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Objective To explore the embolization effect of new platinum coils coated with [4COOH-P (DLLA-co-TMC)] biodegradable polymer and released vascular endothelial growth factor (VEGF) into intracranial aneurysms on rat intracranial aneurysms. Methods A total of 54 adult healthy female SD rats were randomly divided into Group Ⅰ with general platinum coils, Group Ⅱ with polymer-coated platinum coils and Group Ⅲ with platinum coils modified with VEGF (n=18).The right common carotid arteries (CCA) of rats in each group were exposed; and the 8 mm lengths of platinum coil segments were inserted into the ligated right CCA of rats. The distal right CCA was performed ligation and restored the blood flow; 6 rats each time at 15,30 and 90 d after the surgery were chosen;and the distal right CCA was used as aneurysm models,and the left CCA without the coil placement or surgical disruption in Group I with general platinum coil was chosen as normal control.The proliferation and fibrosis of endothelial cells were observed by HE staining; von Willebrand Factor (vWF) expression was detected by immunohistochemical staining; and VEGF expression was examined by Western blotting. Results Cellular proliferation and fibrosis in Group Ⅲ with platinum coils modified with VEGF enjoyed significantly higher grade than those in Group Ⅰ with general platinum coils 10,60 and 90d after the surgery (P＜0.05); Cellular proliferation and fibrosis in Group Ⅲ with platinum coils modified with VEGF enjoyed significantly higher grades than those in Group Ⅱ with polymer-coated platinum coils 30 d after the surgery (P＜0.05).Pathological observations showed that the massive intimal hyperplasia and substantial clot completely occluded the aneurysm lumen in Group Ⅲ with platinum coils modified with VEGF; New small blood vessels having vwf-positive expression were noted in the fiberized tissues;the thrombosis in Group Ⅰ with general platinum coils and Group Ⅱ with polymer-coat platinum coils were not fully organized and showed loose hyperplasia structure with a large number of internal spaces.Western blotting indicated that the VEGF level in Group Ⅲ with platinum coils modified with VEGF were significantly higher than that in other groups 15 and 30 d after the operation,however,the VEGF level in Group Ⅲ with platinum coils modified with VEGF 90 d after the surgery was decreased because the lumen completed fibration and degradation of 4COOH-P (DLLA-co-TMC). Conclusion The VEGF-eontaining biodegradable polymer,by slowly releasing VEGF to modify the surface of platinum coils, could enhance the cellular proliferation, thrombosis and formation of dense fibrous tissue in aneurysm lumen; as compared with general platinum coils,these new platinum coils could occlude the rat aneurysm faster and more completely.
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Objective To explore the effects of Levamlodipine on viability,proliferation and apoptosis of neural stem cells after hypoxia-ischemia injury in adult rats. Methods Hypoxic-ischemic damage to adult neural stem cells was simulated in the established oxygen/glucose deprivation (OGD)models.Four groups were designed according to Levamlodipine concentrations:0 μmol/L,0.5 μmol/L,1.0 μ mol/L and 5.0 μmol/L. Effects of Levamlodipine at 4 different concentrations on the viability,proliferation and apoptosis of hippocampal neural stem cells after OGD were tested by CCK-8 assay,Edu fluorescence staining and flow cytometry (Annexin V-FITC/PI),respectively. Results Models of hypoxia-ischemia damage to hippocampus neural stem cells were successfully established by OGD for 6hours.Compared with control group (0 μmol/L),the viability of hippocampal neural stem cells was significantly increased in the other 3 groups (0.5 μmol/L,1.0 μmol/L and 5.0 μmol/L) (P＜0.05).The proportion of proliferating cells after OGD was significantly increased at S phase in 5.0 μmol/LLevamlodipine group (P＜0.05).The proportion of apoptotic cells after OGD was significantly decreased in 1.0 μ,mol/L and 5.0μ mol/L Levamlodipine groups (P＜0.05). Conclusion Levamlodipine may protect neural stem cells from hypoxic-ischemic injury in adult rats.
