| Background:Central post stroke pain(CPSP)is a central neuropathic pain syndrome,which is one of the most common sequelae of stroke,that can occur after an acute focal lesion of central nervous system(CNS)by a cerebrovascular cause,the most common lesion site is the thalamus,and the incidence rate is about 8-11%.A recent imaging clinical studies showed that posterolateral and inferior thalamic regions and anterior,medial,and lateral pulvinar nucleus have been reported to be more common among patients with CPSP compared with thalamic stroke patients without pain.The most common forms of chronic post-stroke pain are spontaneous pain,hyperalgesia,and allodynia.CPSP lasts for a long time after stroke,which exerts siginificant affects on the life quality during the recovery period,and there is no effective treatment.Therefore,to elucidate the pathophysiological mechanism of CPSP,and seek an effective intervention to alleviate or prevent the development of CPSP has become a critical clinical problem Epoxyeicosatrienoic acid(EETs)is one of the biologically active substances of arachidonic acid(AA)metabolized via cytochrome P450(CYP450).There are four regioisomers of EETs,including 5,6-,8,9-,11,12-,14,15-EET,which are mainly degraded into to dihydroxyeicosatrienoic acids(DHETs)by the enzyme soluble epoxide hydrolase(sEH).In recent years,accumulated studies have found that EETs have multiple neuroprotection effect.EETs attenuates cerebral ischemia and hypoxia injury by increasing astrocyte-releasing vascular endothelial growth factor EETs can regulate cerebral blood flow and reduce cerebral ischemia-induced injury.14,15-EET inhibits BV2 microglial inflammatory response induced by oxygen glucose deprivation/reoxygenation.14,15-EET can inhibit brain microvascular smooth muscle cells apoptosis through activating JNK/c-Jun and m-TOR signaling pathway.In addition,studies have shown that EETs or sEH inhibitors exhibit analgesic effect on inflammatory pain via reducing the expression of COX-2(cyclooxygenase-2),and increasing the level of steroid acute regulatory protein(StAR)that promote neurosteroid synthesis then activate gamma-aminobutyric acid type A receptors.And exogenous sEH inhibitor TPPU can alleviate diabetic peripheral neuralgia via alleviating endoplasmic reticulum stress.Moreover,sEH inhibitors produce anti-inflammatory and analgesic effects by downregulating the expression of TNF-a,IL-1β and COX-2 in the dorsal root ganglion DRG of rat model of sciatica.These previous studies demonstrated that:1.EETs have multimodal neuroprotective effects on cerebral ischemia-reperfusion injury;2.EETs and sEH inhibitors exert analgesic effect on inflammatory pain,painful diabetic neuropathy and neuropathic pain.However,the role and mechanism of EETs or sEH inhibitors on CPSP is unclear.Therefore,in order to further clarify the role and mechanism of EETs in CPSP,we construct a CPSP rat model,using behavioral,morphological and molecular biology techniques to explore the role and mechanisms of 14,15-EET in the development and maintenance of CPSP after thalamic stroke.These findings provide a theoretical basis that EET as a potential therapeutic for patients who suffer from CPSP.Methods and Results:1.14,15-EET and AP-δGABAAR signaling pathway changes in the thalamus after CPSPMethods:The Sprague Dawley(SD)rats were randomly divided into two groups(n=50/group):sham-operated group(Sham)and central post-stroke pain group(CPSP).The CPSP model was constructed by stereotactic microinjection type IV collagenase into the VPL(ventroposterior lateral nucleus).The von Frey filaments and Hargreave were used to assess the paw withdraw mechanical threshold(PWMT)and paw withdraw thermal latency(PWTL)at day-1,7,14,21 and 28.Then,we assay the content of 14,15-EET in the thalamus through high performance liquid chromatography-mass spectrometry(HPLC-MS).The AP concentration was detect by enzyme linked immunosorbent assay(ELISA),immunofluorescence and western-blot was used to test steroidogenic acute regulatory(StAR)andδ-subunit-containg γ-aminobutyric acid A receptors(δGABAARs)expression.Results:The SD rats exhibited a decrease in PWMT at day 7 after surgery and last at least 28 days,and the PWMT of contralateral paw decreased markedly.The content of 14,15-EET in the VPL was significantly decreased from day 7 to day 28.The results of immunofluorescence and western blot showed that StAR decreased significantly at day 7 after surgery and reached to the lowest at day 