Morphine Induced Neuroprotection in Ischemic Stroke by Activating Autophagy Via mTOR-Independent Activation of the JNK1/2 Pathway.

Morphine (Mor) has exhibited efficacy in safeguarding neurons against ischemic injuries by simulating ischemic/hypoxic preconditioning (I/HPC). Concurrently, autophagy plays a pivotal role in neuronal survival during IPC against ischemic stroke. However, the involvement of autophagy in Mor-induced neuroprotection and the potential mechanisms remain elusive. Our experiments further confirmed the effect of Mor in cellular and animal models of ischemic stroke and explored its potential mechanism. The findings revealed that Mor enhanced cell viability in a dose-dependent manner by augmenting autophagy levels and autophagic flux in neurons subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pretreatment of Mor improved neurological outcome and reduced infarct size in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) at 1, 7 and 14 days. Moreover, the use of autophagy inhibitors nullified the protective effects of Mor, leading to reactive oxygen species (ROS) accumulation, increased loss of mitochondrial membrane potential (MMP) and neuronal apoptosis in OGD/R neurons. Results further demonstrated that Mor-induced autophagy activation was regulated by mTOR-independent activation of the c-Jun NH2- terminal kinase (JNK)1/2 Pathway, both in vitro and in vivo. Overall, these findings suggested Mor-induced neuroprotection by activating autophagy, which were regulated by JNK1/2 pathway in ischemic stroke.

Morphine Preconditioning Alleviates Ischemia/Reperfusion-induced Caspase-8-dependent Neuronal Apoptosis through cPKCγ-NF-κB-cFLIPL Pathway.

BACKGROUND: Perioperative cerebral ischemia/reperfusion injury is a major contributor to postoperative death and cognitive dysfunction in patients. It was reported that morphine preconditioning (MP) can mimic ischemia/hypoxia preconditioning to protect against ischemia/reperfusion injury. However, the mechanism of MP on the ischemia/reperfusion-induced neuronal apoptosis has not been fully clarified. METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) model of mice and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in primary cortical neurons were used to mimic ischemic stroke. In vivo, the infarct size was measured by using TTC staining; NDSS, Longa score system, and beam balance test were performed to evaluate the neurological deficits of mice; the expression of the protein was detected by using a western blot. In vitro, the viability of neurons was determined by using CCK-8 assay; the expression of protein and mRNA were assessed by using western blot, RT-qPCR, and immunofluorescent staining; the level of apoptosis was detected by using TUNEL staining. RESULTS: MP can improve the neurological functions of mice following MCAO/R (P<0.001, n=10 per group). MP can decrease the infarct size (P<0.001, n=10 per group) and the level of cleaved-caspase-3 of mice following MCAO/R (P<0.01 or 0.001, n=6 per group). MP can increase the levels of cPKCγ membrane translocation, p-p65, and cFLIPL, and decrease the levels of cleaved-caspase-8, 3 in neurons after OGD/R or MCAO/R 1 d (P<0.05, 0.01 or 0.001, n=6 per group). In addition, MP could alleviate OGD/R-induced cell apoptosis (P<0.001, n=6 per group). CONCLUSION: MP alleviates ischemia/reperfusion-induced Caspase 8-dependent neuronal apoptosis through the cPKCγ-NF-κB-cFLIPL pathway.

Upregulation of Spinal MDGA1 in Rats After Nerve Injury Alters Interactions Between Neuroligin-2 and Postsynaptic Scaffolding Proteins and Increases GluR1 Subunit Surface Delivery in the Spinal Cord Dorsal Horn.