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Objective To observe the expression of growth factors (vascular endothelial growth factor [VEGF],stromal cell-derived factor-1 [SDF-1 ],basic fibroblast growth factor [bFGF],insulin-like growth factor [IGF-1],transforming growth factor-β [TGF-β],platelet-derived growth factor [PDGF],brain derived neurotrophic factor [BDNF],glial cell line-derived neurotrophic factor [GDNF] and nerve growth factor [NGF]) in rat ischemic brain tissues after intravenous implantation of bone marrow stromal cells (BMSCs) and/or endothelial progenitor cells (EPCs). Methods Healthy adult Wistar rats were randomly divided into 4 groups:vehicle group,BMSCs transplantation group,EPCs transplantation group and BMSCs combined with EPCs transplantation group (n=20). The rats were subjected to middle cerebral artery occlusion (MCAO),and 24 h after that,they were intravenously transplanted with either 3×106 BMSCs,EPCs,BMSCs/EPCs or 1 mL physiological saline.Seven d after transplantation,real time-PCR and Western blotting were employed to detect the expressions of VEGF,SDF-1,bFGF,IGF-1,TGF-β,PDGF-BB,BDNF,GDNF and NGF. Results The mRNA expressions of bFGF,VEGF and BNDF in the BMSCs/EPCs transplantation group were significantly higher as compared with those in the other groups (P＜0.05).BMSCs transplantation group enjoyed the highest mRNA levels of NGF,GDNF and TGF-β among all the groups, significantly higher as compared with those in the other groups (P＜0.05),followed by BMSCs/EPCs transplantation group.EPCs transplantation group enjoyed the highest mRNA levels of PDGF,IGF-1 and SDF-1,significantly higher as compared with those in the other groups (P＜ 0.05), followed by BMSCs/EPCs transplantation group. Conclusion BMSCs combined with EPCs implantation can promote the functional rehabilitation in rats after focal cerebral ischemia, which provides new way for improving the transplantation success rate.
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Objective To establish simple,stable and reliable rat models of oxygen glucose deprivation/reoxgenation(OGD/R)in adult neural stem cells(NSCs)in vitro.Methods The NSCs from adult Fisher344 rats were cultured in serum-free medium and identified using nestin and DAPI immunofluorescent double staining.These cells were washed with a Earle′s balanced salt solution without glucose for 2 times,then,incubated for different periods(2,4,6,8 and 10 h)in a trigas incubator with an atmosphere of 1％ O2,5％CO2 and 94％ N2,98％ humidity at 37 ° C.And then,these cells were removed from the anaerobic incubator,washed,and added DEME/F12 containing bFGF supplement.A normoxic-normoglycemic control group was employed.Morphological assessment of NSCs was performed by light microscopy after re-oxgenation for 24 h; CCK-8 colorimetric method was used to determine the survival and proliferation of NSCs,and flow cytometry was employed to detect the apoptosis of NSCs.Results After the setting of oxygen glucose deprivation for 2 h,the OD value and the survival rate in the OGD cells were increased as compared with those in control group without significant difference(P＞0.05).While the morphological damage of NSCs aggravated gradually and the OD value decreased in OGD cells following the prolongation of times; under the setting of oxygen glucose deprivation for 6 h,the OD value in OGD cells obviously decreased as compared with that in the control group(P＜0.05); under the setting of oxygen glucose deprivation for 6 h,the survival rate obviously decreased and the apoptosis rate significantly increased in OGD cells as compared with that in the control group(P＜0.05); under the setting of oxygen glucose deprivation for 6 h,the apoptosis rate of NSCs excessed to 50％.Conclusion By means oftrigas incubator,simple,stable and reliable models of OGD/R in NSCs in vitro can be successfully established.
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Objective To investigate the feasibility of Catwalk-assisted system in analyzing the behavioral features of rat models of middle cerebral artery occlusion(MCAO).Methods Thirty male SD rats were randomly assigned to experimental group and control group.Suture-occluded method was used to establish the focal cerebral ischemia/reperfusion models in the experimental group.Traditional modified neurological severity scale(mNSS),vibrissae-evoked forelimb placing test and cylinder test were employed to analyze the behavioral characteristics of the SD rats 1,3,7,14 and 28 dafter establishment of rat models.Besides that,Catwalk-assisted gait analysis system was employed at the same time points.Results Catwalk-assisted gait analysis results suggest rats from experimental group had smaller intensity,print area and slower walk speed than rats from control group.Besides that,interlimb coordination also changed on the 3rd d of operation and existed until the 28th d of operation.Rotate step patterns like Ra(1.5％)and Rb(2.3％)were detected in experimental group but never seen in control group.Parameters as cadence,stance,swing and other regularity index had no significant changes between the 2 groups.Conclusion Catwalk analysis system,by analyzing such movement parameters of MCAO rats as intensity,speed,print area,step pattern and interlimb coordination,can commendably reflect the damage of brain ischemia and its changes crossing time,which can comprehensively analyze many behavioral features of the models.
