravertebral muscles. The rabbits were randomly divided into three groups with 48 rabbits per group. In group 1, 0.3 ml human amniotic fluid, and in group 2, 0.3 ml saline were injected underneath the perichondrial grafts. Group 3 formed the control group in which no treatment was given. Histologically, neochondrogenesis was evaluated in terms of cellular form and graft thickness at 2, 4, 6, and 8 weeks after surgery. In group 1, the mature cartilage was generated quickly and the cartilage plate in this group was significantly thick and extensive when compared with groups 2 and 3 at 8 weeks ( p<0.05 ANOVA). In conclusion, our study shows that human amniotic fluid enhances neochondrogenesis from free perichondrial grafts. The rich content of hyaluronic acid and growth factors possibly participate in this result.     

ULTRASTRUCTURAL INVESTIGATION ON THE ARTICULAR CARTILAGE FROM RABBIT KNEE JOINT AFTER IMMOBILIZATION IN CAST

The articular cartilages from the left knee of 30 rabbits immobilized in casts in functional position were studied under transmission electron microscope. The pathological changes which begin at early stage of immobilization included both degeneration and repair of cartilage. The striking morphological features observed were: (1) most chondrocytes in various layers of cartilage underwent progresive degeneration; (2) the concomitant abnormal proliferation of some chondrocytes happened in a brief period. The changes in mechanical stress caused by immobilization affected the nutrition of cartilage and finally led to its reactive structural reformation.     

大肠癌旁粘膜细胞的PCNA和AgNORs表达观察

用增殖细胞核抗原（ＰＣＮＡ）和核仁组成区嗜银蛋白（ＡｇＮＯＲｓ）对３５例大肠癌旁粘膜进行细胞增殖表达与观察。结果表明：癌旁粘膜的ＰＣＮＡ标记指数增多，标记增强，排列紊乱。ＡｇＮＯＲｓ在癌旁粘膜中，颗粒增多、增大，形态多样，０￣１ｃｍ和２￣３ｃｍ组的标记指数与４￣５ｃｍ组的标记指数相比较，有显著或非常显著差异（Ｐ〈０．０５或Ｐ〈０．０１）。在ＤｕｋｅＡ、Ｂ期中，细胞标记指数增高增强，与癌的发生和患者     

Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue.

Until now methods using tetramethylbenzidine (TMB) for electron microscopy (TMB-EM methods) are all unable to provide a maximum demonstration of transported horseradish peroxidase (HRP) while maintaining good ultrastructural tissue preservation. In order to solve this problem, we have attempted to adapt a newly developed, highly sensitive TMB method using sodium tungstate (ST) as the stabilizer (TMB-ST method) for HRP electron microscopic retrograde and anterograde fiber tracing. The present study shows that the TMB-ST method combined with diaminobenzidine-cobalt (DAB-Co) is more sensitive than existing TMB-EM methods and that ultrastructural details are well preserved with this combined method. The resultant reaction product complex after osmication is stable and is observed as characteristic crystal-like structures which are extremely electron dense and often aggregated into clumps. In contrast, the TMB-ST method without the DAB-Co step frequently produces a moderate electron-dense reaction product. Therefore, we recommend the TMB-ST method combined with DAB-Co for HRP electron microscopy.     

静脉内毒瘾者HIV感染的转归及影响因素

对ＨＣＶ血清标记阳性的静脉内毒瘤者进行半年和１５个月随访，并调查影响转归的部分因素。结果显示，静脉内毒瘾者抗－ＨＣＶ自然转阴率分别为１１．０／和３３．３％。单因素和多因素分析均提示转阴率以婚姻状况良好的已婚者较离婚者高、人均年收入高者较低者高，说明婚姻状况和人们的经济水平对毒瘾者抗－ＨＣＶ转阴有一定的影响。     

高原地区健康老年人重返平原后外周血T细胞亚群变化的观察

用单克隆抗体测定了移居高原的老年人重返平原后的T细胞亚群的变化结果:①移居西宁(2260m)组、天峻(3000m)组在西宁所测的OKT_3、OKT_4、OKT_8及OKT_4/OKT_8值与在苏州所测的当地老年人无差异。②移居西宁组急返平原后OKT_3、OKT_8水平明显低于返回平原后居住一年以上者(以下简称返回组)(P<0.01～0.001)但OKT_4/OKT_8无差异。③返回组与世居苏州老年人相比,前者OKT_3、OKT_4、OKT_8及OKT_4。OKT_8增高,其中OKT_3、OKT_4增高明显(P<0.01～0.02),作者认为长期移居高原返回平原后T细胞亚群也可能存在一“脱适应”阶段,即机体重新调整重新平衡的一种形式,这一阶段可能需一年以上。     

维生素E对小鼠胸腺和脾脏T淋巴细胞转化及MΦ诱导IL－1的影响

ＶＥ缺乏组、１／３需要量组、需要量组、３倍需要量组、２０倍需要量组的初断奶小鼠喂养８周后观察Ｔ淋巴细胞转化及Ｍ中诱导ＩＬ－１反应。结果表明，ＶＥ增强半合适、合适剂量ＣｏｎＡ诱导的胸腺、脾脏Ｔ淋巴细胞转化，且半合适剂量诱导的反应高于合适剂量，ＶＥ增强合适剂量ＰＨＡ诱导的脾Ｔ细胞反应，但不影响其诱导胸腺Ｔ细胞反应。ＶＥ还提高ＭΦ诱导ＩＬ－１生成。上述实验以缺乏组反应最弱，３倍量组反应最强，而２０倍量组反有下降趋势。