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.

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.

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.

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.

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.

[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.

Reciprocal control of ADAM17/EGFR/Akt signaling and miR-145 drives GBM invasiveness.

INTRODUCTION: Glioblastoma multiforme (GBM) is one of the most devastating brain malignancies worldwide and is considered to be incurable. However, the mechanisms underlying its aggressiveness remain unclear. METHODS: The expression of ADAM17 in tissue samples was detected by immunohistochemistry. Knockdown and rescue experiments were used to demonstrate the regulatory effect of ADAM17 on the invasion ability of GBM cells. Western Blot and qPCR were used to detect the expression of related proteins and RNAs. Moreover, a luciferase reporter assay was performed to verify whether miR-145 directly binds to the 3'-UTR of ADAM17. RESULTS: We revealed that ADAM17 was overexpressed in GBM tissues and correlated positively with poor prognosis. The knockdown of ADAM17 obviously suppressed the invasiveness of GBM cell lines. Furthermore, we found that knockdown of ADAM17 decreased activation of EGFR/Akt/C/EBP-ß signaling, and consequently upregulated miR-145 expression in GBM cell lines. Notably, miR-145 directly targeted the ADAM17 3'-UTR and suppressed expression levels of ADAM17. CONCLUSIONS: Our findings define an ADAM17/EGFR/miR-145 feedback loop that drives the GBM invasion. Reciprocal regulation between ADAM17 and miR-145 results in aberrant activation of EGFR signaling, suggesting that inhibition of ADAM17 expression can be an ideal therapeutic strategy for the treatment of GBM.

The dual role of KCNQ/M channels upon OGD or OGD/R insults in cultured cortical neurons of mice: Timing is crucial in targeting M-channels against ischemic injur ies.

KCNQ/M potassium channels play a vital role in neuronal excitability; however, it is required to explore their pharmacological modulation on N-Methyl- d-aspartic acid receptors (NMDARs)-mediated glutamatergic transmission of neurons upon ischemic insults. In the current study, both presynaptic glutamatergic release and activities of NMDARs were measured by NMDAR-induced miniature excitatory postsynaptic currents (mEPSCs) in cultured cortical neurons of C57 mice undergoing oxygen and glucose deprivation (OGD) or OGD/reperfusion (OGD/R). The KCNQ/M-channel opener, retigabine (RTG), suppressed the overactivation of postsynaptic NMDARs induced by OGD and then NO transient; RTG also decreased OGD-induced neuronal death measured with MTT assay, suggesting the beneficial role of KCNQ/M-channels for the neurons exposed to ischemic insults. However, when the neurons exposed to the subsequent reperfusion, KCNQ/M-channels played a differential role from its protective effect. OGD/R increased presynaptic glutamatergic release, which was further augmented by RTG or decreased by KCNQ/M-channel blocker, XE991. Reactive oxygen species (ROS) were produced partly in a NO-dependent manner. In addition, XE991 decreased neuronal injuries upon reperfusion measured with DCF and PI staining. Meanwhile, the addition of RTG upon OGD or XE991 upon reperfusion can reverse OGD or OGD/R-reduced mitochondrial membrane potential. Our present study indicates the dual role of KCNQ/M-channels in OGD and OGD/R, which will decide the fate of neurons. Provided that activation of KCNQ/M-channels has differential effects on neuronal injuries during OGD or OGD/R, we propose that therapy targeting KCNQ/M-channels may be effective for ischemic injuries but the proper timing is so crucial for the corresponding treatment.

Immediate remote ischemic postconditioning reduces cerebral damage in ischemic stroke mice by enhancing leptomeningeal collateral circulation.

Remote ischemic postconditioning (RIPC) is a promising neuroprotective strategy for ischemic stroke. Here, we employed a focal ischemic stroke mouse model to test the hypothesis that poststroke collateral circulation as a potent mechanism of action underlying the therapeutic effects of immediate RIPC. During reperfusion of cerebral ischemia, the mice were randomly assigned to receive RIPC, granulocyte colony-stimulating factor (G-CSF) as a positive control, or no treatment. At 24 hr, we found RIPC and G-CSF increased monocytes/macrophages in the dorsal brain surface and in the spleen, coupled with enhanced leptomeningeal collateral flow compared with nontreatment group. Blood monocytes depletion by 5-fluorouracil (5-FU) significantly limited the neuroprotection of RIPC or G-CSF treatment. The protein expression of proangiogenic factors such as Ang-2 was increased by ischemia, but treatment with either RIPC or G-CSF showed no further upregulation. Thus, immediate RIPC confers neuroprotection, in part, by enhancing leptomeningeal collateral circulation in a mouse model of ischemic stroke.

