Mechanisms Of Transient Receptor Potential Channels Involved In Diabetic Mechanical Allodynia

【Background and aims】Diabetes mellitus (DM) is the commonly occured endocrinal and metabolicdisordered disease that is characterized by prominent diabetic neuropathic pain (DNP) insome patients. The pathogenesis of DNP is unclear yet, but the pathological changes ofperipheral nerves under the long-term hyperglycemia may be one of the reason. DNP isone of the most serious complications which the patients have to face every day. One formof DNP is the strong painful sensation when the patients’ skin encounters innoxiousmechanical stimuli, a phenomenon named diabetic mechanical allodynia (DMA). DMAseriously affects life quanlity of patients who suffer from it, but presently there is no idealand effective treatment for it because the molecular mechanism underlying DMA has notfully been elucidated. Thus, in the present study, based on the rat model of DMA inducedby streptozocin (STZ), we investigated the spatio-temporal expression of somemechanosensitive transient receptor potential (TRP) channels (TRPV1, TRPV4, TRPC1,TRPC6and TRPA1) in the pain signal “pacemaker” dorsal root ganglia (DRG) and spinal dorsal horn by combined using of molecular biology and morphology. We also evaluatedtheir functional involvement in DMA by employing behavioral pharmacology. Our studyaimed to uncover the molecular mechanism of DMA and provide new strategy andexperimental evidence for clinical treatment.【Methods】(1) Establishment of STZ-induced DM rat model;(2) The paw withdrawal threshold(PWT) of DM model rats was measured by von Frey filaments to select the successfulDMA model rats;(3) The paw withdrawal latency (PWL) of DM model rats was detectedwith Hargreaves method;(4) Quantitative real-time PCR was performed to detect themRNA level of TRPV1in the DRG of DMA model rats;(5) Western blot was used tomeasure the protein level of TRPV1in DRG and spinal dorsal horn of DMA model rats;(6)Immunofluorescent staining was preformed to detect the expression of TRPV1in DRGand spinal dorsal horn of DMA model rats;(7) Immunofluorescent double staining wasused to observe the colocalization of TRPV1with the commonly used neuronal phenotypemarkers, neurofilament200(NF200), calcitonin gene-related peptide (CGRP) and lectinI-isolectin B4(IB4), respectively;(8) Intrathecal implantation and single injection ofvarying doses of ruthenium red (RR) or capsazepine (CPZ) were performed on DMA14dto investigate the PWT of DMA model rats at different time point, and calculate theeffective dose of RR that caused50%of maximal antinociceptive effect (ED50);(9)Intrathecal implantation and multiple injection of varied doses of RR or CPZ from DMA14d to20d (once daily) to assess the PWT of DMA model rats;(10) Quantitativereal-time PCR was performed to detect the mRNA level of TRPV4, TRPC1, TRPC6andTRPA1in DRG of DMA model rats;(11) Western blot assay was used to measure theprotein level of TRPV4, TRPC1, TRPC6or TRPA1in DRG and spinal dorsal horn ofDMA model rats;(12) Immunofluorescent staining was preformed to detect the cellularlocalization of TRPV4, TRPC1, TRPC6and TRPA1in DRG and spinal dorsal horn ofDMA model rats;(13) Immunofluorescent Double staining was used to observe thecolocalization of TRPV4, TRPC1, TRPC6and TRPA1with NF200, CGRP or IB4, respectively;(14) Intrathecal implantation and single injection of three varied doses ofSKF96365on DMA7d were performed to measure the PWT of DMA model rats andcalculate the ED50of SKF96365;(15) According to the ED50value of RR and SKF96365per se, the ED50of combined application RR with SKF96365was calculated andintrathecal implantation and single injection of two drugs were fulfilled to assess the PWTof DMA model rats;(16) PCR was performed to detect the mRNA level of TRPV1,TRPV4, TRPC1, TRPC6and TRPA1in DRG of DM model rats which solely showedthermal hyperalgesia without mechanical allodynia;(17) Intrathecal implantation andsingle injection of RR or SKF of ED50dose were performed on DMA14d to observe thechanges of PWL of DM model rats that only showed thermal hyperalgesia.