Neuronal MeCP2 in the dentate gyrus regulates mossy fiber sprouting of mice with temporal lobe epilepsy.

Sprouting of mossy fibers, one of the most consistent findings in tissue from patients with mesial temporal lobe epilepsy, exhibits several uncommon axonal growth features and has been considered a paradigmatic example of circuit plasticity that occurs in the adult brain. Clarifying the mechanisms responsible may provide new insight into epileptogenesis as well as axon misguidance in the central nervous system. Methyl-CpG-binding protein 2 (MeCP2) binds to methylated genomic DNA to regulate a range of physiological functions implicated in neuronal development and adult synaptic plasticity. However, exploring the potential role of MeCP2 in the documented misguidance of axons in the dentate gyrus has not yet been attempted. In this study, a status epilepticus-induced decrease of neuronal MeCP2 was observed in the dentate gyrus (DG). An essential regulatory role of MeCP2 in the development of functional mossy fiber sprouting (MFS) was confirmed through stereotaxic injection of a recombinant adeno-associated virus (AAV) to up- or down-regulate MeCP2 in the dentate neurons. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to identify the binding profile of native MeCP2 using micro-dissected dentate tissues. In both dentate tissues and HT22 cell lines, we demonstrated that MeCP2 could act as a transcription repressor on miR-682 with the involvement of the DNA methylation mechanism. Further, we found that miR-682 could bind to mRNA of phosphatase and tensin homolog (PTEN) in a sequence specific manner, thus leading to the suppression of PTEN and excessive activation of mTOR. This study therefore presents a novel epigenetic mechanism by identifying MeCP2/miR-682/PTEN/mTOR as an essential signal pathway in regulating the formation of MFS in the temporal lobe epileptic (TLE) mice. SIGNIFICANCE STATEMENT: Understanding the mechanisms that regulate axon guidance is important for a better comprehension of neural disorders. Sprouting of mossy fibers, one of the most consistent findings in patients with mesial temporal lobe epilepsy, has been considered a paradigmatic example of circuit plasticity in the adult brain. Although abnormal regulation of DNA methylation has been observed in both experimental rodents and humans with epilepsy, the potential role of DNA methylation in this well-documented example of sprouting of dentate axon remains elusive. This study demonstrates an essential role of methyl-CpG-binding protein 2 in the formation of mossy fiber sprouting. The underlying signal pathway has been also identified. The data hence provide new insight into epileptogenesis as well as axon misguidance in the central nervous system.

Isoflurane-induced reduction in neurogenesis derived from the tertiary dentate matrix.

Anesthetics-induced disruption of dentate neurogenesis in the young brain is strongly suggested to contribute to delayed neurocognitive deficit. In postnatal rodents, the neurogenesis of the dentate gyrus (DG) is sequentially derived from the secondary dentate matrix, tertiary dentate matrix and subgranular zone (SGZ). However, the effects of anesthetics on the dentate neurogenesis derived from specific sites are poorly understood. To trace the new cells generated from the postnatal secondary dentate matrix, peak stage of the tertiary dentate matrix and early stage of the SGZ after isoflurane exposure, mice at postnatal day 1 (P1), P7 and P31 were injected with BrdU at 12 h before the exposure. We found that isoflurane exposure significantly reduced the numbers of proliferating cells (1 day old), immature granule cells (21 days old) or mature granule cells (42 days old) derived from the peak stage of the tertiary dentate matrix and postnatal secondary dentate matrix, but not from the SGZ. Quantitative assessment of BrdU-/BrdU+NeuN-positive cells and cleaved caspase-3 level in the DG indicated that the reduction was correlated with cell loss rather than neuronal differentiation. Mechanistically, we demonstrated that the PI3K/Akt/GSK-3ß pathway enriched by mRNA-sequencing is a requirement for the isoflurane-induced loss of 1-day-old proliferating cells generated from the tertiary dentate matrix. In addition, this study demonstrated that P1 and P7 mice, but not P31 mice exposure to isoflurane resulted in subsequent deficits in performance of the tasks of the Morris Water Maze.

Identification of microRNA/target gene in the dentate gyrus of 7­day­old mice following isoflurane exposure.