Interleukin-17A-mediated alleviation of cortical astrocyte ischemic injuries affected the neurological outcome of mice with ischemic stroke.

We previously reported that astrocytes are the main sources of interleukin (IL)-17A production that could aggravate neuronal injuries in ischemic stroke. However, the effects of IL-17A on ischemic astrocytes themselves and the underlying molecular mechanism are still unclear. In this study, we found that recombinant mouse (rm) IL-17A could significantly (P < 0.05 or <0.001) alleviate 1-hour oxygen-glucose deprivation (OGD)/reoxygenation (R) 24-hour-induced ischemic injuries in cortical astrocytes with a dose-dependent manner (n = 6 per group). The Western blot and cell cycle analysis results revealed that rmIL-17A significantly ( P < 0.05) inhibited procaspase-3 cleavage without affecting cell proliferation in 1-hour OGD/R 24-hour-treated cortical astrocytes (n = 6 per group). Among the five IL-17 receptor subunits (IL-RA, -RB, -RC, -RD, and -RE), only IL-17RA ( P < 0.01) and -17RC ( P < 0.05) membrane translocation (not messenger RNA and protein) levels were downregulated in cortical astrocytes following 1-hour OGD/reperfusion 24 hours, and rmIL-17A could significantly ( P < 0.05 or <0.001) inhibit this downregulation (n = 6 per group). To further verify the impact of IL-17A on the neurological outcome of ischemic stroke, we found that the intracerebroventricular injection of IL-17A neutralizing monoclonal antibody remarkably ( P < 0.001) reduced the astrocyte activation and improve neurological function ( P < 0.05 or <0.01) of mice following 1-hour middle cerebral artery occlusion/reperfusion (R) 3 to 7 days (n = 6 or 8 per group). These results suggested that IL-17A-mediated alleviation of cortical astrocyte ischemic injuries could affect the neurological outcome of mice with ischemic stroke, which might be mainly dependent on the cell apoptosis pathway through inhibiting the downregulation of IL-17RA and -17RC membrane translocations.

Downregulation of neuroligin1 ameliorates postoperative pain through inhibiting neuroligin1/postsynaptic density 95-mediated synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor GluA1 subunits in rat dorsal horns.

Neuroligin1 is an important synaptic cell adhesion molecule that modulates the function of synapses through protein-protein interactions. Yet, it remains unclear whether the regulation of synaptic transmission in the spinal cord by neruoligin1 contributes to the development of postoperative pain. In a rat model of postoperative pain induced by plantar incision, we conducted Western blot study to examine changes in the expression of postsynaptic membrane of neuroligin1, postsynaptic density 95 (PSD-95), and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor GluA1 and GluA2 subunits in the spinal cord dorsal horn after injury. The interaction between neuroligin1 and PSD-95 was further determined by using coimmunoprecipitation. Protein levels of neuroligin1 and GluA1, but not GluA2 and PSD-95, were significantly increased in the postsynaptic membrane of the ipsilateral dorsal horn at 3 h and 1 day after incision, as compared to that in control group (naïve). A greater amount of PSD-95 was coimmunoprecipitated with neuroligin1 at 3 h after incision than that in the control group. Intrathecal administration of small interfering RNAs (siRNAs) targeting neuroligin1 suppressed the expression of neuroligin1 in the spinal cord. Importantly, pretreatment with intrathecal neuroligin1 siRNA2497, but not scrambled siRNA or vehicle, prevented the upregulation of GluA1 expression at 3 h after incision, inhibited the enhanced neuroligin1/PSD-95 interaction, and attenuated postoperative pain. Together, current findings suggest that downregulation of spinal neuroligin1 expression may ameliorate postoperative pain through inhibiting neuroligin1/PSD-95 interaction and synaptic targeting of GluA1 subunit. Accordingly, spinal neuroligin1 may be a potential new target for postoperative pain treatment.

cPKCγ-Modulated Sequential Reactivation of mTOR Inhibited Autophagic Flux in Neurons Exposed to Oxygen Glucose Deprivation/Reperfusion.

