Objective To explore the effect of chondroitin sulfate enzyme ABC (chABC) on glial scar in rat models of brain traumatic injury (TBI). Methods Thirty-eight Wistar rats were randomly divided into 5 groups, including normal control group (n=2), model group (rat models of TBI,n=9), 1.0 U/mL chABC treatment group (n=9), 2.5 U/ml chABC treatment group (n=9) and 5.0 U/ml chABC treatment group (n=9). After performing TBI by free falling in the later 4 groups, rats of the model group were given no treatment, while those of the other 3 groups were administrated with different concentrations of chABC by local injection respectively. One, 2 and 4 w after TBI, HE staining was performed on the brain tissues of these rat models;and immunohistochemical assay and Western blotting were employed to evaluate the secreting of chondroitin sulfate proteoglycans (CSPGs) and the therapeutic effect of chABC on glial scar. Data were statistically analyzed using t-test. Results Pathological test revealed the scars in the treatment groups were significantly fewer than those in the model group 2 w after TBI, with 5.0 U/mL chABC treatment group enjoying the fewest level (P<0.05). Immunohistochemical assay showed that the secreting of CSPGs in the treatment groups and model group was significantly increased than that in normal control group 2 w after TBI (P<0.05);the 5.0 U/ml chABC treatment group showed an obvious reduction of CSPGs secreting as compared with the model group (P<0.05). Western blotting indicated that the treatment groups showed an obvious reduction of CSPGs secreting as compared with the model group 1, 2 and 4 w after TBI (P<0.05);an obvious gradual reduction of CSPGs secreting in the model group, 2.5 and 5.0 U/ml chABC treatment groups was noted 1, 2 and 4 w after TBI (P<0.05). Conclusion ChABC could degrade the glial scar by degrading the CSPGs molecules and improve the microenvironment of local axonal regeneration after TBI;In this experiment, the highest concentration of chABC (5U/ml) shows the best effect on removing the glial scar.

Objective To explore the feasibility of recently developed nucleofection method in delivering plamid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into primary bone marrow stromal cells (BMSCs) of rabbit. Methods Rabbit BMSCs were harvested by means of density gradient centrifugation following a thighbone puncture. The primary BMSCs were cultured and either transfected with pEGFP-C2 by nucleofector technology (as EGFP group) or uninfected (as control) in vitro. Compared with the control, the cellular viability, adhesive rates and the growth curves of the labeled cells were respectively analyzed. Transfection efficiencies were evaluated through the detection of EGFP expression. Results EGFP were successfully expressed 24 h after nucleofection. Similar morphological development, adhesive rates and growth curves were found in the 2 groups. The positive EGFP expression was enhanced gradually alone with the prolonged culture time, and showed the strongest 6 d after marked, with about 47.8% of EGFP-positive cells in the total BMSCs. The EGFP did not attenuate even 1 month after the marking. Conclusion Neuclofection of pEGFP-C2 shows no significant effect on the proliferation of rabbit BMSCs. EGFP plays an important role in stable gene marking of rabbit BMSCs. Nucleofection is an efficient nonviral gene transfer method for the introduction of genes into primary rabbit BMSCs.

Objective To explore the effect of edaravone (ED) on the neurological functionaldeficits, apoptosis and expression of caspase-3 protein following focal cerebral ischemia/reperfusion(I/R)injury in rats. Methods A total of 24 male Sprague-Dawley (SD) rats were randomly allocated intothe sham-operation group, cerebral I/R group, normal saline treatment group and ED treatment group, 6rats in each group. Rat models with focal cerebral I/R injury induced by middle cerebral artery occlusion(MCAO) were established using a modified suture method. ED (3mg/kg) or equal volume of normalsaline was injected intraperitoneally immediately after cerebral ischemia and 12 h after reperfusion in thetreatment groups;the rats in sham-operation group underwent the same modeling procedure withoutischemia by nylon suture. The neurological behavioral deficits were evaluated 24 h after I/R injury;,immunohistochemical staining and Western blot assay were applied to detect the change in the expressionof caspase-3 protein; in situ TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay was used tostudy the change in neuronal apoptosis. Results The scores of neurological behavioral deficit scale,the positive cells and expression of caspase-3 protein, and the apoptotic cells in the ED treatment groupwere significantly decreased, compared with that of the I/R group and normal saline treatment group(P<0.05 for each comparison). Conclusion ED may effectively reduce neuronal apuptosis andneurological functional deficits after cerebral I/R injury, which might be related with the inhibition of thecaspase-3 protein expression.

