Peripheral Analgesic Effect And Its Mechanism Of Methylcobalamin In The Rat Model Of Chronically Compressed DRG

Peripheral neuropathic pain symptoms in clinical, which were induced by the lumbardisc herniation, spinal stenosis, external force damage and oncothlipsis, could be wellimitated by the chronic dorsal root ganglion (CCD) model. Hyperexcitability of DRGneurons is generated due to the changes in the expression and dynamic properties ofmultiple ion channels. The amount of afferent information increased greatly along withspontaneous activities (SA), which leads to persistant excitability and sensitization in thespinal cord and supraspinal structures of central nervous system. Those changescontributed to the neuropathic pain symptom. The index of SA has become an effectiveway to evaluate analgesic drugs and to investigate analgesic mechanisms. In a latest trial, intramuscular injection of MeCbl produced a significant improvementon the pain scores in patients with chronic non-specific low back pain. Vitamin B12withvitamins B1and B6decreased allodynia induced in the SNL rat and thermal hyperalgesiain the models of CCD and CCI. However, in a2years follow-up study, lumbar spinalstenosis induced pain was not improved significantly in the methylcobalamin (MeCbl)group. The analgesic effect of MeCbl remains incompletely documented. And it is alsoperplexed about the effect of MeCbl on SA and excitibility of primary sensory neurons.The CCD model was used in our study to investigate the effect and mechanisms of MeCblon neuropathic pain.I_hcurrent is defined as a slowly activated inward current evoked by hyperpolarization. Itplays an important role in regulating neurons excitability by limiting membranehyperpolarization and facilitating depolarization. And it was reported that I_hcurrentdensity of medium DRG neurons was obviously increased in the CCD model. But therewas not any evidence about the effect of MeCbl on I_hcurrent density of medium DRGneuons.Main results1. CCD rats exhibited apparently bilateral mechanical allodynia and unilateral thermalhyperalgesia.Mechanical paw withdrawal threshold of bilateral sides were investigated in bothCCD group (n=6) and sham group for21days after surgery. Compared to sham group,bilateral mechanical paw withdrawal thresholds were significantly decreased.(ipsilateralside: P<0.001; contralateral side: P<0.001, Repeated measure ANOVA). Compared withsham group, the thermal paw withdrawal latency (TWL) of CCD group decreasedobviously in the ipsilateral side but not the contralateral side（Ipsilateral side: P<0.001;contralateral side: P>0.05，Repeated measure ANOVA）.2. Successive intraperitoneal injection of MeCbl excerted an analgesic effect on allodyniain CCD rats, but not TWL.Short term and long term effects of intraperitoneal injection of MeCbl on allodynia and TWL in CCD rats were examined. It was showed that MeCbl had no effect on the left andright mechanical allodynia and TWL of the CCD and sham group following the short termtreatment (P=1.00, Repeated measure ANOVA). In the long-term observation, there weresignificant differences in the effect of MeCb1on ipsilateral and contralateral mechanicalallodynia on5d,10d,18d,23d and28d five points (ipsilateral side: P<0.05;contralateral side: P<0.01, Repeated measure ANOVA). Alldynia of bilateral hind paws ofCCD rats were significantly depressed from the5th day after repeated injection of MeCbl（2.5mg/kg）and presented the time cumulative effect.And in the long term investigation,the TWL of two sides in CCD group emerged stable. MeCbl had no effect on them.3. MeCbl significantly inhibited the SA of CCD DRG A-type neurons.Among62single fibers of A-type dorsal root with stabled SA.66.1%were sensitive toMeCbl. MeCb1inhibited SA of A-type DRG neurons significantly (P<0.05, paired t-test)and83.3%was reversed to the baseline after10min washout. MeCb1inhibited SA fromDRG A-type neurons in a dose-dependent manner from3to300μM. The frequencies ofboth non-periodic and periodic pattern discharges were inhibited greatly in the presence ofMeCbl (300mol/L), and there was no difference. With the whole-cell patch clamptechnique, the effect of MeCbl was further tested on the SA of DRG A-type neurons invitro. The SA was eliminated in60s and restored in10min washout.4. MeCbl depressed the excitabilities of medium DRG neurons from CCD ratsapparently.Compared with sham group, the AP amplitude and the Max-rise slope of AP risedobviously in CCD medium DRG neurons (P<0.05, one-way ANOVA). The AP amplitude,afterhyperpolarization (AHP) amplitude, the Max-rise slope, the Max-decay slope weredecreased significantly by MeCbl (P<0.05, n=34, paired t-test), and the half-widthincreased obviously (P<0.05, n=34, paired t-test)5. MeCbl significantly decreased the I_hcurrent density of CCD medium DRG neurons.Slowly activated, inward non-selective cation ion currents (I_h) were recorded under ahyperpolarizing potential. About65%medium DRG neurons of CCD (13/20) and sham(11/17) groups expressed it. Compare to the sham group, the I_hcurrent density of medium DRG neurons was increased markedly following CCD (P<0.05, Repeated measureANOVA). I_hcurrent density was significantly decreased in the CCD rat compared withthat of sham group after the administration of MeCb1(P<0.05, Repeated measureANOVA).Main conclusion1. Long-term intraperitoneal injection of MeCbl inhibited the bilateral mechanicalallodynia significantly, but not TWL of CCD rats.2. MeCbl inhibited the SA of A-type DRG neurons in the CCD rat in vivo and vitro. Inaddition, there is no difference of the inhibitory effect of MeCbl (300mol/L) ondifferent neuronal discharge patterns in vivo.3. The excitabilities of medium DRG neurons were decreased markedly by MeCbl,MeCbl decreased AP amplitude, the Max-rise slope, the Max-decay slope and AHPamplitude in the CCD rat，it also increased the half-width of AP parently.4. The I_hcurrent density of CCD medium DRG neurons was decreased obviously byMeCbl.