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Background: Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. The neuroprotective effect of CART on ischemic brain injury is determined. Methods: Primary cultured rat NSCs were utilized as the research subject. In vitro oxygen glucose deprivation OGD treatment was employed, and NSCs were extracted from SD pregnant rats following previous experimental protocols and identified through cell immunofluorescence staining. Subsequently, cellular immunofluorescence and EdU detection experiments further confirmed the findings obtained from CCK-8 analysis. The mechanism maybe through activating the CREB pathway. Keywords: CAMP-response element binding protein; apoptosis; cocaine amphetamine-regulated transcription peptide; neural stem cells; oxygen glucose deprivation. Abstract Background: Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. Grants and funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

TEXAS TECH UNIVERSITY

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Official websites use. Share sensitive information only on official, secure websites. We previously found that cocaine abuse could increase microRNA miR levels in the hippocampus; yet the roles of miR in cocaine-related abnormal psychiatric outcomes remain unknown. In this study, using the cocaine-induced conditioned place preference CPP mice model, we found that mice exhibit enhanced anxiety-like and depression-like behaviors during the cocaine extinction CE period of CPP, accompanied by obviously increased miR levels and decreased levels of 19 genes that are associated with synaptic plasticity, glia activity, and neurochemical microenvironments, in the ventral hippocampus vHP. Knockdown of miR in vHP in vivo reversed the changes in 15 of 19 potential gene targets of miR and rescued the abnormal anxiety-like and depression-like behavioral outcomes in CE mice. In addition, knockdown of miR suppressed the CE-enhanced microglia activity, inflammatory, apoptotic, and oxidative stress statuses in the vHP. With the data taken together, miR may be involved in cocaine-associated psychiatric problems, potentially via regulating the expressions of its gene targets that are related to synaptic plasticity and neurochemical microenvironments. Cocaine addiction is characterized by a loss of control over drug intake and a high rate of relapse. In the present study Figure 1 A , we propose a link between abnormal behaviors and the miR in cocaine-exposed mice. Managing the miR signal pathway in the vHP might be an effective approach for the treatment of abnormal behaviors caused by cocaine use. A Proposed model of the present study. B Experimental design and timeline. Saline-extinction SE -treated mice were used as controls. To assess the cell types in the vHP that miR is mainly expressed, the BaseScope method combined with immunostaining of specific cell markers was used in naive mice. A Illustration of miRexpressed cell types in the vHP of native mice left. Ratio of colocalization with miR and biomarkers to miRpositive cells right. Middle panels: injection site of virus green; scale bars, 1 mm. In this study, the elevated plus maze EPM and open field test OFT were used to assess anxiety-like behaviors, while the tail suspension test TST and forced swim test FST were used to examine depression-like behaviors in mice. A EPM. B OFT. C TST. D FST. The density of dendritic spine and synapse and the levels of synaptic proteins were examined to assess the synaptic plasticity in the vHP of mice. A Dendritic spines. Red arrows indicate the spines. B Synapses. Red arrows indicate synapses. In this study, GFAP and Iba-1 were used as marks for astrocyte and microglia, respectively, and the neurochemical microenvironments—including pro-inflammation factors, apoptotic molecules, and oxidative stress status—were examined in the vHP. Cocaine addicts with high anxiety and depression levels are likely to relapse to drug use. Local knockdown of miR expression in vivo rescued the abnormal behaviors and reversed CE-induced changes in target genes, synaptic plasticity, glia activity, and microenvironment in the vHP of mice. Several studies demonstrated that miR expressions are significantly increased 17 , 23 , 24 , 25 or decreased 7 , 26 in nervous diseases, such as seizure, ischemic stroke, and neuropathic pain. Blockade or activation of miR could suppress brain damage, 15 , 27 suggesting that targeting miR signaling has potential therapeutic effects on brain disorders. Previously, we found a remarkable upregulation of the miR level in the intact hippocampus by cocaine exposure. Consistent with previous results, CE significantly triggered miR expression in the vHP, accompanied by enhanced anxiety-like and depression-like behaviors in mice. A recent study reports that silencing miR in the brain alleviates depression-like behavior induced by chronic stress exposure in animals. Drug-induced neurotoxicity depends on changes in gene expression. Importantly, knockdown of vHP miR in vivo could restore 15 gene levels of them, indicating that these 15 genes were regulated by miR, which was involved in CE-induced abnormal behavioral outcomes. It has been well established that miRNAs act by binding to target mRNAs and initiating either cleavage or a reduction in the translational efficiency of the target mRNA, depending on the degree of sequence complementarity. Studies also show that Creb-1 and Bdnf are important molecular mechanisms underlying the plasticity deficit that are regulated by miR In addition, we also identified other genes regulated by miR Some of them are closely involved in synaptic plasticity and transmission, such as Snap23 , Sypl2 , Stxbp5 , Stxbp1 , Nfib , Grik3 , Gnao1 , and Drd5 ; 15 , 32 , 33 some contribute to glia and related microenvironments around neurons, such as Bdnf , Snap23 , Ticam2 , Ndnf , Nanog , and Cdh9 ; and most of them play important roles in psychiatric disorders, including depression, addiction, and epilepsy. We think that these target genes might be downstream of the miR directly regulated genes, such as Bdnf being regulated by Creb1 expression. Until now, no effective therapeutic medicine has been available for the treatment of addiction, partially