Objective To offered some prophase works by preparing Neurocan protein, antiserum, and assaying their characteristics, in order to construct the isopropy-β-D-thiogalactoside (IPTG)-participated DNA vaccine, which can neutralize the inhibitors in the injured CNS following the immune administration and then promote the nerve regeneration. Methods Neurocan gene was syntbetized with HisTag label in beginning and enzyme-cut sites at amphi- of the sequence. The prokaryotic expression plasmid, PET30a-Neurocan, was constructed as usual, converted into the Escherichia coli, and induced by IPTG to express positively. The interest protein was identified by SDS-PAGE and Western blot respectively. The preimmune serum was as the negative control during the ELISA assay of antiserum valency. The immune serum was as the first antibody, and the goat-anti-rabbit labeling with alkaline phosphatase (AP) was employed as the second one. Coloration was with NBT/BCIP method. Results The correct sequence of the synthetic Neurocan gene was clearly showed by identification with enzyme-cut, PCR and sequencing. The Neurocan protein expressed by prokaryotic showed its molecular weigh as 55 000 following the SDS-PAGE identification, and it could specifically bind with anti-HisTag, which implied the interesting protein just as the expression product of Neurocan gene. The valency of antiserum was shown by ELISA as 1:1 000 000, the purpose strap of which was confirmed by Western blot. Conclusions Neurocan protein could be successfully expressed in prokaryotic, the antibody of which could be specifically obtained by immune administration to the rabbit. The Neuroncan antibody could bind with the Neurocan protein specifically.

Objective To analyze the gene expression profiles in relation to the migration ability of adult human bone marrow-derived neural stem cells (Md-NSCs), and identify the genetic basis of the high migration potential of Md-NSCs in the central nervous system (CNS). Methods Adult human bone marrow stromal celIs(BMSCs) obtained from adult healthy volunteers were induced to differentiate into Md-NSCs in vitro, and the expressions of the genes related to cell invasiveness and metastasis were investigated by microarray analysis. Quantitative real-time PCR (RT-PCR) was used to verify the microarray results. Results The results of microarray analysis revealed significant overexpressions of the genes MMP1, MMP2, MMP17, ITGA3, RhoB, RhoC and RhoD in the Md-NSCs as compared with the expressions in fresh normal human adult bone marrow cells depleted of red blood cells. Quantitative RT-PCR verified the overexpression ofMMP2 gene by 2.84×100 folds, ITGA3 gene by 2.22×102folds, and RhoC gene by 4.92×100 folds. Conclusion The high migration potential of the Md-NSCs in the CNS is probably associated with the overexpression of the genes that promote cell invasiveness and metastasis. These overexpressed genes are also important oncogenes, and therefore the tumorigenicity of the Md-NSCs warrants further investigation.

OBJECTIVETo investigate the effect of N-acetyl-cysteine (NAC) and depakine (DP) on the changes of membrane potential and peroxidate in rat cortex neurons exposed to ferrous chloride (FeCl(2)).

METHODSCultured cortex neurons of newly born SD rats were randomly divided into control group (PBS group), model group (FeCl(2) group), NAC pretreatment group (NAC group), DP pretreatment group (DP group) and NAC+DP pretreatment group (NAC+DP group). In the latter three groups, NAC (0.08 mg/ml) and DP (0.1 mg/ml) were added in the cell culture 2 and 3 h before FeCl(2) (1 mmol/L) exposure, respectively. After exposure to FeCl(2), the membrane potential of the neurons was detected with fluorescent dye DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol), and the peroxidate level with 2,7-dichlorofluorescin diacetate (H(2)DCF) by laser confocal scanning microscope (LCSM) and nuclear factor-KappaB (NF-KappaB) level with immunocytochemistry.

RESULTSCompared with FeCl(2) group, the expression of NF-KappaB and peroxidate level in the neurons were decreased significantly in NAC and NAC+DP groups (P<0.01), but not in DP group (P>0.05). FeCl(2) depolarized the membrane potential and increased the expression of NF-KappaB in the neurons. Compared with FeCl(2) group, significant changes in the membrane potential were observed in DP and NAC+DP groups (P<0.01) but not in NAC or PBS group (P>0.05).

CONCLUSIONBoth NAC and DP can protect the neurons from FeCl(2)-induced damage but through different pathways, and their combined use can significantly alleviate neuronal damages due to FeCl(2) exposure. Antioxidants such as NAC in combination with antiepileptic drugs may produce favorable effect in prevention and treatment of posttraumatic epilepsy.

Acetylcysteine ; pharmacology ; Animals ; Animals, Newborn ; Cells, Cultured ; Cerebral Cortex ; cytology ; metabolism ; physiopathology ; Female ; Ferrous Compounds ; pharmacology ; Male ; Membrane Potentials ; drug effects ; Neurons ; cytology ; metabolism ; physiology ; Neuroprotective Agents ; pharmacology ; Peroxides ; metabolism ; Rats ; Rats, Sprague-Dawley ; Valproic Acid ; pharmacology

OBJECTIVETo study the effect of superparamgnetic iron oxides (ferumoxides) on the survival and proliferation of neural stem cells (NSCs) and determine the optimal ferumoxides concentration for labeling.

