Effect of glutamate on vascular endothelial growth factor mRNA and protein expressions in hypoxic rat astrocytes in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of glutamate on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in cultured rat astrocytes under hypoxia.</p><p><b>METHODS</b>Cultured rat astrocytes were randomly divided control group, glutamate group, hypoxia group and hypoxia+glutamate group. The cells in the control and glutamate groups were cultured under nomoxic condition (95% air and 5% CO(2)), and those in the other two groups under hypoxic condition (94% N(2), 5% CO(2) and 1% O(2)). The total RNA was extracted from the cells at different time points of hypoxic exposure for real-time FQ-PCR and ELISA to detect the expression of VEGF mRNA and protein in cultured astrocytes, respectively.</p><p><b>RESULTS</b>The expressions of VEGF mRNA and protein underwent no significant changes in the control glutamate groups, but increased obviously in both hypoxia and hypoxia+glutamate groups at 2, 4, 6, 8 and 12 h of hypoxic exposure. At these time points, VEGF expressions at both the mRNA and protein levels were significantly higher in hypoxia+glutamate group than in hypoxia group.</p><p><b>CONCLUSION</b>Glutamate at 1 micromol/L can further increase the expression of VEGF mRNA and protein in astrocytes exposed to hypoxia, which may result from the adaptive changes of glutamate receptors in hypoxic astrocytes.</p>

Neurons in the corpus callosum of rats: expression of Cav2.2 and their connection / 中南大学学报(医学版)

OBJECTIVE@#To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection.@*METHODS@#Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine.@*RESULTS@#There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum.@*CONCLUSION@#There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.

A simple method for assessment of RNA integrity in laser capture microdissection samples / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples.</p><p><b>METHODS</b>The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells).</p><p><b>RESULTS</b>When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples.</p><p><b>CONCLUSION</b>A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.</p>

Treatment of scrapie pathogen 263K with tetracycline partially abolishes protease-resistant activity in vitro and reduces infectivity in vivo / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K.</p><p><b>METHODS</b>Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrPSc in brain tissues of the infected animals was detected by PrP specific Western blot assays.</p><p><b>RESULTS</b>Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75 +/- 0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5 +/- 1.73 and 59.5 +/- 0.58 days (P < 0.05).</p><p><b>CONCLUSION</b>Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.</p>

Effect of ligustrazine on cell proliferation in subventricular zone in rat brain with focal cerebral ischemia-reperfusion injury / 中南大学学报(医学版)

OBJECTIVE@#To observe the effect of ligustrazine on cell proliferation in subventricular zone (SVZ) in rat brain with focal cerebral ischemia reperfusion injury.@*METHODS@#Male SD rats were randomly divided into a normal group,a sham operation group,a ligustrazine treatment group, and a control group. The ligustrazine treatment group and the control group were further divided into 5 subgroups: 1d, 3d, 7d, 14d, and 21d reperfusion after 2h middle cerebral artery occlusion (MCAO). The focal cerebral ischemia-reperfusion model was made by MCAO. S phase cells were labelled with BrdU. Immunohistochemistry method was conducted to detect the BrdU positive cells. The total number of BrdU positive cells in the SVZ was measured. The expression of neuro nitric oxide synthase (nNOS) was detected with Western blot method.@*RESULTS@#There was a significant increase of BrdU positive cells in SVZ of ligustrazine treatment in the 1d and 3d group compared with that of the control group (P<0.01). The total number of BrdU positive cells reached a peak in 7d group and declined afterwards. Cells proliferated also in SVZ on the contralateral side, and peaked at 7d. The nNOS expression of ligustrazine administration after the focal cerebral ischemia-reperfusion decreased at 1d and 3d after the reperfusion compared with that of the control group (P<0.05), and increased at 7d, but with no significant difference compared with that of the control group.@*CONCLUSION@#Ligustrazine may promote the cell proliferation in SVZ of adult rats with ischemia-reperfusion injury by decreasing the nNOS expression.

Effect of ligustrazine on nNOS expression and neuranagenesis in adult rats after cerebral ischemia-reperfusion injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of ligustrazine on cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) and nNOS expression in rat brain after cerebral ischemia-reperfusion injury.</p><p><b>METHODS</b>Male SD rats were randomly divided into normal control group, sham operation group, model group and ligustrazine treatment group. The latter two groups were further divided into 5 subgroups for observation at 1, 3, 7, 14 and 21 days after reperfusion following a 2-hour middle cerebral artery occlusion (MCAO). The cells in S phase were labeled with BrdU, and immunohistochemistry was employed to detect BrdU- and nNOS-positive cells. The numbers of BrdU-positive cells in the SVZ and DG were measured. The expression of nNOS was detected by Western blotting.</p><p><b>RESULTS</b>nNOS expression increased significantly in the model group as compared to the sham operation group (P<0.05), and ligustrazine treatment significantly lowered the expression level in comparison with the model group (P<0.05). Compared with the model group, a significant increase in BrdU-positive cells occurred in the SVZ of rats 1 and 3 days after igustrazine treatment (P<0.05), along with an increase of DG BrdU-positive cells.</p><p><b>CONCLUSION</b>Ligustrazine significantly restrains ischemia-reperfusion injury-induced nNOS activity enhancement and promotes cell proliferation in the SVZ and DG of adult rats after ischemia-reperfusion injury.</p>

