The Expression And Related Mechanisms Of NGF In A Chronic Migraine Rat Model

Background: Our previous study showed that acid-sensing ion channel 3(ASIC3)in the trigeminal nucleus caudalis(TNC)is involved in the pathogenesis of recurrent migraine.ASIC3 is regulated by nerve growth factor(NGF),which induces hyperalgesia in various pain disorders.Neutralization of NGF is considered an effective treatment method.However,the contribution of NGF to repeated migraine-like attacks in chronic migraine(CM)remains unclear.Therefore,this study investigated the expression of NGF in the TNC,the effect of NGF on ASIC3 expression and the role of NGF signaling in inflammatory soup(IS)-induced cutaneous hyperalgesia.Method:1.A rat model was established by repeated dural infusions of IS for seven days to simulate CM attacks.The pain thresholds of periorbital region and hind paws were evaluated with the von Frey test.2.The mRNA and protein expression of NGF in the TNC were detected by PCR and Western blot.The co-expression of NGF and TNC neurons was detected by immunofluorescence.3.CM rats were injected with anti-NGF neutralizing antibody(anti-NGF)into the lateral ventricle.The changes of periorbital region and hind paw thresholds were detected by von Frey test.The protein expressions of PKC,ASIC3 and CGRP in the TNC were detected by Western blot.The mean fluorescence intensity of CGRP and the number of immunopositive cells of c-Fos in the TNC were detected by immunofluorescence.4.PKC inhibitor chelerythrine chloride(CHE)was injected into the lateral ventricle of CM rats.The thresholds of periorbital region and hind paw were measured by von Frey test.The protein expression of ASIC3 and CGRP in the TNC were detected by Western blot.The mean fluorescence intensity of CGRP and the number of immunopositive cells of c-Fos in the TNC were detected by immunofluorescence.Results:1.Compared with the Sham group,the periorbital and hind paw thresholds of the CM group were significantly decreased,indicating that IS induced cutaneous hyperalgesia in the CM group.2.PCR and Western blot showed that the mRNA and protein levels of NGF in the TNC of CM rats were significantly increased than the Sham group.Immunofluorescence results showed that NGF was co-expressed with TNC neurons and mainly expressed in the cytoplasm of neurons.3.An intracerebroventricular injection of anti-NGF significantlyincreased the pain thresholds of periorbital and hind paw of CM rats.Western blot results showed that compared with the Sham group,the protein levels of PKC,ASIC3 and CGRP in the TNC of CM rats were significantly increased.The intracerebroventricular injection of anti-NGF significantly reduced the protein level of PKC,ASIC3 and CGRP in the TNC of CM rats.The administration of anti-NGF also significantly decreased the mean fluorescence intensity of CGRP and the number of c-Fos positive cells in the TNC of CM rats.4.Intracerebroventricular injection with the PKC blocker CHE alleviated IS infusion-induced hyperalgesia and reduced protein level of ASIC3,CGRP,the mean fluorescence intensity of CGRP and the number of c-Fos positive cells in the TNC of CM rats.Conclusion:These results indicate that NGF might regulate ASIC3 expression via PKC activity in the TNC following repeated IS dural stimulation,and this signaling pathway might participate in IS-induced cutaneous hyperalgesia.