Studies on rodents and nonhuman primates suggest that exposure to anesthetics, particularly in the young brain, is associated with neuronal apoptosis as well as hippocampal­dependent cognitive dysfunction. Disruption of the development of dentate gyrus may play an important role in anesthetics­induced neurotoxicity. However, the anesthetics triggered molecular events in the dentate gyrus of the developing brain are poorly understood. By integrating two independent data sets obtained from miRNA­seq and mRNA­seq respectively, this study aims to profile the network of miRNA and potential target genes, as well as relevant events occurring in the dentate gyrus of isoflurane exposed 7­day­old mice. We found that a single four hours exposure to isoflurane yielded 1059 pairs of differently expressed miRNAs/target genes in the dentate gyrus. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis further indicates that dysregulated miRNAs/target genes have far­reaching effects on the cellular pathophysiological events, such as cell apoptosis, axon development, and synaptic transmission. Our results would greatly broaden our functional understanding of the role of miRNA/target gene in the context of anesthetics­induced neurotoxicity.

Neuronal Glypican4 promotes mossy fiber sprouting through the mTOR pathway after pilocarpine-induced status epilepticus in mice.

In temporal lobe epilepsy (TLE), abnormal axon guidance and synapse formation lead to sprouting of mossy fibers in the hippocampus, which is one of the most consistent pathological findings in patients and animal models with TLE. Glypican 4 (Gpc4) belongs to the heparan sulfate proteoglycan family, which play an important role in axon guidance and excitatory synapse formation. However, the role of Gpc4 in the development of mossy fibers sprouting (MFS) and its underlying mechanism remain unknown. Using a pilocarpine-induced mice model of epilepsy, we showed that Gpc4 expression was significantly increased in the stratum granulosum of the dentate gyrus at 1 week after status epilepticus (SE). Using Gpc4 overexpression or Gpc4 shRNA lentivirus to regulate the Gpc4 level in the dentate gyrus, increased or decreased levels of netrin-1, SynI, PSD-95, and Timm score were observed in the dentate gyrus, indicating a crucial role of Gpc4 in modulating the development of functional MFS. The observed effects of Gpc4 on MFS were significantly antagonized when mice were treated with L-leucine or rapamycin, an agonist or antagonist of the mammalian target of rapamycin (mTOR) signal, respectively, demonstrating that mTOR pathway is an essential requirement for Gpc4-regulated MFS. Additionally, the attenuated spontaneous recurrent seizures (SRSs) were observed during chronic stage of the disease by suppressing the Gpc4 expression after SE. Altogether, our findings demonstrate a novel control of neuronal Gpc4 on the development of MFS through the mTOR pathway after pilocarpine-induced SE. Our results also strongly suggest that Gpc4 may serve as a promising target for antiepileptic studies.

The neurotoxic effect of isoflurane on age-defined neurons generated from tertiary dentate matrix in mice.

INTRODUCTION: Recent animal studies showed that isoflurane exposure may lead to the disturbance of hippocampal neurogenesis and later cognitive impairment. However, much less is known about the effect of isoflurane exposure on the neurons generated form tertiary dentate matrix, even though a great increase of granule cell population during the infantile period is principally derived from this area. METHODS: To label the new cells originated from the tertiary dentate matrix, the mice were injected with BrdU on postnatal day 6 (P6). Then, the mice were exposed to isoflurane for 4 hr at 1, 8, 21, and 42 days after BrdU injection, and the brains were collected 24 hr later. The loss of newly generated cells/neurons with different developmental stage was assessed by BrdU, BrdU + DCX, BrdU + NeuN, or BrdU + Prox-1 staining, respectively. RESULTS: We found that the isoflurane exposure significantly decreased the numbers of nascent cells (1 day old) and mature neurons (42 days old), but had no effect on the immature (8 days old) and early mature neurons (8 and 21 days old, respectively). CONCLUSION: The results suggested isoflurane exposure exerts the neurotoxic effects on the tertiary dentate matrix-originated cells with an age-defined pattern in mice, which partly explain the cognitive impairment resulting from isoflurane exposure to the young brain.

Status epilepticus induced Gadd45b is required for augmented dentate neurogenesis.

