The Function Of N-methyl-D-aspartate(NMDA) Receptor On Keratinocyte In Hyperalgesia Of Complex Regional Pain Syndrome(CRPS)

Introduction:Complex regional pain syndrome(CRPS)is a chronic,refractory clinical condition featured by limb hyperalgesia,which gravely undermines patients’life quality.It has been previously reported that activated keratinocytes participate in nociception and a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid(AMPA)receptor on keratinocytes plays a role in postherpetic neuralgia.Furthermore,N-methyl-D-aspartate(NMDA)receptor,which shares structural similarities with AMPA receptor,is also expressed on keratinocytes.This study is aimed to explore the function of NDMA receptor on keratinocyte in the hyperalgesia of CRPS,with special attention to its effect on proinflammatory cytokine release,nociceptive transmission and glia activation.Methods:① Grouping and interventions:chronic post-ischemia pain(CPIP)model,a well recognized animal model of CRPS,was used in this study.NMDA receptor agonist NMDA and antagonist MK801 were intraplantarly injected to study the receptor function.Male Sprague-Dawley rats were divided into acute and chronic group.The acute group was given drugs for consecutive 3 days before modeling,while drug administration was started from the 7th day after modeling to the 14th day in chronic group.Both groups were subdivided into CPIP and sham group.In CPIP groups,an O-ring(internal diameter=0.65 cm)was placed tightly on the ankle of the right limb of rats and removed 3 hours later.Contrarily,a cut O-ring was loosely placed in sham groups.The CPIP groups were further divided into 3 groups,given NMDA(1MM),MK801(1mM)and saline 100 μl/day,respectively·② Behavior and physiological tests:electronic von-Frey was used to test mechanical sensitivity and heat-plate was used to test thermal sensitivity.The difference of bilateral skin temperature was measured by infrared thermometer.The tests were performed at the 6th,12th,18th and 24th hour after modeling in acute groups and on the 2nd,6th,10th,14th day after modeling in chronic groups.③Tissue extraction:right hindpaw skin and L2—L4 spinal cord was harvested on the 1st day after modeling in acute groups and the 14th day in chronic groups.④Western Blot:the expression of NR1 in skin was studied.The release of tumor necrosis factor-α(TNF-a)and interleukin-1β(IL-1β)in skin and spinal cord was also measured.⑤Immunofluorences:keratinocyte marker pan-keratin and obligatory subunit of NMDA receptor NR1 were co-stained in skin samples.The marker of nociceptive stimulus conduction,c-fos,the marker of microglia,ionized calcium binding adaptor molecule 1(Ibal)and the marker of astrocyte,glial fibrillary acidic protein(GFAP)were labeled in spinal cord samples.Results:① Behavior and physiological tests:the comparison of von-Frey and heat-plate tests in both acute and chronic phase was CPIP+NMDA<CPIP+saline<CPIP+MK801<sham.Skin temperature was higher in CPIP groups than sham groups only in acute phase and there was no significant change between CPIP groups.② Skin immunofluorence and Western Blot:in both acute and chronic phase,the expression of NMDA receptor on keratinocytes was increased in CPIP groups.The comparison of TNF-α and IL-1β release in acute phase was CPIP+NMDA>CPIP+saline>CPIP+MK801/sham.This trend failed to persist in chronic phase.③ Spinal cord immunofluorenece and Western Blot:in CPIP+saline group,c-fos was up-regulated in both acute and chronic phase,while Ibal and GFAP were only overexpressed in chronic phase.All of those 3 markers could be suppressed in CPIP+MK801 group.No significant change of TNF-a and IL-1β release was observed in acute phase,but there was significant trend in chronic phase(CPIP+NMDA>CPIP+saline>CPIP+MK801/sham).Conclusions:① CRPS is characterized by cutaneous inflammation in acute phase and central sensitization in chronic phase.② NMDA receptor on keratinocytes is up-regulated in both acute and chronic phase.③ In acute phase,NMDA receptor on keratinocytes participates in the release of proinflammatory cytokines in skin and nociceptive conduction to spinal cord.④ In chronic phase,NMDA receptor on keratinocytes could regulate glia activation and proinflammatory cytokine release in the dorsal horn of spinal cord.