Previous studies suggested that postsynaptic neuroligin-2 may shift from inhibitory toward excitatory function under pathological pain conditions. We hypothesize that nerve injury may increase the expression of spinal MAM-domain GPI-anchored molecule 1 (MDGA1), which can bind to neuroligin-2 and thereby, alter its interactions with postsynaptic scaffolding proteins and increase spinal excitatory synaptic transmission, leading to neuropathic pain. Western blot, immunofluorescence staining, and co-immunoprecipitation studies were conducted to examine the critical role of MDGA1 in the lumbar spinal cord dorsal horn in rats after spinal nerve ligation (SNL). Small interfering ribonucleic acids (siRNAs) targeting MDGA1 were used to examine the functional roles of MDGA1 in neuropathic pain. Protein levels of MDGA1 in the ipsilateral dorsal horn were significantly upregulated at day 7 post-SNL, as compared to that in naïve or sham rats. The increased levels of GluR1 in the synaptosomal membrane fraction of the ipsilateral dorsal horn tissues at day 7 post-SNL was normalized to near sham level by pretreatment with intrathecal MDGA1 siRNA2308, but not scrambled siRNA or vehicle. Notably, knocking down MDGA1 with siRNAs reduced the mechanical and thermal pain hypersensitivities, and inhibited the increased excitatory synaptic interaction between neuroligin-2 with PSD-95, and prevented the decreased inhibitory postsynaptic interactions between neuroligin-2 and Gephyrin. Our findings suggest that SNL upregulated MDGA1 expression in the dorsal horn, which contributes to the pain hypersensitivity through increasing the net excitatory interaction mediated by neuroligin-2 and surface delivery of GluR1 subunit in dorsal horn neurons.

Functional changes in fusional vergence-related brain areas and correlation with clinical features in intermittent exotropia using functional magnetic resonance imaging.

To explore the functional changes of the frontal eye field (FEF) and relevant brain regions and its role in the pathogenesis of intermittent exotropia (IXT) children via functional magnetic resonance imaging (fMRI). Twenty-four IXT children (mean age, 11.83 ± 1.93 years) and 28 normal control (NC) subjects (mean age, 11.11 ± 1.50 years) were recruited. During fMRI scans, the IXT children and NCs were provided with static visual stimuli (to evoke sensory fusion) and dynamic visual stimuli (to evoke motor fusion and vergence eye movements) with binocular disparity. Brain activation in the relevant brain regions and clinical characteristics were evaluated. Group differences of brain activation and brain-behavior correlations were investigated. For dynamic and static visual disparity relative to no visual disparity, reduced brain activation in the right FEF and right inferior occipital gyrus (IOG), and increased brain activation in the left middle temporal gyrus complex (MT+) were found in the IXT children compared with NCs. Significant positive correlations between the fusional vergence amplitude and the brain activation values were found in the right FEF, right IPL, and left cerebellum in the NC group. Positive correlations between brain activation values and Newcastle Control Scores (NCS) were found in the left MT+ in the IXT group. For dynamic visual disparity relative to static visual disparity, reduced brain activation in the right middle occipital gyrus, left cerebellum, and bilateral IPL was found in the IXT children compared with NCs. Significant positive correlations between brain activation values and the fusional vergence amplitude were found in the right FEF and right cerebellum in the NC group. Negative correlations between brain activation values and NCS were found in the right middle occipital gyrus, right cerebellum, left IPL, and right FEF in the IXT group. These results suggest that the reduced brain activation in the right FEF, left IPL, and cerebellum may play an important role in the pathogenesis of IXT by influencing fusional vergence function. While the increased brain activation in the left MT+ may compensate for this dysfunction in IXT children.

Sevoflurane induces neurotoxic effects on developing neurons through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway.