In animal models with temporal lobe epilepsy (TLE), the status epilepticus (SE) leads to a dramatic increase in number of newly born neuron in the subgranular zone (SGZ) of dentate gyrus. How the SE confers a modulation in the dentate neurogenesis is mostly unknown. Gadd45b is involved in epigenetic gene activation by DNA demethylation. This study was performed to present a novel mechanism underling SE-induced dentate neurogenesis. A transient induction (12 hrs to 3 days) of Gadd45b was observed in dentate gyrus of mice after pilocarpine-induced SE. Labeling the dividing cells with BrdU, we next found that the induction of Gadd45b was required to increase the rate of cell proliferation in the dentate gyrus at 7 and 14 days after SE. Afterward, the DNA methylation levels for candidate growth factor genes critical for the adult neurogenesis were assayed with Sequenom MassARRAY Analyzer. The results indicated that Gadd45b was necessary for SE-induced DNA demethylation of specific promoters and expression of corresponding genes in the dentate gyrus, including brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2). Using Timm staining, we further suggested that SE-induced Gadd45b might contribute to the subsequent mossy fiber sprouting (MFS) in the chronically epileptic hippocampus via epigenetic regulation of dentate neurogenesis at early stage after SE. Together, Gadd45b links pilocarpine-induced SE to epigenetic DNA modification of secreted factors in the dentate gyrus, leading to extrinsic modulation on the neurogenesis.

Identification of microRNA-target genes in mice hippocampus at 1 week after pilocarpine-induced status epilepticus.

MicroRNAs (miRNA) are believed to play a crucial role in the cause and treatment of temporal lobe epilepsy (TLE) by controlling gene expression in different stages of the disease. To investigate role of miRNA in the latent stage following status epilepticus, we first compared microRNA expression profiles in mice hippocampus at 1 week after pilocarpine-induced status epilepticus (SE) vs. controls in hippocampal tissues using Exiqon miRCURY LNA™ miRNAs Array. Then, the target genes of altered miRNAs were predicted using both TargetScan 7.1 and miRDB V5, and were further selected by intersecting with another independent mRNA expression profile dataset from the samples at the same time point. We found out 14 common genes as down miRNA target (up-mRNA) and 4 common genes as up miRNA target (down mRNA) in SE mice. miR-669m-3p-TRHR (thyrotropin releasing hormone receptor), miR-669m-3p-B3galt2 (ß-1,3-Galactosyltransferase 2), miR-105-PDPN (Podoplanin) and miR-883b-3p-CLEC-2 (C-type-lectin-like-2) were found to be potential molecular mechanisms to modulate the calcium signaling pathway, glycosylation pathways and chemokine mediated inflammatory processes in mice hippocampus at 1 week after pilocarpine-induced SE, respectively. Our results offered potential novel insights into the cellular events in the mice hippocampus mediated by miRNASs-target genes that shape SE-evoked epileptogenesis.

Forced Physical Training Increases Neuronal Proliferation and Maturation with Their Integration into Normal Circuits in Pilocarpine Induced Status Epilepticus Mice.

Increased number of newly-born neurons produced at latent stage after status epilepticus (SE) contribute to aberrant rewiring of hippocampus and are hypothesized to promote epileptogenesis. Although physical training (PT) was reported to cause further increase in neurogenesis after SE, how PT affect their integration pattern is still elusive, whether they integrate into normal circuits or increase aberrant integrations is yet to be determined. To understand this basic mechanism by which PT effects SE and to elaborate the possible role of neuronal integrations in prognosis of SE, we evaluated the effect of 4 weeks of treadmill PT in adult male mice after pilocarpine-induced SE on behavioral and aberrant integrations' parameters. Changes in BDNF gene methylation and its protein level in hippocampus was also measured at latent stage (2-weeks) to explore underlying pathways involved in increasing neurogenesis. Our results demonstrated that although PT increased proliferation and maturation of neurons in dentate gyrus, they showed reduced aberrant integrations into hippocampal circuitry assessed through a decrease in the number of ectopic granular cells, hilar basal dendrites and mossy fiber sprouting as compared to non-exercised SE mice. While SE decreased the percentage methylation of specific CpGs of BDNF gene's promoter, PT did not yield any significant difference in methylation of BDNF CpGs as compared to non-exercised SE mice. In conclusion, PT increases hippocampal neurogenesis through increasing BDNF levels by some pathways other than demethylating BDNF CpGs and causes post SE newly-born neurons to integrate into normal circuits thus resulting in decreased spontaneous recurrent seizures and enhanced spatial memory.

Newly generated neurons at 2 months post-status epilepticus are functionally integrated into neuronal circuitry in mouse hippocampus.