We have reported that conventional protein kinase Cγ (cPKCγ)-modulated neuron-specific autophagy improved the neurological outcome of mice following ischemic stroke through the Akt-mechanistic target of rapamycin (mTOR) pathway. However, its detailed molecular mechanism remains unclear. In this study, primary cortical neurons from postnatal one-day-old C57BL/6J cPKCγ wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice suffering oxygen glucose deprivation/reperfusion (OGD/R) were used to simulate ischemia/reperfusion injury in vitro. A block of autophagic flux was observed in cPKCγ+/+ neurons under OGD/R exposure, characterized by accumulation of p62. Immunofluorescent results showed a decrease in colocalization between LC3 and Atg14 or Stx17 in cPKCγ+/+ neurons when compared with cPKCγ−/− neurons after OGD/R. However, the colocalization between LC3 and Lamp2 was barely decreased, indicating the presence of autolysosomes. The larger lysotracker-positive structures were also significantly increased. These results suggest that cPKCγ-induced inhibition of autophagy occurred at the stages of autophagosome formation, Stx17 anchoring, and the degradation of autolysosomes in particular. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2481 was dependent on the site of Ser 2448, which may have blocked autophagic flux. cPKCγ-modulated sequential reactivation of mTOR inhibited autophagic flux in neurons exposed to OGD/R, which may provide endogenous interventional strategies for stroke, especially ischemia/reperfusion injury.

cPKCγ-mediated down-regulation of UCHL1 alleviates ischaemic neuronal injuries by decreasing autophagy via ERK-mTOR pathway.

Stroke is one of the leading causes of death in the world, but its underlying mechanisms remain unclear. Both conventional protein kinase C (cPKC)γ and ubiquitin C-terminal hydrolase L1 (UCHL1) are neuron-specific proteins. In the models of 1-hr middle cerebral artery occlusion (MCAO)/24-hr reperfusion in mice and 1-hr oxygen-glucose deprivation (OGD)/24-hr reoxygenation in cortical neurons, we found that cPKCγ gene knockout remarkably aggravated ischaemic injuries and simultaneously increased the levels of cleaved (Cl)-caspase-3 and LC3-I proteolysis product LC3-II, and the ratio of TUNEL-positive cells to total neurons. Moreover, cPKCγ gene knockout could increase UCHL1 protein expression via elevating its mRNA level regulated by the nuclear factor κB inhibitor alpha (IκB-α)/nuclear factor κB (NF-κB) pathway in cortical neurons. Both inhibitor and shRNA of UCHL1 significantly reduced the ratio of LC3-II/total LC3, which contributed to neuronal survival after ischaemic stroke, but did not alter the level of Cl-caspase-3. In addition, UCHL1 shRNA reversed the effect of cPKCγ on the phosphorylation levels of mTOR and ERK rather than that of AMPK and GSK-3ß. In conclusion, our results suggest that cPKCγ activation alleviates ischaemic injuries of mice and cortical neurons through inhibiting UCHL1 expression, which may negatively regulate autophagy through ERK-mTOR pathway.

Sevoflurane neurotoxicity in neonatal rats is related to an increase in the GABAA R α1/GABAA R α2 ratio.

Exposure of neonatal rat to sevoflurane leads to neurodegeneration and deficits of spatial learning and memory in adulthood. However, the underlying mechanisms remain unclear. The type A γ-aminobutyric acid receptor (GABAA R) is a target receptor for sevoflurane. The present study intends to investigate the changes in GABAA R α1/α2 expression and its relationship with the neurotoxicity effect due to sevoflurane in neonatal rats. After a dose-response curve was constructed to determine minimum alveolar concentration (MAC) and safety was guaranteed in our 7-day-old neonatal rat pup mode, we conducted two studies among the following groups: (A) the control group; (B) the sham anesthesia group; and (C) the sevoflurane anesthesia group and all three groups were treated in the same way as the model. First, poly(ADP-ribose) polymerase-1 protein (PARP-1) expression was determined in the different brain areas at 6 hr after anesthesia. Second, the expression of PARP-1 and GABAA R α1/GABAA R α2 in the hippocampus area was tested by Western blotting at 6 hr, 24 hr, and 72 hr after anesthesia in all three groups. After 4 hr, with 0.8 MAC (2.1%) sevoflurane anesthesia, the PARP-1 expression was significantly higher in the hippocampus than the other brain areas (p < .05). Compared with Groups A and B, the expression of PARP-1 in the hippocampus of Group C significantly increased at 6 hr after sevoflurane exposure (216% ± 15%, p < .05), and the ratio of the α1/α2 subunit of GABAA R surged at 6 hr (126% ± 6%), 24 hr (127% ± 8%), and 72 hr (183% ± 22%) after sevoflurane exposure in the hippocampus (p < .05). Our study showed that sevoflurane exposure of 0.8 MAC (2.1%)/4 hr was a suitable model for 7-day-old rats. And the exposure to sevoflurane could induce the apoptosis of neurons in the early stage, which may be related to the transmission from GABAA R α2 to GABAA R α1.

Galanin suppresses proliferation of human U251 and T98G glioma cells via its subtype 1 receptor.