Children repeatedly exposed to anaesthesia have a high risk of cognitive impairment, but the mechanism of its regulation in this context is unknown. The objective of this study was to investigate the possible toxic mechanism of sevoflurane through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway. The hippocampal neuronal HT22 cell line was used in this study. The intervention group was treated with the WNK1 inhibitor WNK-463, CaN inhibitor FK506 and Drp-1 inhibitor Mdivi-1 respectively in the medium for 30 min before sevoflurane anaesthesia. The sevofluane group and all intervention group treated with 4.1% sevoflurane for 6 h. Compared with the control group, sevoflurane treatment decreased cell viability and increased cellular apoptosis. Our study found that WNK-463, FK506 and Mdivi-1 can all alleviate the sevoflurane-induced reduction in cell viability, decrease the cell apoptosis. In addition, WNK-463 pretreatment could inhibit the increase of WNK1 kinase and NKCC1 protein concentration caused by sevoflurane. Further, sevoflurane anaesthesia causes intracellular calcium overload, increases the expression of CaN and induces the dephosphorylation of Drp-1 protein at ser637, while CaN inhibitor FK506 pretreatment could reduce the dephosphorylation of Drp-1. Therefore, the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway plays an important role in sevoflurane-related neurotoxicity. Reducing intracellular calcium influx may be one of the important mechanism to ameliorate sevoflurane toxicity.

Activin A alleviates neuronal injury through inhibiting cGAS-STING-mediated autophagy in mice with ischemic stroke.

Activin A plays an essential role in ischemic stroke as a well-known neuroprotective factor. We previously reported that Activin A could promote white matter remyelination. However, the exact molecular mechanism of Activin A in neuronal protection post-stroke is still unclear. In this study, the middle cerebral artery occlusion/reperfusion (MCAO/R)-induced ischemic stroke mouse model and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated primary neurons were used to explore the molecular mechanism of Activin A-mediated neuroprotection against ischemic injuries. We found that Activin A significantly inhibits cGAS-STING-mediated excessive autophagy through the PI3K-PKB pathway, but not mTOR-dependent autophagy. Consequently, Activin A protected neurons against OGD/R-induced ischemic injury and improved cell survival in a dose-dependent manner. In addition, Activin A improved neurological functions and reduced infarct size of mice with MCAO/R-induced ischemic stroke by inhibiting autophagy. Furthermore, Activin A depended on ACVR1C receptor to exert neuroprotective effects in 1 h MCAO/R treated mice. Our findings showed that Activin A alleviated neuronal ischemic injury through inhibiting cGAS-STING-mediated excessive autophagy in mice with ischemic stroke, which may suggest a potential therapeutic target for ischemic stroke.

cPKCγ-Modulated Autophagy Contributes to Ischemic Preconditioning-Induced Neuroprotection in Mice with Ischemic Stroke via mTOR-ULK1 Pathway.

Neuron-specific conventional protein kinase C (cPKC)γ mediates cerebral hypoxic preconditioning (HPC). In parallel, autophagy plays a prosurvival role in ischemic preconditioning (IPC) against ischemic stroke. However, the effect of cPKCγ on autophagy in IPC still remains to be addressed. In this study, adult and postnatal 1-day-old C57BL/6 J wild-type (cPKCγ+/+) and knockout (cPKCγ-/-) mice were used to establish in vivo and in vitro IPC models. The results showed that IPC pretreatment alleviated neuronal damage caused by lethal ischemia, which could be suppressed by autophagy inhibitor 3-MA or bafilomycin A1. Meanwhile, cPKCγ knockout blocked IPC-induced neuroprotection, accompanied by significant increase of LC3-I to LC3-II conversion and Beclin 1 protein level, and a significant decrease in p62 protein level. Immunofluorescent staining results showed a decrease of LC3 puncta numbers in IPC-treated cPKCγ+/+ neurons with fatal ischemia, which was reversed in cPKCγ-/- neurons. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2448 and ULK1 at Ser 555, rather than p-Thr-172 AMPK, was detected in IPC-pretreated neurons upon lethal ischemic exposure. The present data demonstrated that cPKCγ-modulated autophagy via the mTOR-ULK1 pathway likely modulated IPC-induced neuroprotection.

Identification of brain endothelial cell-specific genes and pathways in ischemic stroke by integrated bioinformatical analysis.