METHODSBone marrow stromal cells (BMSCs) were obtained from rat femoral marrow and cultured in vitro to induce their differentiation into NSCs. Ferumoxides labeling of the NSCs was performed with different final concentrations of ferumoxides, and the labeling efficiency and viability of the labeled NSCs were evaluated by Prussian blue staining, MTT assay, flow cytometry and transmission electron microscope.

RESULTSThe NSCs could be effectively labeled with ferumoxides with a labeling efficiency of around 90%. Prussian blue staining showed numerous fine granules with blue staining in the cytoplasm of the labeled NSCs, and the intensity of the blue staining was in positive correlation with the ferumoxide concentration for labeling. Transmission electron microscopy of the labeled NSCs revealed the presence of numerous vesicles spreading in the cytoplasm and filled with electron-dense magnetic iron particles. The ferumoxides vesicles increased with the labeling concentration of ferumoxides, and at the final concentration exceeding 25 microg/ml, ferumoxides vesicles in the NSCs gave rise to conglomeration which hampered observation of the cellular ultrastructure by transmission electron microscope. The results of flow cytometry and MTT assay demonstrated that the cell viability, proliferation, differentiation and apoptosis of the labeled cells were affected by ferumoxides at the concentration above 25 microg/ml, but such effects could be minimal at lower concentrations.

CONCLUSIONFerumoxides might be feasible for in vitro labeling of the NSCs with the optimal concentration of 25 microg/ml.

Animals ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dextrans ; Ferrosoferric Oxide ; Iron ; pharmacology ; Magnetite Nanoparticles ; Male ; Microscopy, Electron, Transmission ; Neurons ; cytology ; ultrastructure ; Oxides ; pharmacology ; Rats ; Stem Cells ; cytology ; ultrastructure

OBJECTIVETo express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).

METHODSThe 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.

RESULTSThe prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.

CONCLUSIONThe fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

Animals ; Antibodies ; immunology ; isolation & purification ; Antibody Specificity ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; immunology ; Membrane Proteins ; biosynthesis ; genetics ; immunology ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

[Objective]To explore the distribution and expression changes of tight junctional protein JAM-1 in rat models after intracerebral hemorrhage (ICH) and their significance.[Methods]One hundred and twenty-eighty healthy male SD rats were randomly divided into normal control group (n=16) and ICH group (n=112),and the ICH models were induced by stereotactically injecting 75 uL autologous blood into the right caudate nucleus.Seven time points after ICH (6,12,24 and 48 h,and 3,7 and 14 d after ICH,16 rats for each time point) were chosen.BBB permeability was evaluated by Evans blue dye extravasation.The distribution and expression of JAM-1 were detected by immunofluorescence and real-time quantitative PCR.[Results] As compared with that in the normal control group,BBB permeability in the ICH group significantly increased at 24 and 48 h,and 3 and 7 d after ICH (P＜0.05).JAM-1 expression decreased at blood vessels at 12,24 and 48 h after ICH,and JAM-1 expressed at the circulatingleukocytes3 dafterlCH,and abundant JAM-1 positive cells around hematoma were noted in the ED-l-positve macrophages 7 d after ICH.JAM-I mRNA significantly decreased at 12,24 and 48 h after ICH,and significantly increased 7 d after ICH as compared with that in the normal control group (P＜0.05).[Conclusion] JAM-1 experssion changes not only participate in regulation of BBB permeability but also play roles in inflammatory insult after ICH.

Objective To investigate the proliferative differences of adipose-derived stem cells (ADSCs) from neonatal suckling SD rats (5-d-old) and adult ones under the same culture condition.Methods ADSCs were isolated from the subcutaneous adipose tissues of neonatal suckling SD rats and adult ones,and then,type Ⅰ collagenase digestion was employed to obtain the ADSCs; the morphology of these cells was detected.The expressions of such cell surface markers as CD45,CD29 and CD90 were observed. The number of ADSCs on the 4th d of culture under the same condition and with the same planted density was compared between the neonate and adult rats. In vitro culture of the second generation of ADSCs was performed in the 96-well plates, and CCK-8 and alamar blue kit were employed to compare and quantitate the proliferative differences; optical density was observed by microplate reader. Results The ADSCs from neonatal SD rats and adult ones expressed the stem cell biomarkers: the expression of CD45 was negative, and that of CD29 was 98.04％ and 93.17％,respectively,and that of CD90 was 94.92％ and 93.3％,respectively,for neonate SD rat and adult ones.The cell counting results indicated that the number of ADSCs from neonatal rats ([8.87±0.13]×105 cells) was larger than that of adult ones ([4.51±0.36]×105 cells) after being cultured under the same condition and at the same planted density. The optical density value of ADSCs in neonatal rats was significantly higher than that in adult ones on the 6th and 7th d of culturing detected by CCK-8 kit and on the 2nd-7th d of culturing by alamar blue assay. Conclusion The proliferative ability of ADSCs from neonatal rats is greater than that of adult ones.