Adult ; Cerebral Ventricle Neoplasms ; diagnosis ; metabolism ; Diagnosis, Differential ; Glial Fibrillary Acidic Protein ; analysis ; Humans ; Immunohistochemistry ; Lateral Ventricles ; chemistry ; pathology ; Male

OBJECTIVETo evaluate the efficiency of a modified culture method for rat cerebral cortical astrocytes in vitro.

METHODSThe astrocytes derived from the cerebral cortex of 3-day-old Sprague-Dawley rats were first purified as described previously, then the cells were replanted at a low density. The culture flask was changed after 1 hour and substratum was replaced after 24 hours. Cells were syncretized to a monolayer, followed by cell passage. After three passages the cells were cultured in DMEM medium containing 10% fetal serum for a long period. The derivation of the cells was identified by immunofluorescent staining with anti-GFAP polyclonal antibodies.

RESULTSA variety of morphologically distinct astrocytes with many long processes and small cell bodies were obtained. Finally an astrocytic network occurred through cellular process connections. The immunofluorescent staining demonstrated the percentage of GFAP-positive cells was above 98%.

CONCLUSIONSThe modified culture method for astrocytes from rat cerebral tissue is reliable, with a high purity. The cultured astrocytes have a similar morphological development to those in vivo.

Animals ; Astrocytes ; physiology ; Cell Culture Techniques ; Cerebral Cortex ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study influencing factors of detection of bovine central nervous system (CNS) tissue contaminated beef by enzyme immunoassay (EIA), and the method was applied to the detection of imported beef and domestic beef of China.

METHODSRaw beef homogenates containing different concentrations of raw CNS tissue and the same samples which were heated were detected after different time by RIDASCREEN(r) Risk Material 10/5 and RIDASCREEN(r) Probennahme- zubehor Sampling tools kits. PBS suspension and sample dilution buffer (SDB) suspension of bovine brain tissue with the same concentration of the standard were detected. Beef from USA and domestic market of China were then detected by the kits.

RESULTSThe kits could detect both raw and heated CNS tissue in the products with high sensitivity. The absorbance values (AV) increased with the concentrations of CNS in samples. Heating and increasing of time could decrease the absorbance values of the samples which contain CNS tissue. The AV of the PBS suspension of bovine brain tissue was higher than the SDB suspension and the AV of both were higher than the AV of standard of the same concentration. No CNS tissue was detected from all imported beef. No CNS tissue was detected in all samples from domestic market of China except for foxtail.

CONCLUSIONThe EIA method has high sensitivity for detection of bovine CNS tissue contaminated beef with the glial fibrillary acidic protein (GFAP) as accurate target substance. Heating and increasing of time can lead to decreasing of the AV of samples. Improper slaughter process can lead to contamination of bovine products by bovine CNS tissue.

Animals ; Brain ; metabolism ; Brain Chemistry ; Cattle ; Food Contamination ; analysis ; Food Inspection ; methods ; Glial Fibrillary Acidic Protein ; analysis ; Immunoenzyme Techniques ; Meat Products ; analysis

OBJECTIVETo investigate the molecular basis of infantile Alexander disease in a Chinese patient, which may yield useful information for further genetic counseling.

METHODSDNA sequencing analysis and restriction endonuclease analysis were used to detect the mutation of glial fibrillary acidic protein (GFAP) gene in a patient with clinically diagnosed Alexander disease, in her parents and in 50 healthy controls.

RESULTSA 249C>T (R79C) mutation was identified in the exon 1 of the GFAP gene but not in her parents and the controls.

CONCLUSIONThe study on mutation of GFAP gene in Chinese patients with Alexander disease has never been reported previously. The mutation analysis of GFAP gene can provide valuable information for the diagnosis of Alexander disease and can serve as a reliable method of prenatal diagnosis for the family.

Alexander Disease ; diagnosis ; genetics ; Base Sequence ; Child, Preschool ; China ; DNA Mutational Analysis ; Female ; Genetic Predisposition to Disease ; Glial Fibrillary Acidic Protein ; genetics ; Humans ; Mutation ; Polymerase Chain Reaction