BACKGROUND: Ischemic stroke (IS) is a life-threatening condition with limited treatment options; thus, finding the potential key genes for novel therapeutic targets is urgently needed. This study aimed to explore novel candidate genes and pathways of brain microvessel endothelial cells (ECs) in IS by bioinformatics analysis. MATERIALS AND METHODS: The gene expression profiles of brain tissues or brain ECs in IS mice were downloaded from the online gene expression omnibus (GEO) to obtain the differentially expressed genes (DEGs) by R software. Functional enrichment analyses were used to cluster the functions and signaling pathways of the DEGs, while DEG-associated protein-protein interaction network was performed to identify hub genes. The target microRNAs and competitive endogenous RNA networks of key hub genes were constructed by Cytoscape. RESULTS: Totally 84 DEGs were obtained from 6 brain tissue samples and 4 brain vascular EC samples both from IS mice in the datasets GSE74052 and GSE137482, with significant enrichment in immune responses, such as immune system processes and T-cell activation. Eight hub genes filtered by Cytoscape were validated by two other GEO datasets, wherein key genes of interest were verified by reverse transcription-polymerase chain reaction using an in vitro ischemic model of EC cultures. Our data indicated that AURKA and CENPF might be potential therapeutic target genes for IS, and Malat1/Snhg12/Xist-miR-297b-3p-CENPF, as well as Mir17 hg-miR-34b-3p-CENPF, might be RNA regulatory pathways to control IS progression. CONCLUSIONS: Our work identified two brain EC-specific expressed genes in IS, namely, AURKA and CENPF, as potential gene targets for IS treatment. In addition, we presented miR-297b-3p/miR-34b-3p-CENPF as the potential RNA regulatory axes to prevent pathogenesis of IS.

Modulation of Trans-Synaptic Neurexin-Neuroligin Interaction in Pathological Pain.

Synapses serve as the interface for the transmission of information between neurons in the central nervous system. The structural and functional characteristics of synapses are highly dynamic, exhibiting extensive plasticity that is shaped by neural activity and regulated primarily by trans-synaptic cell-adhesion molecules (CAMs). Prototypical trans-synaptic CAMs, such as neurexins (Nrxs) and neuroligins (Nlgs), directly regulate the assembly of presynaptic and postsynaptic molecules, including synaptic vesicles, active zone proteins, and receptors. Therefore, the trans-synaptic adhesion mechanisms mediated by Nrx-Nlg interaction can contribute to a range of synaptopathies in the context of pathological pain and other neurological disorders. The present review provides an overview of the current understanding of the roles of Nrx-Nlg interaction in the regulation of trans-synaptic connections, with a specific focus on Nrx and Nlg structures, the dynamic shaping of synaptic function, and the dysregulation of Nrx-Nlg in pathological pain. Additionally, we discuss a range of proteins capable of modulating Nrx-Nlg interactions at the synaptic cleft, with the objective of providing a foundation to guide the future development of novel therapeutic agents for managing pathological pain.

EphA4 regulates white matter remyelination after ischemic stroke through Ephexin-1/RhoA/ROCK signaling pathway.

Ischemic stroke, which accounts for nearly 80% of all strokes, leads to white matter injury and neurobehavioral dysfunction, but relevant therapies to inhibit demyelination or promote remyelination after white matter injury are still unavailable. In this study, the middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were used to establish the ischemic models. We found that Eph receptor A4 (EphA4) had no effect on the apoptosis of oligodendrocytes using TUNEL staining. In contrast, EphA4 promoted proliferation of oligodendrocyte precursor cells (OPCs), but reduced the numbers of mature oligodendrocytes and the levels of myelin-associated proteins (MAG, MOG, and MBP) in the process of remyelination in ischemic models in vivo and in vitro as determined using PDGFRα-EphA4-shRNA and LV-EphA4 treatments. Notably, conditional knockout of EphA4 in OPCs (EphA4fl/fl + AAV-PDGFRα-Cre) improved the levels of myelin-associated proteins and functional recovery following ischemic stroke. In addition, regulation of remyelination by EphA4 was mediated by the Ephexin-1/RhoA/ROCK signaling pathway. Therefore, EphA4 did not affect oligodendrocyte (OL) apoptosis but regulated white matter remyelination after ischemic stroke through the Ephexin-1/RhoA/ROCK signaling pathway. EphA4 may provide a novel and effective therapeutic target in clinical practice of ischemic stroke.