Emerging evidence has linked chronic temporal lobe epilepsy to dramatically reduced neurogenesis in the dentate gyrus. However, the profile of different components of neurogenesis in the chronically epileptic hippocampus is still unclear, especially the incorporation of newly generated cells. To address the issue, newly generated cells in the sub-granular zone of the dentate gyrus were labeled by the proliferation marker bromodeoxyuridine (BrdU) or retroviral vector expressing green fluorescent protein 2 months after pilocarpine-induced status epilepticus. The newly generated neurons that extended axons to CA3 area or integrated into memory circuits were visualized by cholera toxin B subunit retrograde tracing, and detecting activation of BrdU(+) cells following a recall of spatial memory test at the chronic stage of TLE. We found that the microenvironment was still able to sustain significant neuronal differentiation of newly generated cells at 2 months post-status epilepticus time-point, and newly added neurons into granular cell layer were still able to integrate into neuronal circuitry, both anatomically and functionally. Quantified analyses of BrdU(+) or Ki-67(+) cells demonstrated that there was a reduced proliferation of progenitor cells and diminished survival of newly generated cells in the epileptic hippocampus. Both decreased levels of neurotrophic factors in the surrounding milieu and cell loss in the CA3 area might contribute the decreased production of new cells and their survival following chronic epilepsy. These results suggest that decreased neurogenesis in the chronically epileptic hippocampus 2 months post status epilepticus is not associated with altered integration of newly generated neurons, and that developing strategies to augment hippocampal neurogenesis in chronic epilepsy might be protective.

Metabotropic glutamate receptor 5 (mGluR5) regulates proliferation and differentiation of neuronal progenitors in the developmental hippocampus.

Metabotropic glutamate receptor 5 (mGluR5) is involved in neural stem cell self-renewal, proliferation, differentiation and survival. In this study, we aimed to further determine the role of mGluR5 in the development of hippocampus using mGluR5 deficit (mGluR5(-/-)) and wild type (mGluR5(+/+)) mice at different developmental ages. We showed that the number of BrdU, NeuroD and DCX immunopositive cells was reduced significantly in mGluR5(-/-) than in mGluR5(+/+) mice from postnatal 7 days (P7) to P28, but not at P60. The length and intensity of DCX immunopositive apical dendrites in the dentate gyrus of mGluR5(-/-) mice were much shorter and lower than in mGluR5(+/+) mice respectively at P14, P21 and P28. NeuN immunostaining indicated an accelerated maturation of hippocampal neurons in mGluR5(-/-) mice. When mGluR5(+/+) mice were treated with 2-methyl-6-(phenylethynyl) pyridine (MPEP), a selective antagonist of mGluR5, decreased proliferation of progenitor cells was observed in the hippocampus at early postnatal developmental stages. At P14, there were more BrdU(+) cells in the stratum granulosum and subgranular layer of the dentate gyrus in mGluR5(+/+) than in mGluR5(-/-) mice, but the percentage of BrdU(+)+NeuroD(+)/BrdU(+) in the dentate gyrus did not change significantly between the two genotypes of mice. Western Blot study suggested that programmed neuronal death was p53-dependent apoptosis in the developmental hippocampus in mGluR5(+/+) mice.

[Effect of glutamate on vascular endothelial growth factor mRNA and protein expressions in hypoxic rat astrocytes in vitro].

OBJECTIVE: To study the effect of glutamate on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in cultured rat astrocytes under hypoxia. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[A simple method for assessment of RNA integrity in laser capture microdissection samples].

OBJECTIVE: To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

[Neurons in the corpus callosum of rats: expression of Cav2.2 and their connection].

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.

Treatment of scrapie pathogen 263K with tetracycline partially abolishes protease-resistant activity in vitro and reduces infectivity in vivo.

OBJECTIVE: To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K. METHODS: 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. RESULTS: 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). CONCLUSION: Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.

[Effect of ligustrazine on cell proliferation in subventricular zone in rat brain with focal cerebral ischemia-reperfusion injury].

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].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Protein expression of human neuron-specific enolase and its antiserum preparation.

OBJECTIVE: To clone and express human neuron-specific enolase (HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Effects of ligustrazine on hippocampal dentate gyrus cell proliferation after focal cerebral ischemia in adult rats].

OBJECTIVE: To explore the effects of ligustrazine on cell proliferation in hippocampal dentate gyrus subgranular zone (SGZ) after focal cerebral ischemia in adult rats. METHODS: 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. RESULTS: 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. CONCLUSION: Ligustrazine possesses long lasting effect of promoting cell proliferation in the SGZ after focal cerebral ischemia in adult rats.

[Cloning, expression, and antibody preparation of nestin with immunohistochemical analysis].

OBJECTIVE: To obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development. METHODS: 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. RESULTS: 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. CONCLUSION: 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.