Mechanism Of Neuronal Ferroptosis Induced By Iron Overload In The Motor Cortex Following Spinal Cord Injury

Background and ObjectiveSpinal cord injury(SCI)is a devastating and highly disabling disease of the central nervous system,that often leads to varying degrees of neurological dysfunction below the injury level.It not only causes great misery to the life of patients,but imposes a great burden on families and society.Therefore,how to maximize the recovery of patients’ impaired neurological function,especially motor function,is an urgent problem to be solved in SCI research.Although studies about injury mechanism of SCI and treatments target some mechanisms have made some progress in recent years,there is still a lack of effective clinical treatment for the motor dysfunction after SCI.Throughout the previous studies on SCI,most focused on the lesion site of SCI.But,interestingly,previous clinical studies of our group found that SCI not only caused local lesions of spinal cord,but also caused atrophy of the motor cortex.Moreover,the more severe the motor cortex atrophy,the worse the patient’s motor function recovery.It might be the death of motor cortex neurons after SCI,that induced atrophy of the motor cortex eventually.The death of motor cortex neurons inevitably leads to the injury of associated axons(the corticospinal tract),thereby limiting the effectiveness of local therapy for the injured spinal cord.Thus,targeting motor cortex neuron death may provide new strategies for SCI treatment.However,the specific types and mechanisms of SCI induced motor cortex neuron death are still unclear.In 2012,Dixon et al.reported a new kind of programmed cell death.Its characteristic is that it is driven by a highly iron-dependent lipid peroxidation,hence named "ferroptosis".Subsequent studies have reported that ferroptosis also plays an important role in the pathogenesis of various central nervous system diseases,including neurodegenerative diseases.And our previous animal experiments found that SCI would lead to iron overload in multiple brain regions,including the motor cortex.This suggests that ferroptosis probably involved in the death of motor cortex neurons after SCI.However,is iron overload also present in the motor cortex of patients with SCI,how does SCI lead to iron overload in motor cortex,and whether iron overload in the motor cortex triggers neuronal ferroptosis are all unclear.In view of this,present study intends to determine iron overload in the motor cortex by analyzing cranial susceptibility weighted imaging(SWI)of patients with SCI,and further establish the SCI model of rats to dynamically observe the change of iron content in motor cortex.On this basis,we explore the specific mechanism of iron overload in the motor cortex caused by SCI,and whether iron overload in the motor cortex leads to neuronal ferroptosis by in vivo and in vitro experiments,thus exploring new effective targets for clinical treatment of SCI.Materials and Methods1.Effects of SCI on iron content in the motor cortexThe experiment consists of two parts.In the first part,cranial high-resolution SWI data were collected from 17 SCI patients and 16 healthy controls with matched sex and age.Bilateral primary motor cortex was used as the region of interest,and the phase values of the region of interest were analyzed and measured by SPIN software.Finally,the phase value is converted to radian value for statistical analysis.In the other part,the primary motor cortex samples of rats were collected at 1 day,1 week,2 weeks and 4 weeks after the establishment of SCI model for iron content detection.2.Mechanism of iron overload in the motor cortex after SCIThe experiment consists of three parts.In the first part,experimental rats were randomly divided into the following four groups: The Sham group(sham operation group),SCI group(SCI model group),Vehicle group(saline treatment after SCI),and Mino group(minocycline treatment after SCI).The primary motor cortex samples were collected 28 days after SCI for immunofluorescence and Western blot analysis.In the second part,the experimental animals were randomly divided into five groups: Sham group,SCI group,Vehicle group,Mino group and L-NAME group(L-NAME treatment after SCI).Basso,Beattie and Bresnahan(BBB)locomotor test was used to evaluate motor function 28 days after SCI.Then the primary motor cortex samples were collected for testing the levels of nitric oxide(NO)and for measuring iron metabolism-related proteins by Western blot.In the third part,the cortical primary neurons were isolated and cultured in vitro.After the intervention of the primary neurons with NO donor DETA NONOate,the expressions of iron metabolism related proteins in neurons were detected by Western blot.3.The mechanism of neuronal ferroptosis induced by primary motor cortex iron overload after SCIThe experiment consists of two parts.In the first part,experimental rats were randomly divided into the following four groups: Sham group(sham operation group),Vehicle group(0.01% DMSO treatment after SCI),DFO group(deferoxamine(DFO)treatment after SCI),NAC group(n-acetylcysteine(NAC)treatment after SCI)and Fer-1 group(ferrostatin-1(Fer-1)treatment after SCI).The motor function of hind limbs was scored according to the Basso,Beattie and Bresnahan(BBB)scale at 1 day,1 week,2 weeks,3 weeks and 4 weeks