Objective To isolate, culture and identify human neural stem cells from 7-week to 13-week embryonic brains and to study different suitable culture conditions.Methods By using serum-free cell culture and single cell clone test,the neural stem cells were isolated and cultured from 7-week human embryonic forebrains and 13-week human embryonic cerebral cortex in different conditions and immunofluorescence tests were used to identify the cells. Results The human neural stem cells having the abilities of self-renewal and multipotency were successfully isolated and cultured from different embryonic brains. In 7-week brain, the cells could form clones in different ways except in high concentration suspension culture.In 13-week brain, the cells could form clones only in middle concentration suspension culture and high concentration adhere-wall culture. In coated petri dish, the cells couldn't form clones.Conclusions There are neural stem cells in human embryonic brains. With gestational age increasing, the neural stem cells become more and more difficult to be isolated. High concentration adhere-wall culture can be used in embryonic brains with different gestation age.

ObjectiveTo investigate the culture conditions of skin-derived precursors (SKPs) and to explore a new cell source for central nervous system cell transplantation.MethodCells from skins of juvenile and adult mice were isolated and cultured in serum -free medium, and mechanical methods were adapted to passage these cells and ce lls were identified by immunocytochemistry.ResultsA population of SKPs could be isolated from adult and neonatal skins. They co uld be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 8 pas sages, indicating their proliferative capacity. About 50?% of SKPs expressed n estin and the majorities of these cells expressed fibronectin when they were pla ted on polyornithine and laminin coated plates. About 5?% cells showed typical complicated neuronal states and expressed NF-M and NSE when SKPs were plated i n serum-containing medium. These cell could also differentiate into adipocytes and fibroblast-like cells.ConclusionsAdult skin contains stem cells capable of differentiating into neurons, adipocyt es and fibroblast-like cells. SKPs may represent an alternative autologous stem cell source for CNS cell transplantation.

Objective To summarize the clinical features and transsphenoidal microsurgical experiences of pituitary adenomas in the elderly patients over 70 years old Methods Twenty four elderly patients accepted transsphenoidal microsurgery were conductded a retrospective review,whose clinical data including clinical main presentation,histological types,surgical conditions,perioperative treatment and outcome were also analyzed Results There were 16 non functional adenomas (66 7%) and 18 giant macroadenomas(75%) respectively Visual deterioration was the commonest complain in 20 (83 3%) Despite the majority of patients having coexisting medical conditions,we had no mortality and no postoperative adjunctive morbidity with an average hospital stay of 17 8 days For an average follow up of 3 5 years,we did observe major patients(79 2%) vision had improved Conclusions Pituitary adenomas in the elderly are most frequently non functional macroadenomas that present with visual deterioration and hypopituitarism If special care is devoted to the perioperative treatment,in particular to fluid and electrolyte balances,transsphenoidal microsurgery is safe and effective in hands of an experienced team