Galanin is a neuropeptide with a widespread distribution throughout the nervous and endocrine systems, and recent studies have shown an anti-proliferative effect of galanin on several types of tumors. However, whether and how galanin and its receptors are involved in the regulation of cell proliferation in glioma cells remains unclear. In this study, the roles of galanin and its subtype 1 receptor (GAL1) in the proliferation of human U251 and T98G glioma cells were investigated. We found that galanin significantly suppressed the proliferation of U251 and T98G cells as well as tumor growth in nude mice. However, galanin did not exert apoptotic or cytotoxic effects on these two cell lines. In addition, we showed that galanin decreased the proliferation of U251 and T98G cells via its GAL1 receptor. Finally, we found that the GAL1 receptor was involved in the suppressive effects of galanin by activating ERK1/2.

Determination of Brain-Regional Blood Perfusion and Endogenous cPKCγ Impact on Ischemic Vulnerability of Mice with Global Ischemia.

Conventional protein kinase C (cPKC)γ participated in cerebral hypoxic preconditioning-induced neuroprotection and affected the neurological outcome of ischemic stroked mice. As an independent predictor of ischemic stroke, the internal carotid artery occlusion (ICAO)-caused brain-regional ischemic injury may worsen the neurological outcome of patients. However, the brain-regional ischemic vulnerability and its underlying mechanism remain unclear. In this study, the bilateral ICAO (BICAO) model was applied in cPKCγ wild type (WT) and knockout (KO) mice to determine the cPKCγ impact on brain-regional ischemic vulnerability. The arterial spin labeling (ASL) imaging results showed that 7 days BICAO-induced global ischemia could cause significant blood perfusion loss in prefrontal cortex (69.13%), striatum (61.69%), hypothalamus (67.36%), hippocampus (69.82%) and midbrain (40.53%) of WT mice, along with neurological deficits. Nissl staining and Western blot results indicated that hypothalamus and midbrain had more severe neural cell loss than prefrontal cortex, striatum and hippocampus, which negatively coincided with endogenous cPKCγ protein levels but not blood perfusion loss and cPKCγ membrane translocation levels. Furthermore, we found that cPKCγ KO significantly aggravated the neuron loss in prefrontal cortex, striatum and hippocampus and abolish the regional ischemic vulnerability by using immunofluorescent staining with neuron-specific marker NeuN. Similarly, cPKCγ KO also significantly increased Caspase-3, -8 and -9 cleavage levels in prefrontal cortex, striatum, hippocampus, hypothalamus and midbrain of mice with 24 h BICAO. These results suggested that hypothalamus and midbrain are more vulnerable to ischemia, and endogenous cPKCγ affects the regional ischemic vulnerability through modulating Caspase-8 and -9 dependent cell apoptosis.

Overactivation of corticotropin-releasing factor receptor type 1 and aquaporin-4 by hypoxia induces cerebral edema.

Cerebral edema is a potentially life-threatening illness, but knowledge of its underlying mechanisms is limited. Here we report that hypobaric hypoxia induces rat cerebral edema and neuronal apoptosis and increases the expression of corticotrophin releasing factor (CRF), CRF receptor type 1 (CRFR1), aquaporin-4 (AQP4), and endothelin-1 (ET-1) in the cortex. These effects, except for the increased expression of CRF itself, could all be blocked by pretreatment with an antagonist of the CRF receptor CRFR1. We also show that, in cultured primary astrocytes: (i) both CRFR1 and AQP4 are expressed; (ii) exogenous CRF, acting through CRFR1, triggers signaling of cAMP/PKA, intracellular Ca(2+), and PKCε; and (iii) the up-regulated cAMP/PKA signaling contributes to the phosphorylation and expression of AQP4 to enhance water influx into astrocytes and produces an up-regulation of ET-1 expression. Finally, using CHO cells transfected with CRFR1(+) and AQP4(+), we show that transfected CRFR1(+) contributes to edema via transfected AQP4(+). In conclusion, hypoxia triggers cortical release of CRF, which acts on CRFR1 to trigger signaling of cAMP/PKA in cortical astrocytes, leading to activation of AQP4 and cerebral edema.

Conventional protein kinase Cß-mediated phosphorylation inhibits collapsin response-mediated protein 2 proteolysis and alleviates ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

We previously reported that conventional protein kinase C (cPKC)ß participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCß interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCß, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCß-mediated CRMP2 phosphorylation via the cPKCß-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCß activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCß modulates CRMP2 phosphorylation and proteolysis, and cPKCß activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (cPKC)ß-mediated phosphorylation could inhibit CRMP2 proteolysis and alleviate ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.