cPKCγ Deficiency Exacerbates Autophagy Impairment and Hyperphosphorylated Tau Buildup through the AMPK/mTOR Pathway in Mice with Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus (T1DM)-induced cognitive dysfunction is common, but its underlying mechanisms are still poorly understood. In this study, we found that knockout of conventional protein kinase C (cPKC)γ significantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles, but did not affect the activities of GSK-3ß and PP2A in the hippocampal neurons of T1DM mice. cPKCγ deficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice. Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγ deficiency in T1DM mice. Moreover, cPKCγ deficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro. The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor. In conclusion, these results indicated that cPKCγ promotes autophagy through the AMPK/mTOR signaling pathway, thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.

Morphine pretreatment protects against cerebral ischemic injury via a cPKCγ-mediated anti-apoptosis pathway.

It has been reported that morphine pretreatment (MP) can exert neuroprotective effects, and that protein kinase C (PKC) participates in the initiation and development of ischemic/hypoxic preconditioning in the brain. However, it remains unknown whether PKC is involved in MP-induced neuroprotection. The aim of the present study, which included in vivo and in vitro experiments, was to determine whether the conventional γ isoform of PKC (cPKCγ) was involved in the protective effects of MP against cerebral ischemic injury. The present study included an in vivo experiment using a mouse model of middle cerebral artery occlusion and an in vitro experiment using neuroblastoma N2a cells with oxygen-glucose deprivation (OGD). Furthermore, a cPKCγ antagonist, Go6983, was used to determine the involvement of cPKCγ in the protective effects of MP against cerebral ischemic injury. In the in vivo experiment, neurological deficits, ischemic infarct volume, neural cell damage, apoptosis and caspase-3 activation were evaluated. In the in vitro experiment, flow cytometry was used to determine the activation of caspase-3 in N2a cells with OGD. It was found that MP protected against cerebral ischemic injury. However, intracerebroventricular injection of the cPKCγ antagonist before MP attenuated the neuroprotective effect of MP and increased the activation of cleaved caspase-3. These findings suggested that MP may provide protection against cerebral ischemic injury via a cPKCγ-mediated anti-apoptosis pathway.

Herkinorin negatively regulates NLRP3 inflammasome to alleviate neuronal ischemic injury through activating Mu opioid receptor and inhibiting the NF-κB pathway.

Herkinorin is a novel opioid receptor agonist. Activation of opioid receptors, a member of G protein coupled receptors (GPCRs), may play an important role in Herkinorin neuroprotection. GPCRs may modulate NOD-like receptor protein 3 (NLRP3)-mediated inflammatory responses in the mechanisms of inflammation-associated disease and pathological processes. In this study, we investigated the effects of Herkinorin on NLRP3 and the underlying receptor and molecular mechanisms in oxygen-glucose deprivation/reperfusion (OGD/R)-treated rat cortex neurons. First, Western blot analysis showed that Herkinorin can inhibit the activation of NLRP3 and Caspase-1, decrease the expression of interleukin (IL)-1ß, and decrease the secretion of IL-6 and tumour necrosis factor α detected by enzyme-linked immunosorbent assay in OGD/R-treated neurons. Then we found that Herkinorin downregulated NLRP3 levels by inhibiting the activation of nuclear factor kappa B (NF-κB) pathway, reducing the phosphorylation level of p65 and IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Instead, both the mu opioid receptor (MOR) inhibitor, ß-funaltrexamine, and MOR knockdown reversed the effects of Herkinorin on NLRP3 (p < .05 or .01, n = 3 per group). Further, we found that the level of ß-arrestin2 decreased in the cell membrane and increased in the cytoplasm after Herkinorin pretreatment in OGD/R-treated neurons. In co-immunoprecipitation experiments, Herkinorin increased the binding of IκBα with ß-arrestin2, decreased the ubiquitination level of IκBα, and ß-arrestin2 knockdown reversed the effects of Herkinorin on IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Our data demonstrated that Herkinorin negatively regulated NLRP3 inflammasome to alleviate neuronal ischemic injury through inhibiting NF-κB pathway mediated primarily by MOR activation. Inhibition of the NF-κB pathway by Herkinorin may be achieved by decreasing the ubiquitination level of IκBα, in which ß-arrestin2 may play an important role.

