The Role Of Intracranial Iron Overload In Central Pain After Spinal Cord Injury

IntroductionAround 60%–90% of patients with spinal cord injury(SCI)develop central pain(CP),which is defined as pain hypersensitivity resulting from central nervous system injury.Pain in the lower limbs is often persistent and difficult to endure,making CP a maj or problem for patients and physicians.It is thought that structural and functional remodeling of the brain after SCI can cause CP;however,the mechanisms of CP development are still poorly understood.In our previous work,we used magnetic susceptibility weighted imaging to investigate the functional remodeling of the brain in SCI patients.We found that iron-overloaded regions—including the sensory regions of the cortex,thalamus,and cingutate(unpublished data)—were remodeled,implying that intracranial iron overload is involved in the pathogenesis of CP.Intracranial iron overload plays an crucial role in the occurrence and development of Alzheimer's and Parkinson's disease and cerebral hemorrhage.The toxicity of iron may be linked to oxidative stress injury induced by the Fenton reaction.Iron overload can activate brain microglia,which initiate and amplify neuronal damage.Activated microglia also regulate the levels of cytokines such as interleukin-1?,tumor necrosis factor(TNF)-?,and nitrous oxide;the ensuing formation of reactive oxygen species and amplification of the proinflammatory cytokine cascade can lead to neuronal damage or loss.In addition,activated microglia may induce nuclear factor(NF)-?B signaling in neurons.Objective:The present study investigated the causes of intracranial iron overload and its relationship with CP pathogenesis.Our findings provide a basis for the treatment of CP following SCI through the use of iron-chelating agents and NF-?B and microglia inhibitors.Material and methodsThe present study include four parts:1.Building and evaluation of CP after SCIFemale Sprague-Dawley rats(n = 75)were randomly divided into five groups(n = 15 each): sham operation(laminectomy only without SCI or drug treatment);control(use the Allen hitting method made rats SCI model without drug treatment);NOS inhibitors treatment(L-arginine by i.p.injection after surgery);iron chelator treatment(deferoxamine by i.p.injection after surgery);and microglia activation inhibitors treatment(minocycline by i.p.injection after surgery).Assessment of spontaneous pain:Following surgery,rats were observed for spontaneous pain behaviors,including trimming,scratching,licking of the hindlimbs and tail,and vocalizations.Determination of evoked pain:The latency of thermal pain hypersensitivity was measured after surgery.2.The accumulation of brain iron after spinal cord injury.The iron content of the whole rat brain and hindlimb sensory areas,thalamus,and cingulate gyrus was determined by atomic absorption spectrophotometry.3.The mechanism of brain iron overload after spinal cord injury.TfR1 mRNA expression in various brain regions,including the hindlimb sensory cortex,cingulate gyrus,and thalamus was determined by RT-PCR.TfR1 and Fn levels in various brain regions,including the hindlimb sensory cortex,cin gulate gyrus,and thalamus was determined by enzyme-linked immunosorbent assay(ELISA).IRP1,Fn,Lf,and NF-?B protein levels in the hindlimb sensory cortex of rats in each group were determined by western blotting.4.The mechanism of Microglia activation after spinal cord injuryMicroglia and IRP1 in the hindlimb sensory cortex of rats were observed by immunohistochemical staining method.Data are presented as mean ± standard deviation.Univariate analyses were used to compare group means.P < 0.05 was considered as statistically significant.Results1.Spontaneous pain in animal models is characterized by scratching and biting below the level of injury.After SCI,rats were observed to scratch,bite,and excessively groom body parts below the level of injury,i.e.,hindlimbs and tail,which was not exhib ited by rats in the sham operation group.Thermal pain threshold was higher for sham-operated animals than for the other groups(P < 0.05),while that of the SCI group was decreased relative to the other groups(P < 0.05).The sham operation group had the h ighest latency values,while the SCI group had the lowest values,at the time points examin ed.2.There were no differences in whole brain iron content among groups.However,the iron contents of the hindlimb sensory area,cingulate gyrus and thalamus were lower in the sham operation as compared to the other groups(P < 0.05),while the brain iron contents of the SCI and minocycline treatment groups was higher than those of the other groups(P < 0.05).3.Determination of Western Blot found IRP1 and NF-?B levels were lower,while Fn levels were higher,in the hindlimb sensory cortex of sham-operated rats as compared to other groups(P < 0.05).IRP1 levels were lower,while those of NF-?B and Fn were higher,in the SCI than in the sham operation,or arginine or DFX treatment groups(P < 0.05).These results suggest that arginine treatment decreases IRP1 and NF-?B and increases Fn levels after SCI,whereas DFX treatment decreases NF-?B and increases Fn levels but does not affect IRP1 expression.Minocycline treatment had no effect on the expression of any of these proteins,and there were no differences in LF level among groups.Determination of ELISA found Tf R1 and Fn levels in the hindlimb sensory cortex,thalamus,and cingulate gyrus TfR1 levels were lower,whereas Fn levels were higher,in sham-operated animals than in the other groups(P < 0.05)(Fig.4).In the SCI group,TfR1 expression was higher,whereas Fn expression was lower,than in the sham operation and arginine and DFX treatment groups(P < 0.05).Arginine treatment lowered Tf R1 expression(P < 0.05)and increased Fn levels(P < 0.05)after SCI.DFX treatment increased Fn levels(P < 0.05)but had no effect on TfR1 expression,while minocycline had no effect on Tf R1 or Fn levels(P > 0.05).TfR1 gene expression in the rat hindlimb sensory area,thalamus,and cingulate gyrus was determined by RT-PCR.TfR1 levels in the various brain regions were lower in sham-operated rats as compared to the other groups(P < 0.05),but were higher in the SCI than in the sham and arginine treatment groups(P < 0.05).These results indicate that arginine and DFX treatment decrease TfR1 gene expression after SCI,with the former showing a greater effect.4.The number and activation of microglia in the hindlimb sensory co rtex was markedly increased after SCI.Treatment with arginine,DFX,and minocycline,decreased microglia number and activation.Accordingly,the number of IRP1-positive cells was increased after SCI,an effect that was abrogated in the presence of arginine.Conclusion:1.Allen hitting method is advisable for establishment of the CP rat model of spinal cord injury because it can induce central neuropathic pain under injured level;2.There were no differences in whole brain iron content after SCI.The contents of IRP1,TFR1 and iron of the hindlimb sensory area,cingulate gyrus and thalamus were higher,while Fn were lower.The number and activation of microglia in the hindlimb sensory cortex was markedly increased after SCI.3.Using NOS inhibitors can reduce the contents of IRP1?TfR1?and iron of the hindlimb sensory area,cingulate gyrus and thalamus,while increase Fn content.Alleviate activation of microglia in the hindlimb sensory cortex and improve thermal pain threshold under injured level.4.Using iron chelator can reduce the iron contents of the hindlimb sensory area,cingulate gyrus and thalamus,while increase Fn content.Alleviate activation of microglia in the hindlimb sensory cortex and improve thermal pain threshold under injured level.5.Using microglia activation inhibitors can alleviate activation of microglia in the hindlimb sensory cortex and improve thermal pain threshold under injured level.6.The results presented here indicate that after SCI,activation of IRP can lead to intracranial iron overload,which activates microglia via the NF-?B signaling pathway.The proinflammatory cytokines secreted by these microglia causes neuronal damage and loss,leading to CP.7.Our result suggest that NOS inhibitor,iron-chelating agent or microglia inhibitor,can effectively relieve CP in SCI patients.