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<p><b>OBJECTIVE</b>To report a case of critical illness polyneuropathy (CIP) with Parkinson disease and discuss the development, clinical features and early diagnosis of this condition.</p><p><b>METHODS</b>The clinical data of a patient with CIP and Parkinson's disease and the relevant literature were reviewed.</p><p><b>RESULTS</b>This case showed no typical disease course of sepsis, and the condition exacerbated rapidly. The patient presented initially with abnormal homeostasis, followed by rapid onset of respiratory muscle weakness to require mechanical ventilation, but no limb weaknesses were detected. Intravenous antibiotics and aggressive treatment of sepsis did not produce any positive responses to wean from mechanical ventilation. Examinations of creatine kinase and cerebrospinal fluid showed no abnormalities. Electromyography and nerve conduction studies demonstrated declined nerve conduction velocity and decreased sensory and motor muscle action potentials, suggesting the possibility of CIP.</p><p><b>CONCLUSION</b>In patients with Parkinson disease, the occurrence of sepsis with prolonged mechanical ventilation and limb weakness indicates the necessity of neurophysiological examination, muscle biopsies and laboratory tests, which may help detect CIP in the early phase. Proper interventions of sepsis may reduce the likeliness of CIP. Elimination of the risk factors and aggressive management of sepsis can be effective measures for preventing CIP.</p>
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Aged , Humans , Male , Parkinson Disease , Polyneuropathies , Diagnosis , Respiration, Artificial , Respiratory Insufficiency , SepsisABSTRACT
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.
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Objective To investigate the method for inducing OABAergic neurons from adipose-derived mesenchymal stem cells (ADSCs) and observe the effect of transplantation of these neurons in the treatment of parkinsonian rats. Methods ADSCs isolated from rat inguinal fat pads were digested with collagenase, cultured and passaged in vitro, from which neural stem cells were induced using the neural stem cell culture medium prepared by our institute and identified for the stem cell markers. The neural stem cells obtained were further induced using the GABAergic neuron culture medium. After identification for the marker GAD65, the GABAergic neurons or the neural stem cells were stereotaxically transplanted into the subthalamic nucleus of the Parkinsonian rats, and the behavioral changes of the rats were observed at 2, 4 and 8 weeks after the cell transplantation. Results The neural stem cells differentiated from the ADSCs expressed the stem cell markers including nestin and neuron-specific enolase. After the second induction, the cells were positive for GAD65 as identified by immunofluorescence staining. Four weeks after transplantation of the neural stem cells and GABAergic neurons into the subthalamic nucleus, the parkinsonian rats exhibited significantly improved rotational behavior induced by apomorphine, and the improvement was especially obvious in rats with GABAergic cell transplantation. Conclusion GABAergic neurons can be induced from the rat ADSCs and transplantation of these neurons into the subthalamic nucleus can produce obvious behavioral improvement in rat models of Parkinson disease.
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Objective To investigate the photodynamic effect mediated with 5-aminolevulinic acid (5-ALA) on U251 human glioma cells. Methods Fluorescence microscope and confocal laser scanning microscope were used to detect the localization of Pp Ⅸ in U251 human glioma cells. The cells with/without 5-ALA were irradiated at the wavelength of 635 nm. MTT assay was used to measure the cell survival after laser irradiation. Results 5-ALA cocultured with U251 cells successfully produced endogenous Pp Ⅸthat was observed distributively in the cytoplasm, but not in nuclear region. The overall survival rates of the U251 glioma cells photodamaged by ALA-PDT decreased as the incubation time went by or the 5-ALA concentration increased, while peaked at the incubation time of 6 h and the 5-ALA concentration of 2.0mmol/L. Without one of 5-ALA and light irradiation, the survival rate of the cells had no significant difference compared with that of cells of the control group. Conclusion The 5-ALA-induced PDT appears to be a promising therapy for human glioma. The optimal incubation time may be 6 h and the optimal 5-ALA concentration be 2.0 mmol/L.
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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.
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<p><b>OBJECTIVE</b>To investigate telomerase activity in rabbit bone marrow stromal cells (BMSCs) during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor (GDNF) on the expression of telomerase.</p><p><b>METHODS</b>BMSCs were acquired from rabbit marrow and divided into control group, GDNF (10 ng/ml) group. Cytokine.NSCs medium (prepared by our lab, Patent No. ZL02134314. 4) supplemented with 10 percent fetal bovine serum (FBS) was used to induce BMSCs differentiation along neural pathway. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neuron-specific endase (NSE), and gial fibrillary acidic protein (GFAP). The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activities were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA).</p><p><b>RESULTS</b>BMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomerase activities were undetectable in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomerase expression were observed in the two groups.</p><p><b>CONCLUSIONS</b>Rabbit BMSCs do not display telomerase activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomerase activity of BMSCs.</p>