Activation Of Gliacells And JAK/STAT3 Pathway In Trigeminal Ganglion Involves The Trigeminal Mechanical Allodynia Of Rats

Objective The trigeminal neuralgia model was established by chronic constriction injury of the infraorbital nerve(CCI-ION)or IL-6 injection in SD rats.The change of mechanical sensitivity threshold of rats was recorded by behavioral examination.The role of glia cells and JAK/STAT3 pathway in trigeminal ganglion of rat trigeminal neuralgia model was investigated by molecular biology experiments.Methods Normal SD rats(120-150 g)were choosed and fed adaptively and stimulated for 5 days,then the normal rats with calm and healthy response to a certain amount of stimulation.Phase I experiment: experimental rats were randomly divided into four groups: the IL-6 injection group and the corresponding control group,the NS injection group,and the CCI-ION group and the corresponding control group,the Sham group.Von Frey hair was used to measure and record the mechanical sensitivity threshold of the vibrissa pad in four groups of rats within 7 days after modeling.Experimental rats were killed and ipsilateral trigeminal ganglion was removed respectively at the 48 hour,third day and senventh day.Western blot,PCR and Immunofluorescence technique were used to detect the expression of p STAT3 and glial cell markers in four group of rats,meanwhile,the cell type of which the p STAT3 fluorescent staining located in.Second phase experiment: according to the principle of random grouping,trigeminal neuralgia model rats were administrated AG490(the JAK/STAT3 pathway inhibitor)through ipsilateral suborbital foramina or DMSO(as control)and were divided into CCI-ION group + AG490,IL-6 injection group + AG490,CCI-ION group + DMSO,IL-6 injection group + DMSO.The mechanical sensitivity threshold of four groups of rats were monitored during administration.The experimental rats were killed and the ipsilateral trigeminal ganglion was removed on the 7th day after the establishment of the model(i.e.,the 8th day after the administration of the drug).The expression levels of p STAT3 protein,SOCS3 m RNA,satellite glial cell and microglial cell markers of the experimental rats were detected by Western blot and PCR.Finally,these data were statistically analyzed.Results 1.The results showed that the mechanical sensitivity thresholds of CCI-ION group were significantly lower than that of Sham group,Similarly,the mechanical sensitivity thresholds of IL-6 injetction group were significantly lower than that of NS injection group and both CCI-ION group and IL-6 injection group reached the lowest value on the 7th day.2.The results of immunofluorescence assay showed that there were almost no p STAT3 fluorescent labeling in the ipsilateral trigeminal ganglia of Sham group rats or NS injected rats,however,a mass of p STAT3 immunoreactive staining was found at 48 hours after CCI-ION surgery or IL-6 injection.Then the markers of ITGAM for microglia,GFAP for SGCs and Neu N for neurons were used to conduct double-labeling immunohistofluorescent experiments.As a result,in the ipsilateraltrigeminal ganglion of the CCI-ION group and the IL-6 injection group,a large amount of co-immunostaining between p STAT3 and ITGAM was observed,indicating that p STAT3 was located in microglia cells.Moreover,western blot was used to detect the expression levels of p STAT3 protein.As a result,the protein level of p STAT3 was significantly upregulated in the TG of CCI-ION group on the third day compared with the sham group.And similar changes of significantly upregulated p STAT3 protein levels were also found in the TGs of IL-6injected rats on the third day.3.Western blot and RT-PCR was used to detect the expression levels of GFAP protein(a satellite glial marker)and ITGAM m RNA(a microglia marker),As a result,the expression levels of GFAP protein and ITGAM m RNA in the trigeminal ganglion of CCI-ION group were higher than Sham group.Similarly,higher expression levels of GFAP protein and ITGAM m RNA were found in the IL-6 injection group compared with the NS injection group.4.The trigeminal neuralgia rats was grouped according to the principle of random grouping,the mechanical threshold was detected on the eighth day after giving inhibitor AG490 or DMSO.As a result,compared with the CCI-ION/DMSO group rats,the mechanical threshold of CCI-ION/AG490 group rats is significantly upregulated at the fourth day after delivery and lasted until the eighth day.In the same way,the mechanical threshold of IL-6/AG490 group compared with IL-6/DMSO group is also significantly upregulated(from the fourth day to the eighth day after administration).5.After administration the experiment results of Western blot and RT-PCR analysis showed that,the p STAT3 and GFAP protein levels and SOCS3 m RNA concentration of CCI-ION rats treated with DMSO was both significantly higher than that of sham group rats and CCI-ION rats treated with AG490 on the senventh day.Meanwhile,similar changes of significantly higher expression levels of p STAT3 and GFAP protein and ITGAM m RNA were also observed in the TGs of IL-6/DMSO group rats compared with NS injected rats or IL-6/AG490 group rats.Nevertheless,there was no statistically significant change of ITGAM m RNA levels after administration of AG490 in both CCI-ION or IL-6 injection group.Conclusion In the trigeminal neuralgia model of rats established by chronic contractile injury of the infraorbital nerve or IL-6 injection,both glial cells and JAK/STAT3 pathway were activated,and the activating STAT3 protein was located in microglia cells.Meanwhile,after administration the JAK/STAT3 pathway inhibitor AG490,the expression levels of p STAT3 protein and SOCS3 m RNA of trigeminal neuralgia model rats were significantly decreased compared with the significantly reduced mechanical sensitivity thresold.These results suggest that glial cells and JAK/STAT3 signaling pathway may be involved in the occurence and development of trigeminal neuralgia in rats,and that JAK/STAT3 pathway may be involved in the activation of satellite glial cells.