after SCI.And the electrophysiological tests of hind limbs were performed at 4 weeks after SCI.Then the primary motor cortex of rats was collected for following tests: the ROS content in the primary motor cortex of rats was detected by flow cytometry,the malondialdehyde(MDA)assay was used to assess lipid peroxidation,mitochondrial morphology of neurons was observed by transmission electron microscopy,the expressions of ferroptosis related genes were detected by q RT-PCR,and neurons in primary motor cortex were tested by Nissl’s staining.In the second part,cortical primary neurons were isolated and treated with ferric ammonium citrate(FAC)to construct in vitro iron overload model,and treated with DFO,NAC or Fer-1 respectively.Flow cytometry was used to detect the ROS content and lipid peroxidation of neurons in each group.The mitochondrial morphology of each group was observed by transmission electron microscopy,and the expressions of ferroptosis-related genes in each group were detected by q RT-PCR.Results1.Effects of SCI on iron content in the motor cortex1.1 By analyzing high-resolution SWI data,we found phase values increased in primary motor cortex of SCI patients compared with healthy controls.1.2 Iron content in primary motor cortex of rats after SCI increased gradually over time,and was significantly higher than that in sham operation group.2.Mechanism of iron overload in the motor cortex after SCI2.1 Microglia were significantly activated in primary motor cortex of rats after SCI,and minocycline treatment significantly reduced microglia activation.2.2 After SCI,activated microglia in the primary motor cortex of rats secreted a large amount of NO,and both minocycline and nitric oxide synthase inhibitor L-NAME treatment significantly reduced NO content.2.3 The expressions of iron regulatory protein 1(IRP1),divalent metal transporter 1(DMT1),transferrin receptor 1(Tf R1)in the primary motor cortex of rats was up-regulated after SCI.While the expression of ferritin was down-regulated.The expression of ferroportin1(Fpn1)was not significantly changed.Both minocycline and L-NAME treatment significantly reduced the expressions levels of IRP1,DMT1 and Tf R1,and increased the expression levels of ferritin.2.4 The motor function of rats was significantly impaired after SCI,and treatment with minocycline or nitric oxide synthase inhibitor L-NAME significantly improved motor function recovery.2.5 In vitro cell experiments showed that NO could significantly up-regulate the expressions of IRP1,DMT1 and Tf R1,and significantly down-regulate the expression of ferritin,but had no significant effect on the expression of Fpn1.3.The mechanism of neuronal ferroptosis induced by primary motor cortex iron overload after SCI3.1 ROS content in primary motor cortex of rats increased significantly after SCI,leading to obvious lipid peroxidation.Treatment with DFO,NAC or Fer-1 could significantly reduce ROS production and alleviate lipid peroxidation.3.2 The mitochondrial morphology of neurons in the primary motor cortex of rats changed abnormally after SCI,such as shrinkage or reduction of mitochondrial cristae.While DFO,NAC or Fer-1 treatment significantly alleviated mitochondrial morphological abnormalities in primary motor cortex neurons.3.3 The expressions of ferroptosis related genes in primary motor cortex of rats were disregulated,showing that Ptgs2 expression was up-regulated,Gpx4 expression was downregulated,and Fsp1 expression had no significant difference.DFO,NAC or Fer-1 treatment significantly inhibited the expression of Ptgs2 and up-regulated the expression of Gpx4.3.4 FAC intervention led to the production of a large number of ROS in primary neurons,and the accumulation of ROS could further cause lipid peroxidation.DFO,NAC or Fer-1treatment significantly reduced ROS production induced by FAC and alleviated lipid peroxidation.3.5 FAC intervention led to abnormal morphological changes of mitochondria in primary neurons,manifested as shrinkage or reduction of mitochondrial cristae.DFO,NAC or Fer-1treatment significantly alleviated the mitochondrial morphological abnormalities caused by FAC.3.6 FAC intervention led to disregulated expression of ferroptosis related genes in primary neurons,showing that Ptgs2 expression was up-regulated and Gpx4 expression was down-regulated,while Fsp1 expression had no significant difference.DFO,NAC or Fer-1treatment significantly inhibited the expression of Ptgs2 and up-regulated the expression of Gpx4.Conclusion1.SCI leades to iron deposition in the motor cortex,and iron deposition shows a trend of increasing with time.Iron overload in motor cortex may be an important reason for the structural and functional changes of motor cortex in patients with SCI.2.SCI could induce the activation of microglia in the primary motor cortex,which caused the abnormal expression of iron metabolism related proteins in neurons by secreting excessive NO,and finallly led to the gradual increase of iron content in the motor cortex.3.Iron overload in the primary motor cortex after SCI could cause a large amount of ROS accumulation in neurons,leading to lipid peroxidation,abnormal mitochondrial morphogenesis and disregulated expression of ferroptosis-related genes in neurons,which ultimately triggered neuronal ferroptosis,thus inhibiting the recovery of motor function in SCI rats.