Effect Of Transient Receptor Potential Vanilloid4on Cerebral Ischemia/Reperfusion Injury And Its Underlying Molecular Mechanisms

IntroductionWith the acceleration of global population aging, the incidence of ischemic cerebrovascular disease increases accordingly. Because of its high morbidity, disability and mortality, ischemic cerebrovascular disease has become one of the most critical health problems, which brings individuals, families and society tremendous mental stress and economic burden. Prevention and treatment of cerebral ischemic injury has been the forefront and focus of neuroscience research field. At p5esent, the principle of the treatment of cerebral ischemia in the clinic is to restore the blood and oxygen supply as early as possible through thrombolytic therapy to prevent neuronal injury and rescue its function. However, ischemia-reperfusion often further aggravates brain injury, i.e. ischemia/reperfusion injury, which eventually leads to neuronal necrosis or apoptosis. Therefore, it is of great significance to explore the pathogenesis and to search effective therapeutic targets to improve the cure rate and living quality of patients with cerebrovascular disease.The mechanisms underlying cerebral ischemia/reperfusion injury are complex, mainly including glutamate excitatory toxicity, intracellular calcium ([Ca2+]i) overload, free radicals-and nitric oxide-induced injury, cerebral edema, etc. Among them, glutamate excitatory toxicity has been proved to play a vital role in cerebral ischemic injury induced neural death. However, direct blocking glutamate receptors failed in the treatment of cerebral ischemic stroke in the clinical trials. Therefore, it will be very meaningful to explore mechanisms underlying intracellular calcium overload-induced neuronal injury that is independent of glutamate excitatory toxicity.Transient receptor potential (TRP) family is an important class of non-selective cation channels. TRP vanilloid4(TRPV4) is widely expressed in the central nervous system, including hippocampus, cortex, thalamus, and cerebellum. As a calcium-selective cation channel, TRPV4can lead to inward currents (mainly Ca2+influx) when it is activated. Recently, there is accumulating evidence that TRP family members play an important role in the pathogenesis of cerebral ischemia/reperfusion injury. For example, TRP melastatin2, TRP melastatin7, TRP canonical6and some other TRP members have been reported to be involved in cerebral ischemic neuronal injury. TRPV4has become one of hotspots in the biological research field since it was discovered, because it can be activated by multiple stimuli such as hypotonic stimulation, cell swelling, mild heat, arachidonic acid (AA) and its metabolism epoxyeicosatrienoic acids (EET). During cerebral ischemia, energy metabolism failure can lead to cellular edema, which can activate TRPV4through changes in membrane mechanical tension. Besides this, energy metabolism failure can increase the production of arachidonic acid (AA), which can also activate TRPV4through its metabolism epoxyeicosatrienoic acids, suggesting that the ischemia/reperfusion injury may affect the expression and function of TRPV4. It is reported that blockage of TRPV4can rescue the injury of hippocampal astrocytes exposed to oxidative stress, as well as the hippocampal CA1neuronal death caused by oxygen glucose deprivation, which suggesting blockage of TRPV4may alleviate the brain injury caused by cerebral ischemia.We previously reported that activation of TRPV4had effects on voltage-gated ion channels and TRPV1receptor through modulating intracellular signaling pathways (such as PKA, PKC and PKG, etc) to increase the primary sensory neuronal excitability. The study carried out on retinal ganglion cells found that TRPV4agonist colud dose-dependently enhance [Ca2+]i, increase cellular excitability and lead to cell apoptosis. Activation of TRPV4can increase the frequency of miniature excitatory postsynaptic current in cultured hippocampal neurons, indicating that activation of TRPV4may increase the release of glutamate.In this study, we firstly study whether TRPV4is involved in the pathogenesis of cerebral ischemia/reperfusion injury, and then explore the underlying mechanisms to provide the new target and strategy for the clinical treatment of cerebral ischemia/reperfusion injury.Objective1. To study the involvement of TRPV4in the pathogenesis of cerebral ischemia/reperfusion injury.2. To explore the molecular mechanisms underlying TRPV4-mediated cerebral ischemia/reperfusion injury.Part I Involvement of TRPV4in the pathogenesis of cerebral ischemia/reperfusion injuryMaterials and Methods1. Preparation for focal cerebral ischemia/reperfusion model:focal cerebral ischemia model was induced by occlusion of the middle cerebral artery (MCAO) for60min using intraluminal filament technique.2. