BACKGROUND: There is few studies addressing the long-playing dynamic observation of cysteinyl aspartate specific protease 3 (Caspase-3) expression following cerebral ischemia/reperfusion.OBJECTIVE: To investigate the effect of transplantation of the neural stem cells (NSCs) modified with gene of glial cell line-derived neurotrophic factor (GDNF) on expression of Caspase-3 in adult Sprague Dawley rats with transient cerebral ischemia.DESING: Randomized controlled animal study.MATERIALS: Sixty Sprague Dawley rats were divided randomly into normal control group (N, n =5), ischemia/reperfusion group (IR, n=5), neural stem cell group (NSCs, n=25) and NSCs modified with gene of GDNF group (GDNF/NSCs, n =25). Several clean neonatal Sprague-Dawley rats were selected to harvest NSCs.METHODS: With the exception of normal control group, models of transient cerebral ischemia were created by modified suture method in other groups. At day 3 following reperfusion, 20 μL NSC suspension containing (4.0-5.0)×10~5 NSCs was infused into rats of the NSC group via right lateral ventricle. An equal volume of GDNF-modified NSC suspension was injected into rats of the GDNF/NSC group. 20 μL saline was infused into the rats of the ischemia/reperfusion group. Animals were anesthetized and sacrificed at week 1 following ischemia/reperfusion in the normal control and ischemia/reperfusion groups. Animals were anesthetized and sacrificed at weeks 1, 2, 3, 5, 7 following ischemia/reperfusion in the NSC and GDNF/NSC groups, 5 rats in each time point.MAIN OUTCOME MEASURES: The strept avidin-biotin immunostaining method was used to observe the distributive characteristics of Caspase-3 in the hippocampus and frontal parietal cortex.RESULTS: Immunohistochemical method (SP) showed that positive capase-3 products expressed in nucleus, cytoplasm and partial neurite. In hippocampus, number of Caspase-3-positive cells was decreased in NSC and GDNF/NSC groups. With the exception of at 1-week reperfusion, number of Caspase-3-positive cells was significantly lessened in the GDNF/NSC group compared with the NSC group at other time points (P < 0.05). In frontoparietal cortex, number of Caspase-3-positive cells was reduced in the NSC and GDNF/NSC groups over time. Except 1 and 2 weeks following ischemia/reperfusion, number of Caspase-3-positive cells was significantly lessened in the GDNF/NSC group compared with the NSC group (P < 0.05).CONCLUSION: Transplanting NSCs modified with gene of GDNF can improve remarkably neural function by deceasing Caspase-3 expression and reducing the nervous cell apoptosis. The transplantation of NSCs modified with gene of GDNF obtained better outcomes compared with NSC transplantation.

BACKGROUND: In the central nervous system(CNS) of normal adults there are neural stem cells or neural progenitor cells, which are capable of self-renewing and multiple differentiating. In normal physiological conditions, intrinsic neural stem cells are in a resting state. What state will they be in during cerebral ischemia?OBJECTIVE: To observe the distribution, proliferation and differentiation of intrinsic neural stem cells in focal transient ischemia in rats.DESIGN: A randomized controlled exploratory trial based on the rats.SETTING: Neurobiological department, histological and embryological department of a medical college.MATERIALS: The experiment was conducted in the Neurobiological Department of Luzhou Medical College from July 2001 to July 2002. Altogether 41 healthy adult SD rats of either gender, weighting 250 g - 300 g, were selected.INTERVENTIONS: The focal ischemia model was made by blocking middle cerebral artery(MCA) and reperfusing for 0.5 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days and 10 days. Sham-operation group was treated by the same method, but the filament was not long enough to block MCA, and normal rats served as control group. The rats were sacrificed at given time points, and their brains were made into cerebral slices. The single-and double-labeled immunohistochemical staining was employed to detect the proliferation, distribution and differentiation of intrinsic neural stem cells.MAIN OUTCOME MEASURES: The distribution, proliferation and differentiation of intrinsic neural stem cells.RESULTS: Immunoreactivity of proliferating cell nuclear antigen(PCNA)was present in most ependymal cells in ventricular zone(VZ), and PCNA-positive cells were sparsely distributed in the parenchyma in normal and sham-operation groups. At 3 hours of reperfusion, PCNA-labeled cells were first detected in rostral subventricular zone. At 12 hours of reperfusion and onward, PCNA-positive cells appeared in some choroid plexus cells in bilateral lateral VZ. At day 3 to day 10 of reperfusion, PCNA-labeled cells significantly increased in infarct boundary in preoptic area, striatum and deep layer of frontoparietal cortex. PCNA-labeled cells were first detected in subgranular zone of dentate gyrus 3 days after reperfusion, and increased with time. A very small number of double-positive cells expressed with PCNA and glial fibrillary acidic protein(GFAP) were first detected in infract boundary in preoptic area on day 3 and onward. No double-PCNA and NF-positive cells were detected within 10 days of reperfusion.CONCLUSION: Focal cerebral ischemia activates intrinsic neural stem cells, which proliferate and differentiate, and migrate toward ischemic striatum and frontoparietal cortex. This may help clarify the mechanism of functional recovery after ischemia.

