Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice,accompanied by the downregulation of DNA methyltransferases(DNMTs)in the brain.However,the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood.This study aimed to establish an in vitro model of hypoxic preconditioning,using a cultured mouse hippocampal neuronal cell line(HT22 cells),to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning.HT22 cells were divided into a control group,which received no exposure to hypoxia,a hypoxia group,which was exposed to hypoxia once,and a hypoxic preconditioning group,which was exposed to four cycles of hypoxia.To test the ability of hypoxic preadaptation to induce hypoxic tolerance,cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay.Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group.The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry.Compared with the hypoxia group,the expression levels of caspase-3 and spectrin,which are markers of early apoptosis and S-phase arrest,respectively,noticeably reduced in the hypoxic preconditioning group.Finally,enzyme-linked immunosorbent assay,real-time polymerase chain reaction,and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning.The results showed that compared with the control group,hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B.In conclusion,hypoxic preconditioning may exert anti-hypoxic neuroprotective effects,maintaining HT22 cell viability and inhibiting cell apoptosis.These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Potassium bisperoxo(1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases

Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells(HT_(22)) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT_(22) cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT_(22) cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT_(22) cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.

Spatial and temporal plasticity of synaptic organization in anterior cingulate cortex following peripheral inflammatory pain: multi-electrode array recordings in rats

To explore whether experiencing inflammatory pain has an impact upon intracortical synaptic organization, the planar multi-electrode array （MEA） technique and 2-dimensional current source density （2D-CSD） imaging were used in slice preparations of the anterior cingulate cortex （ACC） from rats. Synaptic activity across different layers of the ACC was evoked by deep layer stimulation through one electrode. The layer-localization of both local field potentials （LFPs） and the spread of current sink calculated by 2D-CSD analysis was characterized pharmacologically. Moreover, the induction of long-term potentiation （LTP） and changes in LTP magnitude were also evaluated. We found that under naive conditions, the current sink was initially generated in layer Ⅵ, then spread to layer Ⅴ and finally confined to layers Ⅱ-Ⅲ. This spatial pattern of current sink movement typically reflected changes in depolarized sites from deep layers （Ⅴ-Ⅵ） to superficial layers （Ⅱ-Ⅲ） where intra- and extra- cortical inputs terminate. In the ACC slices from rats in an inflamed state （for 2 h） caused by intraplantar bee-venom injection, the spatial profile of intra-ACC synaptic organization was significantly changed,showing an enlarged current sink distribution and a leftward shift of the stimulus-response curves relative to the naive and saline controls. The change was more distinct in the superficial layers （Ⅱ-Ⅲ） than in the deep site. In terms of temporal properties, the rate of LTP induction was significantly increased in layers Ⅱ-Ⅲ by inflammatory pain. However, the magnitude of LTP was not significantly enhanced by this treatment. Taken together, these results show that inflammatory pain results in distinct spatial and temporal plasticity of synaptic organization in the ACC, which may lead to altered synaptic transmission and modulation.