Cloning, expression, and antibody preparation of nestin with immunohistochemical analysis / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development.</p><p><b>METHODS</b>The nestin cDNA was cloned from human neural stem cells by RT-PCR and ligated to prokaryotic expression plasmid pQE30 for construction of the recombinant vector pQE30-nestin. After sequencing, the recombinant vector was transformed into E.coli M15 and His-tagged nestin was induced by IPTG. The nestin was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blotting. BALB/c mice were immunized with the purified recombinant protein to prepare the antiserum, which was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.</p><p><b>RESULTS</b>The nestin gene was successfully cloned from human neural stem cells, which was identical to that reported in GenBank. After IPTG induction, the E.coli transformed with pQE30-nestin plasmid expressed a 25,000 His-tagged protein, which was successfully purified and identified as nestin by Western blotting. Western blotting, ELISA and immunohistochemistry demonstrated that the antiserum could specifically bind to the recombinant nestin as well as to nestin in fetal human and rat brains.</p><p><b>CONCLUSION</b>We successfully cloned the nestin gene and expressed the nestin, and nestin mAb prepared can specifically recognize not only the recombinant nestin, but also nestin from human and rats brain tissues.</p>

Effects of ligustrazine on hippocampal dentate gyrus cell proliferation after focal cerebral ischemia in adult rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of ligustrazine on cell proliferation in hippocampal dentate gyrus subgranular zone (SGZ) after focal cerebral ischemia in adult rats.</p><p><b>METHODS</b>Middle cerebral artery occlusion (MCAO) model was established in adult rats by placement of an intraluminal filament at the origin of the MCA. Ligustrazine was administered intraperitoneally at a daily dose of 80 mg/kg starting at 2 h after MCAO, and BrdU (50 mg/kg daily) was also injected intraperitoneally starting at 4 h after MCAO. BrdU-positive cells in the SGZ were counted 7, 14 and 24 days after MCAO, respectively.</p><p><b>RESULTS</b>Compared with sham operation group, ischemic ipsilateral BrdU-positive cells in the ischemic model group increased 7 days after MCAO, reaching the peak on day 14, and decreased on day 21 (P<0.01). The number of ischemic ipsilateral BrdU-positive cells in ligustrazine group was significantly greater than that in the ischemic model group on days 7, 14 and 21 (P<0.01), and maintained the high level on day 21.</p><p><b>CONCLUSION</b>Ligustrazine possesses long lasting effect of promoting cell proliferation in the SGZ after focal cerebral ischemia in adult rats.</p>

Protein expression of human neuron-specific enolase and its antiserum preparation / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone and express human neuron-specific enolase (HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis.</p><p><b>METHODS</b>HuNSE gene was amplified by RT-PCR and subcloned into a HIS-tagged expression vector pQE30 after sequence verification. HIS-NSE fusion protein expression was obtained in E. coli M15 after IPTG induction followed by purification of the fusion protein by Ni-NTA affinity chromatography. Two male rabbits were immunized for 4 times with the purified protein, and the antiserum against NSE protein was collected and evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemistry.</p><p><b>RESULTS</b>SDS-PAGE assay yielded an approximately 22 kD HIS-NSE fusion protein. The prepared antiserum could recognize both recombinant NSE protein and native NSE protein extracted from the brain tissues of different mammalian species as shown by Western blotting and immunohistochemistry.</p><p><b>CONCLUSION</b>High expression of HuNSE is obtained in E. coli and the prepared antiserum against HuNSE can be used potentially for diagnosis of prion-associated diseases and other nervous degeneration diseases.</p>

Preparation of monoclonal antibodies against prion proteins with full-length hamster PrP / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases.</p><p><b>METHODS</b>Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry.</p><p><b>RESULTS</b>The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc.</p><p><b>CONCLUSION</b>The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.</p>

Dynamic analyses of PrP and PrP(Sc) in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrP(Sc) during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrP(Sc) forms.</p><p><b>METHODS</b>Hamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrP(Sc) were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immuno-golden EM.</p><p><b>RESULTS</b>PrP(Sc) was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrP(Sc) in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions.</p><p><b>CONCLUSION</b>These results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrP(Sc) may indwell in brain tissues during the infection.</p>

Comparison study on clinical and neuropathological characteristics of hamsters inoculated with scrapie strain 263K in different challenging pathways / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.c.), intraperitoneal (i.p.), intragastrical (i.g.), intracardiac and intramuscular (i.m.) approaches.</p><p><b>METHODS</b>Hamsters were infected with crude- or fine-prepared brain extracts. The neuropathological changes, PrP(Sc) deposits, and patterns of PK-resistant PrP were analyzed by HE stain, immnunohistochemistry (IHC) assay and Western blot. Reactive gliosis and neuron loss were evaluated by glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) specific IHC.</p><p><b>RESULTS</b>The animals inoculated in i.m. and i.p. ways with crude PrP(Sc) extracts showed clinical signs at the average incubation of 69.2 +/- 2.8 and 65.5 +/- 3.9 days. Inoculation in i.c. and intracardiac ways with fine PrP(Sc) extracts (0.00035 g) caused similar, but relative long incubation of around 90 days. Only one out of eight hamsters challenged in i.g. way with low dosage (0.01 g) became ill after a much longer incubation (185 d), while all animals (4/4) with high dosage (0.04 g) developed clinical signs 105 days postinfection. The most remarkable spongiform degeneration and PrP(Sc) deposits were found in brain stem among the five challenge groups generally. The number of GFAP-positive astrocytes increased distinctly in brain stems in all infection groups, while the number of NSE-positive cells decreased significantly in cerebrum, except i.c. group. The patterns of PK-resistant PrP in brains were basically identical among the five infection routes.</p><p><b>CONCLUSION</b>Typical TSE could be induced in hamsters by inoculating strain 263K in the five infection ways. The incubation periods in bioassays depend on infective dosage, administrating pathway and preparation of PrP(Sc). The neuropathological changes and PrP(Sc) deposits seem to be related with regions and inoculating pathways.</p>