BACKGROUND:Previous studies have demonstrated that cell transplantation has neuroprotective effect on intracerebral hemorrhage,and some researches have indicated that transplantation of bone marrow stromal cells(BMSCs)can promote neural function recovery after cerebral infarction.OBJECTIVE:To explore whether transplantation of BMSCs-modified by gtial cell line-dedved neurotrophic factor gene(GDNF)gene provides a better therapeutic effect than native BMSCs after stroke.METHODS:Totally 36 SD rats were induced intracerabral hemorrhage models by injecting autologous arterial blood,and then divided into 3 groups(n=6).each group was assigned into 2 sub-groups Rabbits in each group were stereotaxically grafted with 20 μL GDNF/BMSCs,BMSCs or saline respectively.The rats were executed at 1 and 2 weeks after operation,and immunohistochemistry was used to observe the expressions of synaptophysin(Syn)and growth associated protein-43(GAP-43)in the margin of the hemorrhagic focus.RESULTS AND CONCLUSlON:Compared with the BMSCs and control groups.both Syn-immunoreactive and GAP-43-immunoreactive products were significantly increased in the GDNF/BMSCs group(P＜0.05).Present results demonstrate that transplantation of GDNF gene-modified BMSCs provides better neuroprotection than native BMSCs delivery for stroke.

Previous experiments has shown that Ginsenoside Rb1 (GRb1), which is one of the most important active ingredients in ginseng (Panax ginseng C.A. Meyer), reduced infarct and neurologic deficit followed by the transient cerebral ischemia in rats. The mechanism of this neuroprotective function is unclear. In this study, we tested whether the neuroprotective effect of GRb1 is achieved through preventing the neuronal apoptosis and modulating expression of neuronal apoptosis inhibitory protein (NAIP). Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO) in Wistar rats. GRb1 (40 mg/kg, i.p.) was administered immediately after the onset of reperfusion. The rats with neurological deficits were randomly divided into 2 groups: the ischemia and the GRb1 group. Each group was again divided into subgroups according to the various reperfusion time (3 h, 12 h, 1, 2, 3, 5, 10 days, n=4 per time point). Apoptotic cells were analyzed using TUNEL. Immunohistochemical method was used to assess expression of NAIP. This results showed that the number of apoptotic cells elevated at 3 h of reperfusion, and peaked at 24 h, then declined, but the number of apoptotic cells at 10 d after ischemia was significantly more than those of control groups (P＜0.01). Compared with ischemia group, the apoptotic cells decreased at all subgroups of GRb1; however, the significant differences were only found from 12 h to 3 d of reperfusion. In normal and sham groups, NAIP weak immunostaining was diffusely present in the neurons of parenchyma. The number of NAIP-positive cells started to increase in ischemic regions at 3 h after ischemia, peaked at 12 h and declined up to 5 d of reperfusion. At 5 d after ischemia, the number of NAIP-positive cells was less than that of control group (P＜0.05). A few astrocytes strongly expressed NAIP in the ischemic area. In the GRb1 group, the number of NAIP-positive cells from 12 h to 10 d after ischemia was evidently higher than in the ischemia group. Thus, these results suggest that GRb1 has potential ability to prevent apoptosis, the mechanism of which is related to induce expression of NAIP.