because addictive drugs of abuse lead to complicated changes in gene expression and subsequent plasticity in the brain. Schratt et al. Later, Numakawa et al. It is intriguing to explore whether the altered levels of miR in vHP are a neuronal or glial response. In this study, BaseScope data showed that the expression of miR in the vHP was highly restricted in neurons, but not astrocytes or microglia, in naive mice. These results indicated that miR mainly localized in the glutamatergic neurons in the vHP. Studies have shown that miR negatively regulates the development of dendritic spine and synaptic plasticity in the hippocampus. The number of dendritic spines is closely associated with the strength of excitatory transmission. Most importantly, local knockdown of vHP miR in vivo restored the decreased levels of synaptic proteins, accompanied by an enhanced fEPSC in the CA1 of vHP, indicating that miR negatively regulates excitatory transmission in mice during the CE period. Most studies demonstrate the negative regulation of miR on hippocampal dendritic spine size 9 and density. In recent decades, increasing evidence has demonstrated that the microenvironment of the neuron could be damaged by these addictive drugs, always being due to the overactive glial cells, oxidative stress, or inflammatory or apoptotic status around neurons. Microglia, but not astrocytes, were reversed by miR inhibition, indicating that microglia may be potential intervention targets for miR Since we found that miR was mainly expressed in neurons of vHP but not glial cells, there must be a crosstalk mechanism between neurons and glia that miR plays its regulatory roles in the glia activities and neurochemical microenvironments. In summary, our studies link miR with the increased anxiety and depression by CE exposure. A limitation of this study is not identifying the role of miR in the crosstalk between neuron and glial cells under the condition of cocaine abuse. The results of this study demonstrate that the miR signaling pathway in the vHP contributes to the development of drug-related psychiatric problems, mechanically through regulating synaptic plasticity and microenvironment. All mice were allowed to acclimate for 7 days before receiving any experimental manipulation. CPP procedures were performed in all mice. Some cocaine-experienced mice were subjected to local knockdown treatment of miR in the vHP. Brain tissue was collected on day 33 from some mice, and the behavioral tests were carried out from day 34 in the other mice Figure 1 B. Cocaine CPP procedures were performed as described previously. During CPP acquisition conditioning, mice received an injection of saline 0. To train CPP extinction, mice were confined to the drug-paired chamber for 45 min once daily without any drug treatment. During the pre-test, CPP test, and extinction test, mice were allowed to freely access two chambers for 15 min. Time spent in the drug-paired chamber was recorded as the CPP score. The movement status of each mouse was recorded for 6 min. Immobility time during the last 4 min of the tests was measured. The EPM apparatus consists of four elevated arms 70 cm above the floor arranged in a cross pattern with two 1-cm walled arms open arms and two cm walled arms close arms. Mice were released from the central platform individually, and the behaviors were recorded for 5 min with a camera positioned overhead. Between each trail, the maze was cleaned with ethanol and water. The time spent in the open arms was measured. In the TST, each mouse was suspended 40 cm above the tabletop surface with adhesive tape placed 1 cm from the tip of the tail. Movement was recorded using a camera for 6 min, and the immobility duration in the last 4 min was calculated. Mice were considered immobile only when they hung motionless. Immobile time during last 4 min of the 6-min test was scored. Mice were judged to be in an immobile state whenever they remained floating in the water without struggling and only made movements necessary to remain afloat. Latency to immobility was defined as the time between an introduction to the chamber and the first instance of immobility. Mice were anesthetized with isoflurane and injected with 0. The injectors were kept in place for an additional 5 min to allow for virus diffusion. Then, the sections were incubated with the Cy3- and fluorescein isothiocyanate FITC -labeled secondary antibodies dilution, Sigma, St. Negative control sections were performed by replacing the primary antibodies with PBS buffer. The number of double-stained cells was counted by software Leica, Wetzlar, Germany. Then, we proceeded to the normal immunofluorescence assay described previously after performing the RNAscope 2. Other primers of this study are listed in Supplemental Methods. Mice were deeply anesthetized with isoflurane and decapitated, and the vHP region was separated immediately. In this study, dendrites and spines were stained using the Golgi staining method and the GFP-labeled virus method. Dendritic spines can be shown by GFP expression. Mouse brains were removed, and the vHP region was separated immediately on ice. Western blot was performed as described previously. The protocols were performed as described previously. For brain slice preparation, mice were deeply anesthetized with isoflurane and decapitated. Brains were quickly removed and submerged in cutting solution in millimolar as follows: NaCl, 2. After 30 min of stable baseline recording, LTP was induced via four theta burst stimulation TBS trains each with 10 bursts containing 4 pulses at Hz and repeating at 5 Hz , given at s intervals. Slopes of eight consecutive fEPSP responses were averaged as an index for synaptic intensity. Statistical analysis was carried out using GraphPad Prism software. All authors read and approved the final manuscript. The authors report no biomedical financial interests. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Mol Ther Nucleic Acids. Find articles by Yuehan Li. Find articles by Xue Lu. Find articles by Jiaxun Nie. Find articles by Panpan Hu. Find articles by Feifei Ge. Find articles by Ti-Fei Yuan. Find articles by Xiaowei Guan. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

Nanjing where can I buy cocaine