[Changes in microglia number and Iba1 expression level in the prefrontal cortex of type 1 diabetic mice].

The aim of the present study was to observe the activation of microglia in the prefrontal cortex of type 1 diabetes mellitus (T1DM) mice, and the expression of the marker genes of the disease-associated microglia (DAM) associated with neurodegenerative diseases. Sixty healthy adult male C57BL/6J mice were randomly divided into two groups, normal control (CON) group and T1DM group. Streptozocin (STZ) was injected intraperitoneally to induce T1DM mice. The spatial learning and memory function of mice was detected by Morris water maze at the 8th week after the successful model establishment. The number and activation of microglia in the prefrontal cortex of mice were detected by immunofluorescence staining and Western blot. Changes in the mRNA level of several DAM molecular markers were detected by RT-FQ-PCR. The results showed that, compared with CON mice, the fasting blood glucose of T1DM mice increased significantly, while the body weight of T1DM mice decreased remarkably (P < 0.05). The escape latency of water maze in T1DM mice was longer than that in CON mice (P < 0.05). Compared with CON group, the Iba1 protein expression and the number of microglia in prefrontal cortex of T1DM group increased significantly (P < 0.05). In addition, the mRNA levels of several DAM markers in prefrontal cortex of T1DM group were increased significantly (P < 0.05). These results suggest that the microglia are activated and transformed to DAM type in the prefrontal cortex of T1DM mice.

In vivo reduction of hippocampal Caveolin-1 by RNA interference alters morphine addiction and neuroplasticity changes in male mice.

Prescription opioids are powerful pain-controlling medications that have both benefits and potentially serious risks. Morphine is one of the preferred analgesics that are widely used to treat chronic pain. However, chronic morphine exposure has been found to cause both functional and structural changes in several brain regions, including the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and hippocampus (HPC), which lead to addictive behavior. Caveolin-1 (Cav-1), a scaffolding protein of membrane lipid rafts (MLRs), has been shown to organize GPCRs and multiple synaptic signaling proteins within the MLRs to regulate synaptic signaling and neuroplasticity. Previously, we showed that in vitro morphine treatment significantly elevates Cav-1 expression and causes neuroplasticity changes. In this study, we confirmed that chronic morphine exposure can significantly increase Cav-1 expression (P < 0.05) and microtubule-associated protein (MAP-2)-positive neuronal dendritic growth in the hippocampus. Moreover, the rewarding effect and dendritic growth in the HPC induced by chronic morphine exposure were significantly inhibited by hippocampal Cav-1 knockdown. Together, these data suggest that Cav-1 in the hippocampus plays an essential role in the neuroplasticity changes that underlie morphine addiction behaviors.

The emerging role of kainate receptor functional dysregulation in pain.

Pain is a serious clinical challenge, and is associated with a significant reduction in quality of life and high financial costs for affected patients. Research efforts have been made to explore the etiological basis of pain to guide the future treatment of patients suffering from pain conditions. Findings from studies using KA (kainate) receptor agonist, antagonists and receptor knockout mice suggested that KA receptor dysregulation and dysfunction may govern both peripheral and central sensitization in the context of pain. Additional evidence showed that KA receptor dysfunction may disrupt the finely-tuned process of glutamic acid transmission, thereby contributing to the onset of a range of pathological contexts. In the present review, we summarized major findings in recent studies which examined the roles of KA receptor dysregulation in nociceptive transmission and in pain. This timely overview of current knowledge will help to provide a framework for future developing novel therapeutic strategies to manage pain.