【Results】(1) Three days after STZ injection, a marked uprising of blood glucose level andimpaired body weight increase were observed;(2)7days after STZ injection,approximately50%of DM rats showed significant decrease of mechanical withdrawalthreshold (PWT <8g, DMA model rat);(3)7days after STZ injection, about90%DMrats exhibited remarkablely prolonged paw withdrawal latency (PWL>8s, thermalhyperalgesia);(4) Significant increase of TRPV1mRNA level in DRGs was observed onDMA7d and14d;(5) Western blot results revealed that TRPV1protein level wassignificantly increased in DRG neurons’ soma on DMA14d while those in spinal cordwas enhanced from DMA7d and further increased on DMA14d;(6) Immunofluorescentstaining showed that TRPV1-positive neurons and profiles were significantly increased inDRG and spinal dorsal horn, and there is an altered size distribution of TRPV1-positiveDRG neurons with the progression of DMA;(7) Immunofluorescent double labelingshowed that the increased TRPV1-immunoreactive neurons were likely to be CGRP-ergic;(8) Single intrathecal injection of RR or CPZ both displayed dose-dependent analgesiceffects on DMA rats, but the effect of RR was slow-onset and long-term effective whilethat of CPZ was inverse;(9) Multiple intrathecal applications of RR or CPZ at varyingdoses, effectively alleviated DMA, but the effect of the former was more prominent and long-lasting;(10) RT-PCR experiments revealed the up-regulation of othermechanosensitive TRP channels at differential time point: TRPV4on DMA14d, TRPC1on28d, TRPC6on7d, and TRPA1on21d and28d;(11) Western blot analysis showedthat the temporal changes of TRPV4, TRPC6and TRPA1coincided with their mRNAchanges, while that of TRPC1was1w prolonged than its mRNA changes;(12)Immunofluorescent staining suggested that the cellular localization of these four channelswere not the same; TRPC6and TRPA1were evenly distributed within the cell whereasTRPC1and TRPV4were characterized by their subcelluler sediments; the increased TRPprotein by DMA stimulation were more enriched in DRG neurons with bigger size;(13)Immunofluorescent double labeling revealed that neurochamical feature of the increasedTRP-positive neurons were varied; TRPV4-and TRPA1-positive neurons were likely to beCGRP-ergic, TRPC1-positive cells were large myelination NF200-positive neurons andTRPC6-positive neurons were IB4-positive neurons;(14) Single intrathecal injection ofSKF96365displayed the dose-dependent analgesic effect on DMA rats with fast-onset butshort effective feature;(15) Intrathecal co-administration of RR and SKF96365produceda fast onset and long-term effective analgeic effect, suggesting that there was a synergisticaction of these two drugs when combined application;(16) In the DRG of those DMmodel rats which only showed thermal hyperalgesia, an elevated mRNA levels of TRPV1and TRPV4instead of TRPC1, TRPC6or TRPA1were detected;(17) Single intrathecalinjection of RR or SKF96365to these pure thermal hyperalgesic rats, only RR couldreverse the the lowed PWL value whereas SKF96365could not.【Conclusions】(1) The incidences of mechanical allodynia and thermal hyperalgesia in DNP modelrats did not coincide with each other. Among DM model rats, about50%of rats showedboth mechanical allodynia and thermal hyperalgesia,90%of rats exhibited thermalhyperalgesia, and nearly10%of rats were with no pain. Thus, caution should be taken todiscriminate the pain modality and strictly select animal model when performing geneexpression study in case of any overestimation or underestimation. (2) TRPV1expression in DRG and spinal dorsal horn was significantly increased onDMA14d and the non-specific and specific antagonists of TRPV1, RR and CPZeffectively inhibited DMA behavior, suggesting that TRPV1may be an essential channelprotein involved in mechanosensing and transduction.(3) In DRG and spinal dorsal horn of DMA model rats, the expression of TRPV4,TRPC1, TRPC6and TRPA1showed a transient trend of increases at varied time point.Intrathecal delivery of non-specific antagonists of TRPV family, RR and of TRPC family,SKF96365could dose-dependently alleviate the pain behaviors of DMA model rats.These results collectively suggest that the initation and maintenance of DMA is a complexprocess which involves the contribution of five TRP channels at varid time schedule:TRPC6was in the observed early stage, TRPV1and TRPV4in the middle stage, andTRPC1and TRPA1in the late stage. Therefore, similar to the channel spectrumrepsonsible for thermal hyperalgesia, there is also such a molecular spectrum for DRGneurons to detect noxious mechanical stimuli.(4) Unlike the above TRP channel involvement in DMA, the expression of TRPV1aswell as TRPV4but not TRPC1, TRPC6or TRPA1were found to increase in the DRG ofDM rats which solely exhibited thermal hyperalgesia. Consitently, intrathecaladministration of RR other than SKF96365produced prominent analgisic effect. Together,these results suggest that the peripheral mechanisms for diabetic thermal hyperalgesia andmechanical allodynia are essentially different. Thus, individualized treatment consideringthe pain modality, molecular target and pain schedule may be the hopeful strategy tochallenge the clinical dilemma in the treatment of diabetic neuropathic pain.