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot:the levels of TRPV4mRNA and protein expression in hippocampus at different time after reperfusion were examined using RT-PCR and Western blot.3. TTC staining:after24h of MCAO, brain slices were reacted with TTC solution. Normal brain tissues appear red while cerebral infracted tissues appear white. Sum of the white volume were examined at24h after reperfusion of MCAO to determine the neuroprotection, dose-dependence and the effective time window of TRPV4antagonist on cerebral ischemia.4. Toluidine blue staining:Coronal brain sections were stained with toluidine blue until the pyramidal neurons in the hippocampual CA1region was clear.Results1. In ipsilateral hippocampus, the level of TRPV4mRNA was up-regulated within2to72h after reperfusion and TRPV4protein expression was up-regulated within4to48h after reperfusion. The up-regulation of TRPV4protein reached at a peak at18h after reperfusion, gradually decreased after that and reached to the normal level at72h after reperfusion.2. Administration of TRPV4selective antagonist HC-067047, within the dosage ranging from0.1to30μM/2uL/mouse, dose-dependently decreased brain infarction at24h after reperfusion. Treatment with HC-067047(10μM/2μL/mouse) had neuronprotection with at least a12h efficacious time window. I.c.v. injection of TRPV4non-selective antagonist ruthenium red could also decrease brain infarction at24h after reperfusion.3. Administration of TRPV4agonist4α-PDD caused neuronal death in hippocampal CA1area.ConclusionTRPV4protein expression is up-regulated during4-48h after cerebral ischemia/reperfusion, while blockage of TRPV4exerts protection against cerebral ischemia/reperfusion injury. Part Ⅱ Molecular mechanisms underlying blockage of TRPV4-mediated protective effect of cerebral ischemia/reperfusion injuryMaterials and Methods1. Establishing cell model of cerebral ischemia/reperfusion injury:During cerebral ischemia, energy metabolism failure can lead to cellular edema Our early research had proved that cellular edem could actiate TRPV4, so we choose hypotonic stimulation (240mOsm/kg) to mimic the cerebral edema after ischemic injury.2. Field potential recording:When hippocampal slices were prepared sucessfully, the slope of Schaffer collateral-CA1synaptic excitatory post-synaptic potentiation (EPSP) and paired-pulse facilitation (PPF) in CA1neurons were examined.3. Whole cell patch clamp recording:When hippocampal slices were prepared sucessfully, miniature excitatory postsynaptic current (mEPSC) and NMDA-induced current (INMDA) in hippocampal CA1neurons and high voltage-gated calcium current (Ica) in hippocampal CA3neurons were recorded.4. Toluidine blue staining:see part Ⅰ.Results1. Results of field potential recording shows that application of TRPV4agonist 4a-PDD can enhance the Schaffer collateral-CA1synaptic slope of EPSP and reduced PPF in hippocampus slice, indicating that actiation of TRPV4can increase pre-synaptic release of neurotransmitters.When decreasing the osmotic pressure of the hippocampal slice extracellular perfusate to240mOsm/kg (hypotonic stimulation), the Schaffer collateral-CA1synaptic slope in hippocampus was enhanced while PPF reduced. Application of TRPV4selective antagonist HC-067047can block the hypotonic stimulation-enhanced release of pre-synaptic neurotransmitters.2. Results of patch-clamp recording shows that application of hypotonic stimulation increased the frequency and amplitude of mEPSC, indicating that hypertonic stimulation not only increases pre-synaptic neurotransmitters but also enhances the activity of post-synaptic glutamate receptor.3. Hypotonic stimulation almost had no effect on the amplitude, current-voltage curve (Ⅰ-Ⅴ curve), voltage-dependent activation or inactivation curve of lCa. in hippocampal CA1presynaptic pyramidal neurons. Morever, hypotonic stimulation-induced enhancement of EPSP slope could not be attenuated by N-or P/Q-type of Voltage-gated calcium channel (VGCC) antagonists.4. Application of TRPV4agonist4α-PDD can increase the amplitude of INMDAs the effect of4a-PDD, application of hypotonic stimulation increased the amplitude of INMDA, which could be blocked by HC-067047, indicating that hypotonic stimulation through actiating TRPV4enhanced the activity of NMDA receptor.5. As the effect of4a-PDD, the maximal NMDA-activated current was increased by hypotonic stimulation, leaving EC50value unaffected. Ⅰ-Ⅴ curve of INMDA did not change after application of hypotonic stimulation or4α-PDD. These results indicate that hypotonic stimulation through actiating TRPV4could enhance the actiation of NMDA receptor6. Hypotonicity-induced increase in INMDA was markedly attenuated by antagonist of NR2B subunit (ifenprodil), but not antagonist of NR2A subunit (NVP-AAM007). Blockage of CaMKII signaling pathway significantly blocked hypotonicity-increased INMDA, while antagonism of PKC or CKII signaling pathway had no such effect.7.4α-PDD-induced neuronal death in hippocampal CA1area was markedly blocked by pre-application of NMDA antagonist MK801.ConclusionIn cerebral ischemia, overexpression and over actiation of TRPV4caused by cerebral edema enhanced TRPV4medicated Ca2+influx, increased the release of pre-synaptic glutamate, and actiated CaMKII to further enhance the response of NMDA receptor via increasing the level of phosphorylate NR2B subunit. NMDA receptor-medicated Ca2+infux caused intracellular calcium ([Ca2+]i) overload, which activated apoptosis pathways and eventually induced cell dearth. Thus, the present study proved that blockage of TRPV4has preotective effect on cerebral ischemia injury.