BACKGROUND: Spinal cord injury occurs frequently and its consequence is very severe. There is no effective method to rebuild the function of demylinated nerves. Transplantation of a kind of special glial cells in olfactory system of mammal attracts more attention.OBJECTIVE: To observe whether combined transplantation of human fetal olfactory ensheathing cells (human OECs) and rat embryonic spinal cord tissues (rat ECS) possesses synergistic effect in promoting axonal regeneration in the rats following spinal cord transection.transection.DESIGN: Open experiment.SETTING: Cell Room, Third People's Hospital of Wuxi City; Department of Neurology, First Hospital Affiliated to Soochow University; Department of Neurosurgery, Zhongda Hospital Affiliated to Central South University MATERIALS: This experiment was carried out in the cell laboratory of the Third People's Hospital of Wuxi from September 2002 to October 2004. ① Totally 36 adult female SD rats, of clean grade, were selected and randomly divided into 4 groups: human OECs group (n=10), rat ECS group (n=10), combined transplantation group (n=10) and sham-operation group (n=6). ② Fresh 12-week aborted human embryo was used for culture and purification of human OECs (Informed consent was obtained from the parturient). ③One SD rat at embryonic 14 days underwent caesarean operation, and fetal rat and fetal membrane were taken out together and used for preparing new embryonic spinal cord.METHODS: ①Rats of 4 groups were all created into hemisection cavity models. Gelatin sponge and complete culture medium of 8 μL were packed into the injured cavity of rats in the model group, and the same culture medium of 2 μL was injected at 1 mm above or below injure; Human OECs suspension of 8 μL was added to gelatin sponge in human fetal Human OECs group, and human OECs suspension of 2 μL was injected at 1 mm above and below injure; rat embryonic spinal cord tissue of rat ECS group was chipped into pieces, which were packed into the cavity,and gelatin sponge was spread on the injury part. Embryonic spinal cord with the same size was packed into the cavity of combined transplantation group, then 8 μL human OECs suspension was injected into cavity with micro sample injector, and gelatin sponge was spread on the injury part, and then cellular suspension of 2 μL was injected at 1 mm above and below the cavity, and muscular layer skin was sutured layer by layer. ②The rats of each group were performed ethological evaluation periodically. Combined with pathological observation, effect of human OECS and rat ECS on neuronal survival and regeneration was evaluated by performing horseradish peroxidase-tetramethyl benzidine tracer technique.MAIN OUTCOME MEASURES: ①In vitro culture and purification of human fetal human OECs. ② In vitro immunocytochemistrical analysis. ③BBB scoring of motor function of hindlimb of rats. ④ Immunohistochemical detection of implants and injured spinal cord repair⑤ Quantitative analysis on labeled neurons at the cortex and mesencephalic red nucleus ineach group with horseradish peroxidase-tetramethyl benzidine tracer technique.RESULTS: ① Most of human fetal OSCs presented double-polar spindle.Five to seven days after culture, OSCs weaved into net and a lot of mitosis phases were found. The cellular purity was 85%. ② The rate of P75 positive cells was (83±7)%. Glial fibrillary acidic protein was found in about (81±6)% of cells and Vimentin in (91±9)% of cells and the rate of Nestin positive was (77±5)%. ③Three to five days after operation, affected limb of rats of sham-operation group began to contract, the activity of hindlimb of intact side was limited a little. Fewer obvious contraction symptoms were found in the other 3 groups. From 2 weeks after operation,behavioral function recovered significantly fast in each group. BBB scores of combined transplanted group were significantly high than those of human OECs group, rat ECS group and sham-operation group [(6.2±1.13) vs.(5.0±1.15)vs.(3.9±0.88)vs.(3.3±1.03)scores,P ＜ 0.05]. ④In bipolar or multipolar cells, in which basic protein(+)granules were found, P75 and glial fibrillary acidic protein positive were found at the implanted part in the range of 2.0 to 5.0 mm of transplanted region in the human OECs group and combined transplantation group. A great many of small MAP2 positive neurons were found in the spinal defected focus in the rat ECS group and combined transplantation group. Nerve plexus positive fibers were observed in spinal defected region of human OECs group, rat ECS group and combined transplantation group to different extents, especially significantly in the combined transplantation group, but they were not found in the model group. ⑤ Horse radish peroxidase labeling was hardly found in neurons at the injured side of sham-operation group, while the number of labeled neurons at the cortex and nesencephalic red nucleus was significantly higher in the combined transplantation group than in the human OECs group and rat ECS group (P ＜ 0.05).CONCLUSION: Combined transplantation of OECs and ESC can obviously protect injured spinal cord, promote host spinal axonal regeneration and play s a complementary and synergetic effect in speeding up the functional recovery of rats.