Activin A improves the neurological outcome after ischemic stroke in mice by promoting oligodendroglial ACVR1B-mediated white matter remyelination.

Activin A plays important roles in ischemic injury and white matter remyelination, but its mechanisms are unclear. In this study, the adult male C57BL/6 J mice were used to establish the model of 1 h middle cerebral artery occlusion/reperfusion (MCAO/R) 1 d to 28 d-induced ischemic stroke in vivo. We found that the neurological outcome was positively correlated with the levels of myelin associated proteins (include MAG, CNPase, MOG and MBP, n = 6 per group) both in corpus callosum and internal capsule of mice with ischemic stroke. The dynamic changes of Luxol fast blue (LFB) staining intensity, oligodendrocyte (CC1+) and proliferated oligodendrocyte precursor (Ki67+/PDGFRα+) cell numbers indicated demyelination and spontaneous remyelination occurred in the corpus callosum of mice after 1 h MCAO/R 1 d-28 d (n = 6 per group). Activin receptor type I (ACVR1) inhibitor SB431542 aggregated neurological deficits, and reduced MAG, MOG and MBP protein levels of mice with ischemic stroke (n = 6 per group). Meanwhile, recombinant mouse (rm) Activin A enhanced the neurological function recovery, MAG, MOG and MBP protein levels of mice with 1 h MCAO/R 28 d. In addition, the injection of AAV-based ACVR1B shRNA with Olig2 promoter could reverse rmActivin A-induced the increases of CC1+ cell number, LFB intensity, MAG, MOG and MBP protein levels in the corpus callosum (n = 6 per group), and neurological function recovery (n = 10 per group) of mice with 1 h MCAO/R 28 d. These results suggested that Activin A improves the neurological outcome through promoting oligodendroglial ACVR1B-mediated white matter remyelination of mice with ischemic stroke, which may provide a potential therapeutic strategy for ischemic stroke.

IL-17A Neutralization Improves the Neurological Outcome of Mice With Ischemic Stroke and Inhibits Caspase-12-Dependent Apoptosis.

We previously reported that the levels of astrocyte-derived interleukin-17A (IL-17A) increased both in the peri-infarct region and cerebrospinal fluid (CSF) of mice with 1-h middle cerebral artery (MCA) occlusion/12-h reperfusion (1-h MCAO/R 12 h)-induced ischemic stroke. However, the effects of IL-17A neutralization on the neurological outcome of mice with ischemic stroke and its underlying molecular mechanism are unclear. In this study, we found that the intracerebroventricular injection of IL-17A-neutralizing monoclonal antibody (mAb; 2.0 µg) could reduce the infarct volume, alleviate neuron loss, and improve the neurological outcomes of mice with 1-h MCAO/R 24-h- or 3-day-induced ischemic-stroke mice. The IL-17A neutralization could also significantly inhibit the increase of pro-caspase-3 cleavage through caspase-12-dependent cell apoptosis, as well as preventing the decrease of antiapoptotic factor B-cell lymphoma 2 (Bcl-2) and the increase of proapoptotic Bcl-2-associated X protein (Bax) in the peri-infarct region of mice following ischemic stroke. In addition, we confirmed that the recombinant mouse (rm) IL-17A could significantly aggravate 1-h oxygen-glucose deprivation/24-h reoxygenation (1-h OGD/R 24 h)-induced ischemic injuries in cortical neurons in a dose-dependent manner, and the rmIL-17A could also exacerbate neuronal apoptosis through caspase-12 (not caspase-8 or caspase-9)-dependent pathway. These results suggest that IL-17A neutralization could improve the neurological outcome of mice with ischemic stroke through inhibiting caspase-12-dependent neuronal apoptosis.

