The Roles Of Neuron-astrocyte Interactive Regulation In Neuropathic Pain And Antinociception

【Background】Patients suffering from trauma, nerve compression, infection or neurodegenerative diseases often present neuropathic pain, which is essentially intractable to currently available anti-nociceptive therapies. This is in part due to poor understanding on how neuropathic pain is induced and maintained. According to classic pain research, the pain pathway has been described simply as a serial chain of neuronal elements, while immerging research has implicated spinal cord glial cells as key players in the induction and maintenance of neuropathic pain. Previous studies have confirmed the expression of astrocytes and its relation with neuropathic pain, however, lots of questions remained unanswered.In the present study, by using a rat model of neuropathic pain, combined methods including behavioral testing, molecular neurobiology and morphology, we sought to elucidate the influence of neuroimmune response on spinal pain circuits and its contribution to neuropathic pain. In addition, we tried to investigate the analgesic effect of inhibiting neuron-glia interactions after nerve injury. Our study may provide further understanding on pain mechanisms and introduce some novel strategies on neuropathic pain treatment.【Aims】To investigate the role of neuron-astrocyte interactive regulation in the neuropathic pain and related analgesic strategies【Methods】(1) Establishment of spinal nerve ligation (SNL)-induced neuropathic pain model; (2) The rat paw withdrawal threshold after SNL was measured with von Frey filaments; (3) The rat thermal response latencies after SNL was measured with Hargreaves method; (4) Immunofluorescent histochemistry was used to detect the expressions of spinal astrocytic GFAP and glutamate transporter-1 (GLT-1); (5) Double immunofluorescent staining was performed to observe the colocalization of GLT-1 and GFAP; (6) Intrathecal implantation and administration of c-fos antisense oligodeoxynucleotides (ASO) or astrocytic toxin L-α-aminoadipate (L-AA); (7) The rat paw withdrawal threshold after intrathecal injection of c-fos ASO or L-AA was measured with von Frey filaments; (8) Immunofluorescent histochemistry was used to detect the expressions of spinal GFAP and Fos; (9) Double immunofluorescent staining was performed to observe the colocalization of Fos and GFAP or NeuN; (10) Western blot was used to detect the phosphorylation of c-Jun N-terminal kinase (JNK); (11) Double immunofluorescent staining was performed to observe the colocalization of pJNK and GFAP; (12) Intrathecal injection of NMDA, pJNK inhibitor SP600125, NMDA receptor antagonist MK-801, NR2B selective antagonists Ro25-6981 and ifenprodil, neuronal nitric oxide synthase (nNOS) selective inhibitor 7-NINA and guanylate cyclase (GC) selective inhibitor ODQ; (13) von Frey filaments detection was used to measure the behavioral response after drugs administration; (14) Western blot was performed to evaluate the phosphorylation of JNK; (15) Real-time PCR was used to measure the mRNA expressions of JNK-related cytokines and chemokines, including IL-1beta, TNF-alpha, MCP-1 and IL-6 after different treatments; (16) Intrathecal injection of ketamine, LAA or combination of these two drugs; (17) The rat paw withdrawal threshold after drugs treatments was measured with von Frey filaments; (18) Western blot was performed to evaluate the phosphorylation of NMDA receptor NR1 subunit; (19) Immunofluorescent histochemistry was used to detect the expressions of spinal GFAP following ketamine or LAA treatments; (20) The effect of the extract of tripterygium (T4) treatment on rat paw withdrawal threshold was determined by von Frey filaments; (21) The rat thermal response latencies after T4 treatment on SNL rats was measured with Hargreaves method; (22) Immunofluorescent histochemistry was used to detect the expressions of spinal GFAP, OX42 and NeuN; (23) Western blot was performed to evaluate the phosphorylation of MAPK after T4 treatment on SNL rats; (24) Real-time PCR was used to measure the mRNA expressions of pain-related cytokines and chemokines T4 treatment.【Results】(1) Compared to the sham group, SNL induced a marked expression of GFAP in the ipsilateral than in the contralateral spinal cord; (2) SNL also induced a significant change in the expression of GLT-1 within the ipsilateral spinal dorsal horn. Double immunofluorescent staining revealed that GLT-1 immunoreactivity was completely colocalized with GFAP in the spinal cord; (3) GFAP up-regulation was not evident on day 1, significant on day 3, reached a peak on day 7, and remained at high levels on day 21 after SNL; (4) SNL-induced GLT-1 expression displayed an interesting biphasic change, with an initial up-regulation followed by a down-regulation after SNL: increased during the development of neuropathic pain (<5 d) but decreased in the maintenance phase (>7 d), and almost disappeared on day 21 after SNL; (5) SNL