14.The AP content decreased significantly at day 7 and reached to the lowest at day 14 post lesion.The expression of δGABAAR was progressively decreased after lesion.2.14,15-EET mediated AP-δGABAAR signaling pathway exhibit analgesic effect on CPSP ratsMethods:The 14,15-EET solution(0.025,0.05,0.1 μg/μl,1 μl)or vehicle(0.1%DMSO,1 μl)was microinjected into VPL nucleus once daily within the first 3 days after hemorrhagic stroke using a polyethylene catheter(PE-10)connected to amicrosyringe needle via the guide cannula slowly over a period of 5 min.In the EET antagonist treatment group,14,15-EEZE(3.25 ng/μl,1 μl)was administered 15 min before 14,15-EET administration,then the PWMT was tested.The mRNA and protein levels of StAR and δGABAAR from the MT of CPSP rats were detected by the RT-qPCR assay and western blot,AP concentration was detect by ELISAIn order to test the role of AP-δGABAAR pathway in EET-mediated analgesia,we treated the rats with the 5a-reductase inhibitor FIN(10 μg/200 nl/day)or DUT(10μg/200 nl/day)or vehicle(0.1%DMSO)in a combination with 14,15-EET(0.1 μg)once daily within the first 3 days after CPSP as described above,then the PWMT was testedMoreover,to test the role of δGABAR in 14,15-EET mediated analgesic effect,a group of 14,15-EET treated CPSP rats,we administered BIC,the GAB AAR antagonist,at post-lesion day 14 and then measured the PWMT immediately to 4 hours thereafterResults:We found that PWMT in both hind paws were significantly increased to almost 2 folds in CPSP rats treated with 0.1 μg of 14,15-EET,compared with the vehicle group.However,these effects were completely eliminated by pretreatment with the 14,15-EET antagonist 14,15-EEZE(3.25 ng).Compared with the vehicle,14,15-EET dose-dependently upregulated the StAR mRNA and protein expression showed by real-time PCR,western blot and immunostaining.Simultaneously,ELISA analysis show that 0.1 μg 14,15-EET markedly increased the content of AP.Moreover,the δGABAAR mRNA and protein in the MT were increased.However,these increases were completely abolished when co-applied with 3.25 ng 14,15-EEZE.The analgesic effect of 14,15-EET at dose of 0.1 μg,was completely suppressed by FIN/DUT application.The analgesic effect of 14,15-EET at dose of 0.1 μg,was abolished by BIC3.To explore other possible mechanism of 14,15-EET mediated analgesic effect on CPSP ratsMethods:We randomly divided the SD rats into 2 groups,:Sham group and CPSP model group,to observe the inflammatory response and expression of apoptotic related proteins in the CPSP model.The PWMT was measured by von Frey filament at day-1,7,14,21 and 28 after surgery,and astrocyte and microglia activation were observed by immunofluorescence.The pro-inflammatory cytokines(IL-1β,IL-6 and TNF-α)and anti-inflammatory cytokines(IL-4 and IL-10)were detected by ELISA assay,and apoptosis-related proteins Bax,Bcl-2 and cleaved-caspase3 expression were tested by western blot analysis.To investigate the inhibition of inflammation and apoptosis mediated by 14,15-EET on CPSP rats,75 SD rats were randomly divided into 5 groups(n=24/group):CPSP model group,0.025,0.05,0.1,0.1 μg 14,15-EET group,and 0.1 μg.14,15-EET+14,15-EEZE group.The PWMT of rats were measured by von Frey filament at day-1,7,14,21 and 28 after surgery,the activation of astrocyte and microglial were observed by immunofluorescence.Pro-inflammatory cytokines(IL-1β,IL-6 and TNF-α)and anti-inflammatory cytokines(IL-4 and IL-10)were detected by ELISA assay,and apoptosis-related proteins Bax,Bcl-2 and cleaved-caspase3 expression measured by western blot analysis.Results:We observed that the astrocytes and microglia activated,pro-inflammatory cytokines expression increased,anti-inflammatory cytokines decreased contribute to the development of CPSP;and 14,15-EET inhibited astrocyte,microglia activation and inflammatory response,and then alleviated the mechanical allodynia on CPSP rats;The expression of apoptosis-related protein was upregulated in the VPL nucleus contributing to the development of CPSP;and the anti-apoptotic effect mediated by 14,15-EET attenuates the mechanical allodynia of CPSP rats.Conclusion:(1)CPSP rats exhibited reduce of 14,15-EET,StAR expression and AP production in VPL,and δGABAAR levels decreased in MT;(2)14,15-EET attenuated mechanical allodynia on CPSP rats by enhancing thalamic AP-δGABAAR signaling pathway;(3)14,15-EET alleviate mechanical allodynia on CPSP rats via inhibiting inflammation response and apoptosis. |
PDF Full Text Request