Recently the origin of gastrointestinal stromal tumors (GISTs) is thought be the interstitial cells of Cajal or primitive stem cells. This study was performed to evaluate the roles of fine needle aspiration cytology (FNAC), cell block preparation, and immunohistochemistry in the diagnosis of GISTs. Nine cases of GIST in which FNAC was performed were included in this study. Cytologically, the tumor cells characteristically occurred in closely packed cohesive tissue fragments with high cellular density often in bloody background. The tumor cells often formed fascicles with parallel, side-by-side arrangements of the nuclei. Histologically, GISTs were highly cellular spindle or epithelioid tumor with basophilic appearance. Immunohistochemically, GISTs were c-kit positive in all of nine cases, CD34 positive in seven, focally SMA positive in two, and S-100 and GFAP negative in all. Both histologic and cell block sections showed the same histologic and immunohistochemical features. Cytomorphologically GISTs show a broad morphologic spectrum but rarely a significant nuclear pleomorphism and the assessment of malignant potential is difficult based on cytology alone. However, in the appropriate clinical and radiologic setting, a confident diagnosis of primary or metastatic GIST can be established by FNAC, cell block, and immunohistochemistry.
Actins/analysis ; Adult ; Antigens, CD34/analysis ; Biopsy, Needle ; Female ; Gastrointestinal Neoplasms/chemistry/*pathology ; Glial Fibrillary Acidic Protein/analysis ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Paraffin Embedding ; Proto-Oncogene Proteins c-kit/analysis ; S100 Proteins/analysis ; Stromal Cells/*pathology

OBJECTIVEThe purpose of this study was to culture and identify neural stem cells from mouse embryos in vitro using a modified method and provide a basis for further study of the biology of neural stem cells under hypoxia.

METHODSThe cells were isolated mechanically from the front cortex of fetal Institute of Cancer Research (ICR) mice on embryonic day 14. They were passaged by mechanical dissociation and enzymatic digestion. The neurospheres were identified by immunofluorescent staining of nestin. Cell differentiation was induced by 1% fetal bovine serum and then the cells were identified by immunohistochemistry of β-tubulin III and GFAP.

RESULTSThe cells obtained from the front cortex of fetal ICR mice had the capacity of forming neurospheres which showed nestin immunoreactive positivity. After being induced by 1% fetal bovine serum, the cells were differentiated into β-tubulin III-positive cells and GFAP-positive cells.

CONCLUSIONSUsing mechanical dissociation of primary cells and mechanical dissociation with enzymatic digestion of primary cells, the NSCs from the front cortex of mouse embryos can be obtained.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Intermediate Filament Proteins ; analysis ; Mice ; Mice, Inbred ICR ; Nerve Tissue Proteins ; analysis ; Nestin ; Neural Stem Cells ; chemistry ; cytology ; Tubulin ; analysis

BACKGROUNDFunctional electrical stimulation (FES) is known to promote the recovery of motor function in rats with ischemia and to upregulate the expression of growth factors which support brain neurogenesis. In this study, we investigated whether postischemic FES could improve functional outcomes and modulate neurogenesis in the subventricular zone (SVZ) after focal cerebral ischemia.

METHODSAdult male Sprague-Dawley rats with permanent middle cerebral artery occlusion (MCAO) were randomly assigned to the control group, the placebo stimulation group, and the FES group. The rats in each group were further assigned to one of four therapeutic periods (1, 3, 7, or 14 days). FES was delivered 48 hours after the MCAO procedure and divided into two 10-minute sessions on each day of treatment with a 10-minute rest between them. Two intraperitoneal injections of bromodeoxyuridine (BrdU) were given 4 hours apart every day beginning 48 hours after the MCAO. Neurogenesis was evaluated by immunofuorescence staining. Wnt-3 which is strongly implicated in the proliferation and differentiation of neural stem cells (NSCs) was investigated by Western blotting analysis. The data were subjected to one- way analysis of variance (ANOVA), followed by a Tukey/Kramer or Dunnett post hoc test.

RESULTSFES significantly increased the number of BrdU-positive cells and BrdU/glial fibrillary acidic protein double- positive neural progenitor cells in the SVZ on days 7 and 14 of the treatment (P < 0.05). The number of BrdU/doublecortin (DCX) double-positive migrating neuroblast cells in the ipsilateral SVZ on day 14 of the FES treatment group ((522.77 ± 33.32) cells/mm(2)) was significantly increased compared with the control group ((262.58 ± 35.11) cells/mm(2), P < 0.05) and the placebo group ((266.17 ± 47.98) cells/mm(2), P < 0.05). However, only a few BrdU/neuron-specific nuclear protein-positive cells were observed by day 14 of the treatment. At day 7, Wnt-3 was upregulated in the ipsilateral SVZs of the rats receiving FES ((0.44 ± 0.05)%) compared with those of the control group rats ((0.31 ± 0.02)%, P < 0.05) or the placebo group rats ((0.31 ± 0.04)%, P < 0.05). At day 14, the corresponding values were (0.56 ± 0.05)% in the FES group compared with those of the control group rats ((0.50 ± 0.06)%, P < 0.05) or the placebo group rats ((0.48 ± 0.06)%, P < 0.05).