induced a significant increase of Fos and astrocytic GFAP in the ipsilateral spinal dorsal horn; (6) Intrathecal injection of c-fos ASO or astrocytic toxin LAA prevented SNL-induced mechanical allodynia; (7) c-fos ASO prevented SNL-induced up-regulation of spinal GFAP; (8) LAA shortened the duration of spinal Fos expression following SNL; (9) pJNK was significantly increased in the spinal dorsal horn ipsilateral to the SNL; (10) Double immunofluorescent staining revealed that pJNK was completely colocalized with GFAP in the spinal dorsal horn; (11) Intrathecally infusing JNK inhibitor SP600125 (from day 0-7 following SNL) significantly prevented SNL-induced mechanical allodynia from day 3 to day 10; (12) Intrathecal injection of a noncompetitive NMDA receptor antagonist, MK-801, or selective antagonists of NMDA receptor containing the NR2B subunit, Ro25-6981 and ifenprodil inhibited SNL-induced mechanical allodynia and JNK activation; (13) Immunofluorescent double labeling showed that NR2B was almost completely colocalized with neuronal specific marker NeuN, however, no astrocytic NR2B could be detected as there was no remarkable colocalization of NR2B with GFAP; (14) Repeated intrathecal injection of NMDA activated JNK in spinal astrocytes; (15) Blocking nNOS or GC could successfully prevent SNL-induced mechanical allodynia, nNOS but not GC was essential for SNL-induced JNK activation; (16) Blocking NMDA-nNOS prevented JNK-related spinal cytokines and chemokines mRNA expression; (17) Intrathecal ketamine showed an effective and reliable anti-allodynia effect in a dose-dependent manner on SNL-induced neuropathicpain, which was a very quick but short effect; (18) Intrathecal LAA also showed an effective anti-allodynia effect, was a late but persistent effect; anti-allodynia effect of coadministration (ketamine 100μg/kg and LAA 50 nmol) produced a more persistent and effective role; (19) Astrocytic inhibitor LAA could enhance the inhibitory effect of ketamine on NMDA receptor functioning while ketamine could facilitate the inhibitory effect of LAA on astrocytic activation; (20) Our study provided a new potential strategy for treating clinical neuropathic pain: targeting neuronal transmission and astrocytic function; (21) Intraperitoneal injection of the extract of tripterygium (T4) prevented and attenuated SNL-induced mechanical allodynia and thermal hyperalgesia; (22) T4 treatment inhibited SNL-induced microglial and astrocytic activation; (23) SNL-induced spinal MAPK activation was significantly inhibited by T4 treatment; (24) T4 prevented and reversed pain-related spinal cytokines and chemokines mRNA expression.【Conclusions】(1) Following SNL, the dynamic expression changes of astrocytic GFAP and GLT-1 indicate spinal astrocyte is closely related and involved in the development and maintenance of neuropathic pain.(2) SNL induces significant activation of spinal neurons and astrocytes; inhibiting neuronal or astrocytic activation could alleviate pain behavior and simultaneously decrease the activities of the two counterparts. These results suggest that neuronal and astrocytic activations are closely related with the maintenance of neuropathic pain through a reciprocal"crosstalk".(3) After peripheral nerve injury, spinal NMDAR activation increases astrocytic JNK phosphorylation, which is essential for neuropathic pain; NMDAR-nNOS pathway plays an important role in JNK activation, and consequent expression of cytokines, for instance, IL-1beta. Together with our previous studies, it's believed that spinal NMDAR-mediated excitatory transmission (glutamatergic transmission) may cause activation of signaling pathways (for instance, JNK pathway) in astrocytes. Activated astrocytes could synthesize and release inflammatory mediators that further increase activity of dorsal horn neurons. This"glutamate-cytokine"pathway significantly enlarges pain signals in the spinal dorsal horn following peripheral nerve injury.(4) Intrathecal application of NMDA receptor antagonist ketamine alleviates mechanical allodynia with decreased NMDA receptor phosphorylation in a quick but short response, whereas astrocytic cytotoxin LAA relieves mechanical allodynia with attenuated astrocytic activation in a late but persistent manner; combining ketamine with LAA suppresses neuropathic pain in a quick and stable way, whereas, NMDA receptor phosphorylation and astrocytic activation are both much more suppressed than those of either single drug administration. Taken together, combination of NMDA receptor antagonist and astrocytic inhibitor exhibits some additive and complementary analgesic effects on SNL-induced neuropathic pain.(5) Intraperitoneal injection of T4 prevents and attenuates SNL-induced mechanical allodynia and thermal hyperalgesia, inhibiting SNL-induced microglial and astrocytic activation, MAPK phosphorylation and pain-related spinal cytokines and chemokines mRNA expression. These data suggest T4 may provide some novel strategies on pain treatment.