The Two-Pore Domain Potassium Channel TREK-1 Promotes Blood-Brain Barrier Breakdown and Exacerbates Neuronal Death After Focal Cerebral Ischemia in Mice.

Earlier studies have shown the neuroprotective role of TWIK-related K+ channel 1 (TREK-1) in global cerebral and spinal cord ischemia, while its function in focal cerebral ischemia has long been debated. This study used TREK-1-deficient mice to directly investigate the role of TREK-1 after focal cerebral ischemia. First, immunofluorescence assays in the mouse cerebral cortex indicated that TREK-1 expression was mostly abundant in astrocytes, neurons, and oligodendrocyte precursor cells but was low in myelinating oligodendrocytes, microglia, or endothelial cells. TREK-1 deficiency did not affect brain weight and morphology or the number of neurons, astrocytes, or microglia but did increase glial fibrillary acidic protein (GFAP) expression in astrocytes of the cerebral cortex. The anatomy of the major cerebral vasculature, number and structure of brain micro blood vessels, and blood-brain barrier integrity were unaltered. Next, mice underwent 60 min of focal cerebral ischemia and 72 h of reperfusion induced by the intraluminal suture method. TREK-1-deficient mice showed less neuronal death, smaller infarction size, milder blood-brain barrier (BBB) breakdown, reduced immune cell invasion, and better neurological function. Finally, the specific pharmacological inhibition of TREK-1 also decreased infarction size and improved neurological function. These results demonstrated that TREK-1 might play a detrimental rather than beneficial role in focal cerebral ischemia, and inhibition of TREK-1 would be a strategy to treat ischemic stroke in the clinic.

Soluble epoxide hydrolase inhibitor attenuates BBB disruption and neuroinflammation after intracerebral hemorrhage in mice.

Intracerebral hemorrhage (ICH) is a devastating disease with high mortality and morbidity. Soluble epoxide hydrolase (sEH) is the key enzyme in the epoxyeicosatrienoic acids (EETs) signaling. sEH inhibition has been demonstrated to have neuroprotective effects against multiple brain injuries. However, its role in the secondary injuries after ICH has not been fully elucidated. Here we tested the hypothesis that 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), a potent and highly selective sEH inhibitor, suppresses inflammation and the secondary injuries after ICH. Adult male C57BL/6 mice were subjected to a collagenase-induced ICH model. TPPU alleviated blood-brain barrier damage, inhibited inflammatory response, increased M2 polarization of microglial cells, reduced the infiltration of peripheral neutrophils. In addition, TPPU attenuated neuronal injury and promoted functional recovery. The results suggest that sEH may represent a potential therapeutic target for the treatment of ICH.

Exosomes derived from astrocytes after oxygen-glucose deprivation promote differentiation and migration of oligodendrocyte precursor cells in vitro.

BACKGROUND: Excessive release of glutamate, oxidative stress, inflammation after ischemic brain injury can lead to demyelination. Astrocytes participate in the maturation and differentiation of oligodendrocyte precursor cells (OPCs), and play multiple roles in the process of demyelination and remyelination. Here, we studied the role of Astrocyte-derived exosomes (AS-Exo) under ischemic conditions in proliferation, differentiation and migration of OPCs in vitro. METHODS AND RESULTS: Exosomes were collected from astrocytes supernatant by differential centrifugation from control astrocytes (CTexo), mild hypoxia astrocytes (O2R24exo) which were applied oxygen-glucose deprivation for 2 h and reperfusion for 24 h (OGD2hR24h) and severe hypoxia astrocytes (O4R24exo) which were applied oxygen-glucose deprivation for 4 h and reperfusion for 24 h (OGD4hR24h). Exosomes (20 µg/ml) were co-cultured with OPCs for 24 h and their proliferation, differentiation and migration were detected. The results showed that AS-Exo under severe hypoxia (O4R24exo) inhibit the proliferation of OPCs. Meanwhile, all exosomes from three groups can promote OPCs differentiation and migration. Compared to control, the expressions of MAG and MBP, markers of mature oligodendrocytes, were significantly increased in AS-Exo treatment groups. AS-Exo treatment significantly increased chemotaxis for OPCs. CONCLUSIONS: AS-Exo improve OPCs' differentiation and migration, whereas AS-Exo with severe hypoxic precondition suppress OPCs' proliferation. AS-Exo may be a potential therapeutic target for myelin regeneration and repair in white matter injury or other demyelination related diseases.

Tamoxifen promotes white matter recovery and cognitive functions in male mice after chronic hypoperfusion.