SPARC Negatively Correlates With Prognosis After Transarterial Chemoembolization and Facilitates Proliferation and Metastasis of Hepatocellular Carcinoma via ERK/MMP Signaling Pathways.

Background: Transarterial chemoembolization (TACE) represents a widely accepted treatment procedure for intermediate stage or unresectable hepatocellular carcinoma (HCC). However, few studies have evaluated serologic prognosis factors in patients with HCC before TACE. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein affecting tumorigenesis and metastasis, and leading to poor prognosis in HCC. Therefore, to further explore the potential prognosis value of SPARC, the expression levels in the plasma of patients and its potential molecular mechanisms underlying the regulation of HCC were investigated in this study. Materials and Methods: The study population included 43 patients with HCC who underwent TACE. To evaluate the expression of SPARC in different grades of pathological tissues, the immunohistochemistry was performed on tissues from 89 patients with HCC. Lentiviral vectors carrying interference sequences, as well as vectors harboring the complete open reading frame of SPARC for the knockdown or overexpression of SPARC in HuH-7 or HepG2 cells, respectively, allowed us to determine the biological functions of SPARC in vitro and in vivo. We also evaluated the levels of phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) and matrix metalloproteinases 2/9 (MMP2/9) activation. Results: The association between serum levels of SPARC and the survival at different TNM and Barcelona-Clinic Liver Cancer (BCLC) stages in patients with HCC undergoing TACE were evaluated. We observed a significant upregulation of SPARC in high grade HCC tissues, predicting unfavorable prognosis, and suggesting an important tumor-promoting effect of SPARC. Functional studies indicated that downregulation of SPARC contributed to the inhibition of proliferation and metastasis of HuH-7 cells in vitro, whereas its overexpression led to opposite phenotypes. Mechanistically, decreased expression of SPARC resulted in dephosphorylation of ERK1/2 and deactivation of MMP2/9, thereby inhibiting growth and metastasis of HCC. Importantly, low expression levels of SPARC inhibited the formation of subcutaneous tumors in nude mice. Conclusions: SPARC was found to facilitate proliferation and metastasis of HCC via modulation of the ERK1/2-MMP2/9 signaling pathways. Our research has provided a glimpse on the biological mechanism of SPARC and might contribute to the eventual treatment of liver cancer.

Monocular Deprivation Affects Visual Cortex Plasticity Through cPKCγ-Modulated GluR1 Phosphorylation in Mice.

Purpose: To determine how visual cortex plasticity changes after monocular deprivation (MD) in mice and whether conventional protein kinase C gamma (cPKCγ) plays a role in visual cortex plasticity. Methods: cPKCγ membrane translocation levels were quantified by using immunoblotting to explore the effects of MD on cPKCγ activation. Electrophysiology was used to record field excitatory postsynaptic potential (fEPSP) amplitude with the goal of observing changes in visual cortex plasticity after MD. Immunoblotting was also used to determine the phosphorylation levels of GluR1 at Ser831. Light transmission was analyzed using electroretinography to examine the effects of MD and cPKCγ on mouse retinal function. Results: Membrane translocation levels of cPKCγ significantly increased in the contralateral visual cortex of MD mice compared to wild-type (WT) mice (P < 0.001). In the contralateral visual cortex, long-term potentiation (LTP) and the phosphorylation levels of GluR1 at Ser 831 were increased in cPKCγ+/+ mice after MD. Interestingly, these levels could be downregulated by cPKCγ knockout compared to cPKCγ+/++MD mice (P < 0.001). Compared to the right eyes of WT mice, the amplitudes of a-waves and b-waves declined in deprived right eyes of mice after MD (P < 0.001). There were no significant differences when comparing cPKCγ+/+ and cPKCγ-/- mice with MD. Conclusions: cPKCγ participates in the plasticity of the visual cortex after MD, which is characterized by increased LTP in the contralateral visual cortex, which may be a result of cPKCγ-mediated phosphorylation of GluR1 at Ser 831.