Objective To study the clinical significance of the injury and functional change of hypothalamic-pituitary-adrenal (HPA) axis after acute severe traumatic brain injury (TBI) in the rats.Methods A total of 60 adult healthy male Spraque-Dawley rats were randomly (random number) divided into 3 groups (n =20 in each group):sham operation group,model group and treatment group.The TBI models of rats were established by Feeney' s method.A low dose of dexamethasone (0.6 mg/kg) was injected into the abdominal cavity 20 minutes,24 hours and 48 hours after injury in treatment group,while rats of sham operation group and model group received equal volume of normal saline instead.All the rats were injected 1 μg adrenocorticotropic hormone (ACTH) into the abdominal cavity.The related parameters were detected at four time points,3 hours,12 hours,24 hours and 72 hours after cerebral contusion.The plasma corticosterone (CORT) and ACTH levels were measured by chemiluminescence.The hypothalamic,pituitary and adrenal of the rats were taken out for observing interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) expression detecting by immunohistochemical techniques at 72nd hour after TBI.One-way ANOVA and SNK-q test were used to analyze the results with SPSS 17.0 software package.Results The levels of ACTH and CORT on 3rd hour of model group raised remarkably compared with that of sham operation group,then they reduced gradually.The levels of CORT were lower than that of sham operation group at every time points after ACTH stimulation test (P ＜0.05 or P ＜0.01).The levels of CORT at all time points of treatment group were changed remarkably compared with that of model group.However,the ACTH levels of treatment group on 24 h increased slightly than that of model group.And the tendency of them was similar to model group (P ＜ 0.05 or P ＜ 0.01).The number of the hypothalamus and pituitary cells which express IL-6 and TNF-α in model group was more significantly increased when compared with that in sham operation group (P ＜ 0.01),while the number of this kind of cell in treatment group was significantly decreased than that in model group (P ＜ 0.01).The number of the adrenal cortex cells which express IL-6 in treatment group was more significantly decreased when compared with that in model group (P＜ 0.01),while the number of this kind of cell in model group was significantly increased than that in sham operation group (P ＜ 0.01).However,there was no significant difference of the TNF-α between all the groups (P ＞ 0.05).Conclusions Functional change of adrenal occurs early in the severe acute traumatic brain injury rats,and the response of adrenal to ACTH decreased as time goes by.Low-dose,short-course dexamethasone can delay the pathological changes,reduce the inflammatory response of HPA axis and increase the sensitivity of adrenal response to ACTH.

Objective To locally inject human umbilical cord blood (HUCB) mesenchymal stem cells (MSCs) to rat traumatic brain injury (TBI) model to investigate expression of neural markers and neurological functional improvement. Methods HUCB-MSCs were labeled by bis-benzimide for over 24 hours and stereotactically transplanted into the brain of the rats. All rats were divided into four groups, ie, sham injury group, TBI group, control (TBI + PBS) group and treatment (TBI + MSCs) group, Im-munohistochemical methods and immanofluorescence staining were used to observe the survival, migration and differentiation of the transplanted cells. The neurological functional improvement was evaluated by u-sing the neurological severity score (NSS). Results There existed a large number of MSCs survived in local region of the brain that received transplants, when some MSCs differentiated into neurons or astro-cytes and expressed the neurocyte markers including NSE and GFAP around the grafted site. Treatment group had significantly improved scores compared with sham injury group, TBI group and control group. Conclusions HUCB-MSCs transplantation can potentially improve neurological functional after TBI and may be a good alternative to bone marrow cells for stem cell transplantation or cell therapy.