Cerebral white matter lesions (WMLs) induced by chronic cerebral hypoperfusion are one of the major components of stroke pathology and closely associated with cognitive impairment. However, the repair and related pathophysiology of white matter after brain injury remains relatively elusive and underexplored. Successful neuroregeneration is a method for the potential treatment of central nervous system (CNS) disorders. A non-steroidal estrogen receptor modulator, Tamoxifen, is an effective inhibitor of cell-swelling-activated anion channels and can mimic neuroprotective effects of estrogen in experimental ischemic stroke. However, its remains unclear whether Tamoxifen has beneficial effects in the pathological process after WMLs. In the present study, we investigated the efficacy of Tamoxifen on multiple elements of oligovascular niche of the male C57BL/6 mice brain after bilateral carotid artery stenosis (BCAS) - induced WMLs. Tamoxifen was injected intraperitoneally once daily from 1 day after BCAS until 1 day before sacrificed. Following chronic hypoperfusion, BCAS mice presented white matter demyelination, loss of axon-glia integrity, activated inflammatory response, and cognitive impairments. Tamoxifen treatment significantly facilitated functional restoration of working memory impairment in mice after white matter injury, thus indicating a translational potential for this estrogen receptor modulator given its clinical safety and applicability for WMLs, which lack of currently available treatments. Furthermore, Tamoxifen treatment reduced microglia activation and inflammatory response, favored microglial polarization toward to the M2 phenotype, enhanced oligodendrocyte precursor cells proliferation and differentiation, and promoted remyelination after chronic hypoperfusion. Together, our data indicate that Tamoxifen could alleviate white matter injury and play multiple targets protective effects following chronic hypoperfusion, which is a promising candidate for the therapeutic target for ischemic WMLs and other demyelination diseases associated cognitive impairment.

Autophagy inhibition exerts neuroprotection on white matter ischemic damage after chronic cerebral hypoperfusion in mice.

Autophagy plays vital roles in the pathophysiology of many central nervous system diseases. Emerging evidence indicates that autophagy has both detrimental and protective effects in ischemic cerebral injury. This study aimed to investigate the temporal pattern of autophagy activation in the white matter of bilateral common carotid artery stenosis (BCAS) mouse model by immunofluorescence and western blotting. The effect of wortmannin, an autophagy inhibitor, against hypoperfusion induced white matter injury (WMI) was studied by immunofluorescence and eight-arm radial maze test. We found that autophagy was initially activated in the white matter 3 days after BCAS, and then suppressed by day 10, and was activated again at day 30. Administration of wortmannin during the first three days after BCAS revealed protective effects on axon-glia integrity and against the cognitive injury induced by the chronic hypoperfusion. The results indicated the possible link between autophagy and white matter ischemic damage after chronic cerebral hypoperfusion. Modulation of autophagy in a time course dependent manner may broaden the insight on the treatment of WMI.

Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating medical emergency with high mortality and severe neurological deficit. ICH-related poor outcomes are due to a combination of pathological processes that could be complicated by secondary insults. TWIK-related K+ channel 1 (TREK-1) is a two-pore-domain potassium channel that is highly expressed in the mammalian nervous system. Previous studies have shown that TREK-1 channels play important roles in various central nervous system diseases. However, its role in the secondary injuries after intracerebral hemorrhage remains unknown. In this study, we explored the function of TREK-1 in secondary blood-brain barrier injuries and neuroinflammation after intracerebral hemorrhage in mice. METHODS: Adult male TREK-1-/- mice and WT mice were subjected to a collagenase-induced ICH model. Immunostaining, western blot, and enzyme-linked immunosorbent assay were used to assess inflammatory infiltration and neuronal death. Blood-brain barrier compromise was assessed using electron microscopy and Evans Blue dye injection on days 1 and 3 after intracerebral hemorrhage. Magnetic resonance imaging and behavioral assessments were conducted to evaluate the neurologic damage and recovery after intracerebral hemorrhage. RESULTS: Genetic deficiency of TREK-1 channel exacerbated blood-brain barrier impairment and promoted cerebral edema after intracerebral hemorrhage. Meanwhile, TREK-1 deficiency aggravated focal inflammatory featured by the increased recruitment of microglia and neutrophils, the enhanced secretion of proinflammatory factors interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and cell adhesion molecules (CAMs). Furthermore, TREK-1 deficiency promoted neuronal injury and neurological impairment. CONCLUSIONS: These results establish the first in vivo evidence for the protective role of TREK-1 in blood-brain barrier injury and neuroinflammation after intracerebral hemorrhage. TREK-1 may thereby be harnessed to a potential therapeutical target for the treatment of intracerebral hemorrhage.

Inhibition of Astrocyte Connexin 43 Channels Facilitates the Differentiation of Oligodendrocyte Precursor Cells Under Hypoxic Conditions In Vitro.

Oligodendrocyte precursor cells (OPCs) proliferation and differentiation are essential for remyelination after white matter injury. Astrocytes could promote oligodendrogenesis after white matter damage whereas the underlying mechanisms are unknown. In this study, the role of astrocytic connexin43 (Cx43) hemichannels involved in OPC proliferation and differentiation in chronic hypoxia was evaluated. In an astrocyte-OPC co-culture chronic hypoxia model, OPCs became proliferative but failed to mature into oligodendrocytes. Application of astrocytic Cx43 blockers attenuated astrocyte activation, suppressed Cx43 hemichannel uptake activity and glutamate release induced by hypoxia, as well as improved OPC differentiation. Moreover, AMPA but not NMDA glutamate receptor antagonist rescued OPC differentiation in hypoxia. In conclusion, these findings suggested that astrocytic Cx43 hemichannel inhibition could potentially improve OPC maturation by attenuating AMPAR-mediated glutamate signaling. Astrocytic Cx43 hemichannels could serve as a potential therapeutic target for remyelination after chronic hypoxia.