Objective To study the effect of DNA damage induced by H2 O2 on the micronucleus frequency in lymphocytes. Methods Resting lymphocytes were treated with different levels of H2 O2 (10,50,100,1 000 μmol/L).1 000 μmol/L H2 O2 was added into mitogen-stimulated lymphocyte cultures at different time intervals.Then micronucleus rate was examined by the conven-tional culture method.Results There was no significant change of the micronucleus frequency in the experimental groups.Conclu-sion H2 O2 could induce lymphocyte DNA damage rapidly,but exerts no effect on the formation of micronuclei,which may be relat-ed to the type of DNA damage and rapid DNA repair.

Objective To construct a recombinant Escherichia coli ( E. coli) with surface-dis-played lead specific binding protein PbrR and to further study intestinal colonization by the recombinant bac-teria in mice and gastrointestinal tolerance of the bacterial surface-displayed PbrR. Methods Chimeric pro-tein Lpp-OmpA coding sequence was chemically synthesized and inserted into the expression vector pET-21a to construct the outer membrane display vector pLOA. PbrR coding sequence was also obtained by chemical-ly synthesis and inserted into pLOA to generate the outer membrane display plasmid pLOA-pbrr. E. coli BL21 (DE3)pLysS was transformed with pLOA-pbrr and induced by IPTG. The expressed recombinant proteins were analyzed by 15% SDS-PAGE and Western blot assay. Lead adsorption capacity of the cell surface-dis-played PbrR in the simulated intestinal juice and tolerance of the recombinant E. coli to simulated gastric juice were analyzed, respectively. KM mice were orally given the induced recombinant bacteria by gastric lavage for 7 consecutive days and then were continually fed until day 30. The contents of recombinant bacte-ria in stool samples were detected by dilution plate method on day 7, 15 and 30. The recombinant protein with His tag was detected by immunoblotting on day 7 and 15. Results Based on Lpp-OmpA, the PbrR outer membrane display vector was successfully constructed. The recombinant fusion protein Lpp-OmpA-PbrR-His tag was highly expressed in E. coli. The recombinant E. coli strains displaying PbrR on their outer membrane accumulated a significant level of Pb2+ in simulated intestinal juice. Moreover, those strains showed a tolerance to gastric acid in vitro and could colonize in the intestinal tracts of mice via oral infection. The surface-displayed recombinant fusion protein showed a better tolerance to the environment of digestive tract. Conclusion The recombinant E. coli strain displaying PbrR on its surface showed a stronger capabili-ty of lead accumulation from simulated intestinal environment and could colonize in the intestinal tracts of mice. The surface-displayed recombinant PbrR also showed a good tolerance to digestive juice. This study paved the way for further researches on the selective elimination of lead by biosorption based on animal mod-els.

OBJECTIVETo establish a modified method for microculturing whole human blood for cytogenetic analysis.

METHODSA novel tube rack was designed to overcome the drawbacks of directly culturing the cells within centrifuge tubes. The fractions of human plasma, human serum and two commercial fetal bovine sera were analyzed with 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The influence of adding 0%, 5%, 10%, 15%, 20%, 25% and 30% autologous plasma to the culture on lymphocyte transformation rate and mitotic index (MI) was examined.

RESULTSThe SDS-PAGE analysis showed a significant difference between commercial fetal bovine sera, and that the components of human plasma were similar to those of fetal bovine serum. The value of MI in lymphocyte was evidently increased along with addition of autologous plasma. However, this has exerted no significant effect on the transformation rate. With the addition of 10% autologous plasma, the MI value has become much higher than the conventional method.

CONCLUSIONA modified method was established by application of a novel tube inclined rack and optimization of whole blood inoculation. This method is easier and cheaper, and is suitable for application in clinical practice.

Adolescent ; Adult ; Cell Culture Techniques ; methods ; Cytogenetics ; Female ; Humans ; Lymphocytes ; ultrastructure ; Male ; Mitotic Index