Objective To observe the correlation between the changes of neural cell apoptosis arid caspase-3 gene expression in brain tissues following acute severe traumatic injury to brain(TIB).Method A total of 120 adult Spraque-Dawley rats were divided into a control group(n=8)，TIB group(n=56)and TIB with administration of caspase-3 inhibitor group(n=56).TIB models of rats were made with Feeney's method.The z-DEVDfmk(5 μg)，caspase-3 inhibitor，was administered by intracerebral infusion，and the rats were sacrificed 1，6，24，48 hours and 3，7，14 days postinjury(n=8 for each interval).The specimens of the injured cerebral cortex，suhcerticai white matter，hippocampus，dentate gyrus and contrahteral corresponding brain tissues were taken for detecting apoptesis of neural cells by the terminal deoxynucleotidyl transferase mediated DUTP nick end labeling (TUNEL)methods and flow cytomeay.Caspase-3 mRNA and protein expression were detected by using RT-PCR，immunohistochemistry and western blot analysis.The caspase-3 activity was detected by using caspase-3 fluorescent assay kit.Student t-test and Spearman correlation analysis were used to analyze the data with SPSS version 10.1 software package.Results Apoptesis indexes(AI)and the apoptesis percentage(AP)of neural cells in the injured brain regions increased quickly after injury，and reached its peak 24 to 48 hours later，then decreased slowly,but it remained at higher level above that of normal till 14 days later(P＜0.01).The levels of caspase-3 mRNA,eastme-3 protein and caspase-3 activity were increased significantly post injury，and reached its peak at 24 to 48 hours，then it gradually decreased.Compared with control group，the levels ofoptical density of caspase-3 proteins in the injured hippocampus and subcortical white matter at 24 and 48 hours post injury increased 1484% and 1690%，caspase-3 mRNA expressiom increased 1043%and 1180%，and the degreas of caspase-3 activity increased 148% and 183%，respectively.The expression of caspase-3 proenzyme and its P17 subarrit increased.After trealment with caspase-3 inhibitor z-DEVD-fmk，the levels of caspase-3 mRNA，protein expression and caspase-3 activity were significantly decreased.and AI and AP were significantly decreased as well.The correlation between caspase-3 mRNA and level of neural apoptesis was positive(r=0.821，P＜0.01)，and it was likewise between caspase-3 protein and level of neural apoptosis(r=0.638.P＜0.01).Interestingly enough，a positive correlation was found between caspase-3 mRNA and easpase-3 proteins(r=0.945，P＜0.01).Conclusions The activation of caspase-3 leads to apoptosis of neural cells after acute TIB.The expression of caspase-3 are consistent with apoptosis of neural cells following TIB.The regulation of caspase-3 induced by TIB occurs at a ceriain critical link before transduction.Caspase-3 inhibitor can efficiently inhibit apoptosis of neural cells following TIB.

Objective To investigate curative effects of hypothermia on the secondary axotomy of nondisruptive axonal injury (NDAI) after diffuse brain injury (DBI).Methods A total of 16 male Sprague-Dawley rats were randomly and equally divided into hypothermia group (at 32℃ for 6 hours) and control group (at 37.5℃ ).The axonal swelling and axonal balls were detected by means of NF68kD immunochemistry after DBI caused by fluid percussion.The changes of maximal density of axonal swelling and axonal balls in callosum,diencephalon-mesencephalon,pons-oblongata and cerebellum were compared 24 and 72 hours after injury between both groups.Results NF68kD immunochemistry well showed axonal swellings and axonal balls in whole brain.The axonal swelling and axonal balls were significantly decreased 24 hours after DBI in both groups (P<0.05),especially in diencephalon-mesencephalon ,pons-oblongata and cerebellum (P<0.01).While there showed significant decrease of axonal swellings and axonal balls in pons-oblongata and cerebellum in hypothermia group 72 hours after DBI (P<0.05,P<0.01) but insignificant changes in the callosum and the diencephalon-mesencephalon compared with control group (P>0.05 ).Conclusions Hypothermia can retard the progress of mild or severe NDAI at early stage,which would taper with the longer time after injury except for partial mild NDAI.Hypothermia may prevent mild NDAI from secondary axotomy.

Objective To investigate the effect and mechanism of human amniotic epithelial cells (hAECs) transplantation in treatment of cerebral hemorrhage in rabbits.Methods Thirty New Zealand white rabbits were used to induce cerebral hemorrhage.Animals were divided into hAECs group and isotonic saline group according to random number table,with 15 rabbits per group.Before transplanted to rabbits,hAECs were transfected with the retrovirus carrying green fluorescent protein (GFP).Morphologic and behavioral changes in both groups were noted periodically.Survival of transplanted hAECs and expressions of glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP-2)in focal cerebral tissues were observed.Results In hAECs group,the rabbits obtained progressive recovery in walking,supporting and coordinated motion.Restoration period mostly ranged from 2-3 weeks.Most of the rabbits in hAECs group had limb motor function recovered to grade Ⅱ-Ⅲ,while the recovery is slow in isotonic saline group with most ranging from grade Ⅰ-Ⅱ.According to Tarlov score,limb motor function presented significant difference between hAECs group and isotonic saline group (P ＜ 0.05).Immunohistochemical staining showed positive expressions of GFAP and MAP-2 in hAECs group,but no expressions in isotonic saline group (P ＜ 0.05).Conclusion hAECs transplantation effectively improves neural behavior and reduces nerve function impairment in treatment of cerebral hemorrhage in rabbits.