[Effect of Electroacupuncture Stimulation of Sensitized Acupoints on Bowel Dysfunction in Rats with Diarrhea-predominant Irritable Bowel Syndrome].

OBJECTIVE: To investigate the effect of electroacupuncture(EA)stimulation of sensitized acupoints on bowel dysfunction in diarrhea-predominant irritable bowel syndrome(D-IBS)rats. METHODS: Fifty SD rats were randomly divided into control, model, EA sensitized acupoint and EA non-sensitized acupoint groups, with 20 rats in the model group and 10 rats in each of the other 3 groups. The D-IBS model was established by chronic restraint stress and intragastric administration of folium sennae (0.3 g/mL, 10 mL/kg), once daily for 2 weeks, followed by two weeks' restraint stress stimulation. The sensitized acupoints were determined by locating the extravasation points of Evans Blue (EB) dye after tail-intravenous injection, and stimulated with EA (2 mA, 2 Hz) for 30 min, once daily for 7 consecutive days. For rats of the EA non-sensitized acupoint group, bilateral BL 15 were stimulated with the same parameters and same stimulation duration. The rats' bowel mobility was evaluated by Bristol stool scale (BSS), loose stools rate and diarrhea index. RESULTS: After modeling, the BSS, loose stool rate, and diarrhea index were significantly increased in the model group relevant to the control group (P<0.05). After the treatment, the BSS, loose stool rate, and diarrhea index on day 7 were considerably lowered in the EA-BL 25 group (P<0.05) but not in EA-BL15 group (P<0.05), suggesting a better therapeutic effect of EA of the sensitized acupoint. No significant changes were found in the abovementioned 3 indexes on day 4 after the treatment relevant to the model group (P<0.05). CONCLUSIONS: EA stimulation of the sensitized acupoint can improve diarrhea in D-IBS rats.

Soluble epoxide hydrolase inhibition Promotes White Matter Integrity and Long-Term Functional Recovery after chronic hypoperfusion in mice.

Chronic cerebral hypoperfusion induced cerebrovascular white matter lesions (WMLs) are closely associated with cognitive impairment and other neurological deficits. The mechanism of demyelination in response to hypoperfusion has not yet been fully clarified. Soluble epoxide hydrolase (sEH) is an endogenous key enzyme in the metabolic conversion and degradation of P450 eicosanoids called epoxyeicosatrienoic acids. Inhibition of sEH has been suggested to represent a prototype "combination therapy" targeting multiple mechanisms of stroke injury with a single agent. However, its role in the pathological process after WMLs has not been clarified. The present study was to investigate the role of a potent sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on multiple elements in white matter of mice brain after chronic hypoperfusion. Adult male C57BL/6 mice were subjected to bilateral carotid artery stenosis (BCAS) to induce WMLs. Administration of TPPU significantly inhibited microglia activation and inflammatory response, increased M2 polarization of microglial cells, enhanced oligodendrogenesis and differentiation of oligodendrocytes, promoted white matter integrity and remyelination following chronic hypoperfusion. Moreover, these cellular changes were translated into a remarkable functional restoration. The results suggest that sEH inhibition could exert multi-target protective effects and alleviate cognitive impairment after chronic hypoperfusion induced WMLs in mice.

Deficiency of TREK-1 potassium channel exacerbates secondary injury following spinal cord injury in mice.

Spinal cord injury (SCI) involves complex pathological process which can be complicated by secondary injury. TREK-1 is a member of the two-pore domain potassium (K2P) channel family, which can be modulated by a number of physiological and pathological stimuli. Recent studies suggest that TREK-1 plays an active role in depression, pain and neuroprotection. However, its role in the pathological process after SCI remains unclear. In this study, we tested the expression and function of TREK-1 in spinal cord of mice after traumatic SCI. TREK-1 was widely expressed in mice spinal cord, including astrocytes and neurons. Deficiency of TREK-1 significantly exacerbated focal inflammatory responses as indicated by the increased accumulation of microglia/macrophage as well as pro-inflammatory factor interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha expression. Meanwhile, TREK-1 knockout mice showed enhanced reactive astrogliosis, chondroitin sulphate proteoglycans (CSPGs) production and decreased glutamate transporter-1 expression compared to the wide-type mice after SCI. Furthermore, TREK-1 deficiency promoted neurons and oligodendrocytes apoptosis, aggravated demyelination, cavity formation and retarded motor recovery. In summary, our findings provide the first in vivo evidence suggesting that TREK-1 may thereby constitute a promising therapeutic target to treat acute SCI.