CONCLUSIONFES augments the proliferation, differentiation, and migration of NSCs and thus promotes neurogenesis, which may be related to the improvement of neurological outcomes.

Animals ; Bromodeoxyuridine ; metabolism ; Cell Proliferation ; Cerebral Ventricles ; physiopathology ; Electric Stimulation Therapy ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Neural Stem Cells ; physiology ; Neurogenesis ; Rats ; Rats, Sprague-Dawley ; Stroke ; physiopathology ; therapy ; Wnt3A Protein ; analysis

The expression of two intermediate filaments, nestin and vimentin, was studied in spinal cord injury (SCI) to elucidate their roles in the formation of glial scars. Rats were sacrificed 1, 4, and 7 days after induction of compression injury of the spinal cord using an aneurysm clip. The affected spinal cords were studied using antibodies against nestin and vimentin intermediate filaments. One day after spinal cord injury, some clusters of nestin-positive vessels were detected in the center of the injury, but few were seen in other cell types. Vimentin immunostaining was detected in some glial cells in the center and its level of immunoreactivity was enhanced in the ependymal cells of the central canal. On days 4 and 7 after spinal cord injury, astrocytes and some ependymal cells in the central canal were stained positively for nestin and increased expression of nestin was observed in vessels. Vimentin was detected in some macrophages and astrocytes in the lesions. Nestin was co-localized with glial fibrillary acidic protein in some glial cells in SCI. These findings imply that spinal cord cells in adult animals have embryonic capacity, and these cells are activated after injury, which in turn contributes to repair of spinal cord injury through formation of a glial scar.
Animals ; Cicatrix/pathology ; Glial Fibrillary Acidic Protein/analysis ; Immunohistochemistry ; Intermediate Filament Proteins/analysis ; Intermediate Filaments/*physiology ; *Nerve Tissue Proteins ; Neuroglia/*pathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries/*pathology ; Vimentin/analysis

OBJECTIVETo study the effects of miR-124-1 on neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs).

METHODSMSCs cells were assigned into three groups: control (uninfected and untransfected), miR-124-1+ (infected with miR-124-1), and miR-124-1- (transfected with Anti-rno-miR-124* Inhibitor). MSCs were induced by β-mercaptoethanol (β-ME) to differentiate into neurons. The fluorescence expressed by infected MSCs was observed under an inverted fluorescence microscope. MTT method was used to measure cell survival rate after transfection or infection. Immunocytochemistry, RT-PCR and Western blot methods were used to detect the expression of β3 tubulin, MAP-2 and GFAP 6 days after β-ME induction.

RESULTSThe expression of miR-124-1 in the miR-124-1+ group was significantly higher 2 days after infection of lentivirus vector compared with the control group (P<0.01). In the miR-124-1- group, the cell survival rate and the miR-124-1 expression level decreased significantly 24 hrs after transfection of anti-rno-miR-124* inhibitor (P<0.01). After 6 days of β-ME induction, the protein and mRNA expression levels of β3 tubulin and MAP-2 in the miR-124-1+ group were much higher than the other two groups (P<0.01); while the expression levels of β3 tubulin and MAP-2 in the miR-124-1-group were lower than the control group (P<0.01). The expression of GFAP in the three groups was weak (<1%).

CONCLUSIONSmiR-124 might promote neuronal differentiation of rat MSCs.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; physiology ; Microtubule-Associated Proteins ; analysis ; Neurons ; cytology ; Rats ; Rats, Wistar ; Tubulin ; analysis

OBJECTIVETo promote stem cells differentiation into neurons and enhance neuromotor function after brain injury through brain-derived neurotrophic factor (BDNF) induction.

METHODSRecombinant 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.

RESULTSThe 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.

CONCLUSIONBDNF gene can promote the differentiation of the stem cells into neurons rather than glial cells, and enhance neuromotor function after brain injury.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Brain Injuries ; physiopathology ; therapy ; Brain-Derived Neurotrophic Factor ; analysis ; genetics ; Cell Differentiation ; Glial Fibrillary Acidic Protein ; Humans ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Nerve Tissue Proteins ; analysis ; Neurons ; cytology ; Phosphopyruvate Hydratase ; analysis ; Transfection