The Expression Change And Mechanism Of GDF10 In Spinal Cord Of Neuropathic Pain Rat

Neuropathic pain is a refractory disease which caused by central or peripheral nervous system injury.The current treatment methods are mainly temporary pain relief and physical therapy,but did not achieve the purpose of long-term treatment of pain.The study of the basic mechanism of the disease is very important for the development of ne w therapeutic drugs and methods.At present,the main mechanisms of neuropathic pain are neuroinflammation and central sensitization.Transforming growth factor beta(TGF-?)superfamily is a kind of multifunctional and environmental dependent cytokine,which includes TGF-?s,bone morphogenetic proteins(BMPs),activins and growth and differentiation factors(GDFs),these members,such as TGF-?1,have an inhibitory effect on neuroinflammation which can reduce the incidence of neuropathic pain.Growth and differentiation factor 10(GDF10)is a member of the BMPs subfami ly which plays an important role in neurogenesis and axonal regeneration.According to GEO datasets,the expression of GDF10 is upregulated in dorsal ganglion of neuropathic pain rat.However,there is no report of the role of GDF10 in spinal cord in neuropathic pain.The central sensitizationin spinal cord triggeredby activation of N-methyl-D-aspartate(NMDA)receptor plays an important role in the development of neuropathic pain,We further investigate whether GDF10 is associated with activation of NMDA receptor in this disease.Methods:(1)The construction of neuropathic pain model of rats.Neuropathic pain is induced by ligation of left L5 spinal nerves of SD rats.(2)Drugs intrathecal injection.NMDA was injected intrathecally to bring the rats with neuropathic pain,and the NMDAR antagonist MK-801 was intrathecal injection to spinal nerve ligation rats.(3)Behavior test.The paw withdrawal threshold of rats was detected by von Frey filaments to observe the change of mechanical allodynia of rat with spinal nerve ligation or NMDA intrathecal injection and spinal nerve ligation rats with MK-801 intrathecal injection.(4)Western Blotting.The expression change of GDF10 in spinal cord of spinal nerve ligation rats at 1d?3d?10d?21d after surgery was detected by western blotting.The change of GDF10 in spinal dorsal horn after NMDA intrathecal injection and the expression of GDF10 in rats spinal dorsal horn which injected MK-801 intrathecally by western blotting as well.(4)Immunoflouroscence.The expression change of GDF10 in spinal dorsal horn neurons at 3d,10 d,21d after surgery and rats with NMDA intrathecal injection were observed by immunofluorescence.Double staining was used to detect the cellular location of GDF10 in spinal cord.Main results1.The paw withdrawal threshold of the rats with spinal nerve ligation was decreased from 1d after surgery until 3d with obvious differentiation compared with the na?ve rats(P<0.05)and the most obvious decrease is at 10 dand then stabilized at 21 d.The rats with spinal nerve ligation exert obvious increase of mechanical allodynia which indicates the neuropathic pain model was successfully established.2.Double staining of normal rats L5 spinal cord indicated that GDF10 was co-localized with neuronal-specific marker NeuN,but not with astrocyte marker GFAP or the microglia marker Iba-1.The expression of GDF10 in L5 dorsal horn detected by western blotting was reduced after surgery,significantly decreased from 10 duntil more than 21 dafter surgery(P<0.05)in spinal nerve ligation group compared with na?ve group.The expression of GDF10 in spinal dorsal horn neurons is gradually decreased at 3d,10 d,21d by immunofluorescence,and the change is accordance with western blotting.2.NMDA was given twice a day for 3days to normal rats.Rats exhibited significant decrease of paw withdrawal threshold following repeated NMDA injection compared with vehicle group at the same time point(P< 0.05).Western blot results confirmed the decrease of GDF10 expression induced by NMDA compared with vehicle group(P< 0.05).NMDA injection contributes to decrease of GDF10 in spinal dorsal horn neurons as well.3.Intrathecal injection of MK-801 significantly blocked SNL-induced the decrease of paw withdrawal threshold compared with vehicle group(P< 0.05).Western blot analysis confirmed that MK-801 inhibited the decrease of GDF10 in SNL rats compared with vehicle group(P< 0.05).Conclusions(1)The rat neuropathic pain model is successfully established by left L5 spinal nerve ligation.(2)The mechanical hypersensitivity of rats is increased after spinal nerve ligation and the expression of GDF10 in spinal dorsal horn neurons is reduced.(3)NMDA intrathecal injection make the increase of mechanical hypersensitivity of rats and GDF10 in spinal dorsal horn neurons is reduced as well which indicates the decrease of GDF10 is correlated to NMDAR activation.(4)Blocked the activation of NMDAR in spinal cord of spinal nerve ligation rats can alleviate the neuropathic pain and inhibit the decrease of GDF10 in spinal cord.