Spinal HMGB1Upregulation Is Required For The Development And Maintenance Of Neuropathic Pain In Rat

Pain has been recognized as the fifth vital signs in addition totemperature，respiration，pulse and blood pressure by WHO. Neuropathic pain(NP) is the most common and intractable painful disease. InternationalAssociation for the Study of Pain（IASP）has defined neuropathic pain as painarising as a direct consequence of a lesion or disease affecting thesomatosensory system. It is well accepted that proinflammatory factors suchas TNF-α, IL-1β and IL-6play an important role in the development ofneuropathic pain. However, currently available therapeutics againstinflammatory cytokines in the treatment of neuropathic pain is less effective.High mobility group protein (HMGB1), so called “late inflammatory factor”,is a kind of non-histone bingding protein located in the nucleus of eukaryoticcells. In some pathological conditions, HMGB1is secreted from the cell andcause inflammation in the surrounding tissues. In the present study, weevaluated the role of spina l HMGB1in the development and maintenance ofneuropathic pain in rat.Experiment one: Intrathecal injection of recombinant HMGB1 induces long-lasting mechanical allodynia.Obje ctive: Intrathecal injection of HMGB1recombinant protein to testwhether HMGB1induce mechanical allodynia, and the correlation ofHMGB1with neuropathic pain development.Methods: Intrathecal injection is identical to Jasmin’s methods. Male SD ratswere randomly divided into three groups (n=8, each group): the NS group(control group), intrathecal injection of sterile saline; Group A, intrathecalinjection of1μg of HMGB1recombinant protein; Group B, intrathecalinjection of10μg of HMGB1recombinant protein.50%mechanicalwithdrawal threshold (MWT) was measured using von Frey flaments beforeinjection and1h,1d,3d,7d,14d,21d and28d after intrathecal administrationof HMGB1.Results: Compared to the saline group, Intrathecal injection of HMGB1induced long-lasting mechanical allodynia. Mechanical withdrawal threshold(MWT) of group A was significantly reduced7d after the adminastration.MWT of group B was significantly decreased from1h after injection andlasted for at least28d.Experiment two: Double-labeling immunofluorescencedemonstrates that HMGB1was mainly expressed in neurons, buthardly to see in microglia and astrocytes.Obje ctive: To observe the distribution and cellular localization of HMGB1expression in spinal cord dorsal horn.Methods: Double-labeling immunofluorescence histochemistry was used toinvestigate the cellular localization of HMGB1positive components in thespinal dorsal horn. Six healthy SD male rats weighting180g to220g wereused. Rats were perfused with0.9%NaCl and followed by4%paraformaldehyde in0.1M phosphate buffer. The spinal cord was cut intosections on a cryostat. The sections were incubated with rabbit anti-HMGB1 antibody and mouse anti-NeuN, or mouse anti-GFAP, or mouse anti-OX42,followed by Alex488anti-rabbit and Alex594anti-mouse antibody. Thelabeling was visualized by confocal microscopy.Results: Double-labeling immunofluorescence histochemistry demonstratedthat HMGB1was mainly expressed in neurons, but hardly to see in microgliaand astrocytes;Experiment three: Spinal nerve injury induces upregulation ofHMGB1in the spinal dorsal hornObje ctive: To observe whether the spinal expression of HMGB1isupregulated in neuropathic pain conditionMethods: Western blot was used to examine the spinal expression of HMGB1before SNL and at different time points after SNL.42male SD rats weighing180g~220g were randomly divided into control group (n=6) and SNL modelgroup (n=6).The expression of HMGB1in rat spinal cord dorsal horn on1d,3d,7d,14d,21d and28d after the surgery were measured by westernblotting. All rats were rapidly sacrificed and the lumbar spinal cord wasremoved. The protein was extracted under low temperature，loaded onto gels，and electroblotted onto a PVDF membrane. The membrane were placed in ablocking solution which contained5%non-fat milk, then incubated themembrane with rabbit anti-HMGB1and mouse anti-β-actin. Bound primaryantibodies were incubated with HRP-conjugated antibody，HRP-anti-rabbitand HRP-anti-mouse antibody. The reactions were detected by the enhancedchemiluminescence detection method. The densities of protein were analyzedusing Photoshop.Results: Western blotting analysis indicated that the expression of HMGB1was increased in the spinal dorsal horn after SNL, reached the peak at7d，and lasted at least for28d after SNL. But expression of HMGB1incontralateral spinal dorsal horn was not changed. Conclusions: Intrathecal injection of recombinant HMGB1induceslong-lasting mechanical allodynia; HMGB1was mainly expressed in neurons,but not in microglia and astrocytes; Peripheral nerve injury induces upregulationof HMGB1in the spinal dorsal horn. These results suggest that upregulation ofHMGB1may play an important role in the development of mechanical allodyniaafter nerve injury.