Role Of Iron Overload Induced Ferroptosis In Secondary White Matter Injury After Spinal Cord Injury

There is an increasing tendency in the incidence of spinal cord injury(SCI)which always induces parslysis of patients.However,the effective therapy are absent and the mechanism of secondary damage following SCI remains to be elucidated.Our previous study and recent evidences have shown that white matter damage may be more important than grey matter injury after SCI.Past studies have reported that there is a wide range of focal hemorrhage between gray and white matter following SCI and the time law of blood decomposition is highly consistent with oligodendrocytes death.Furthermore,iron chelator deferoxamine can reduce iron overload,alleviate white matter injury,and improve neurological function.Therefore,we hypothesized that SCI may lead to the local increase in the iron content and abnormalities of iron transport,metabolism and regulation proteins,which would cause iron overload of oligodendrocytes,then cell ferroptosis,axon demyelination and Wallerian degeneration.On the basis of our previous research work,this programme will use methods of in vitro and in vivo experiments,combined with morphology,molecular and cell biology,neurophysiology,and ethology,to explore iron generation,distribution and overloading and its role in the white matter secondary injury after SCI.The purpose of this study is to demonstrate that iron overload would result in white matter secondary injury in SCI and provide new targets for SCI treatment and promote the clinical application of the iron chelator.Part Effect of iron overload on ? the secondary white matter injury after spinal cord injury in ratsObjectiveTo investigate the role of iron overload in the secondary white matter injury following SCI and the effect of DFO on experimental spinal cord injury in rats.MethodsWe established the model of spinal cord contusion injury at T10 in female rats.Rats were divided into three experimental groups: Sham group,SCI group and DFO group.The SCI group rats were treated with vehicle and the DFO group received DFO injection treatment.The hindlimb motor and sensory functions of rats were evaluated by BBB score and SEP(evoked potential)detection.Bladder contractile function was evaluated by bladder pressure detection.Iron overload was examined by Perl's staining and spectrophotometric detection.The expression of transferrin receptor(Tf R),divalent metal transporter 1(DMT1),amyloid precursor protein(APP),degraded myelin basic protein(d MBP)was detected via immunohistochemical staining and Western Blot.The degree of demyelination in the white matter of the spinal cord was measured through transmission electron microscopy.The rats in each group were decapitated at designed time points for detection.Results1.The levels of APP and d MBP proteins,which are the markers of white matter injury,were significantly increased in the SCI group compared to the sham group.Immunohistochemical staining showed that oligodendrocyte counts were significantly decreased following SCI.Electron microscopy examination also revealed that the demyelination of white matter axons was more severe in the SCI group than in the sham group.Meanwhile,in the SCI group rats,the BBB score was significantly decreased,the latency of SEP was prolonged evidently,and the bladder pressure was increased at the maximum volume compared with the sham group rats.These results suggest rats develop hindlimb paralysis,sensory disturbance and neurogenic bladder after spinal cord injury,.2.Perl,s staining showed that the iron content in spinal cord tissue was significantly augmented in the SCI group compared to the sham group.In addition,the level of non-heme iron of the SCI group detected by spectrophotometric method was much higher than that of the SHAM group.3.Compared with the spinal cord injury group,DFO can reduce intramedullary iron content and iron overload,which was accompanied by attenuated white matter injury including the reduced expression of APP and d MBP proteins,increased oligodendrocytes count,and reduced axonal demyelination.Meanwhile,neurological function has been improved in the DFO group compared to the SCI group.For example,the BBB score of the hind limb improved,the SEP latency shortened,and the bladder pressure at the maximum urinary flow rate decreased.4.The expression of iron transport-related proteins such as Tf R and DMT1 was significantly enhanced following SCI.Immunofluorescence staining showed that Tf R and DMT-1 was co-expressed with oligodendrocytes markers CC1 respectively.However,after DFO treatment,the expression of Tf R and DMT1 decreased significantly,accompanied by reduced iron overload.5.Cells Ferroptosis had been found in the spinal cord tissue after SCI in rats,which is manifested by condensed mitochondria and increased mitochondrial membrane density.We speculate that Ferroptosis also occurs in oligodendrocytes.Further research is need to demonstrate the hypothesis.Conclusion1.Secondary white matter injury occurs after SCI in rats,which leads to severe neurological dysfunction;2.Iron overload can occur after spinal cord injury;3.DFO can significantly reduce white matter injury and improve neurological function of SCI rats via the alleviation of iron overload;4.The mechanism of iron overload in oligodendrocytes may be related to the enhanced expression of iron transporters Tf R and DMT1 proteins;5.Oligodendrocytes Ferroptosis may be the mechanism of white matter injury caused by iron overload after SCI,which needs further study.Part II Role of Ferroptosis in oligodendrocyte injury induced by iron overloadObjectiveWe established an in vitro oligodendrocyte iron overload model to study the role of Ferroptosis in oligodendrocyte injury.MethodsWe established the oligodendrocyte iron overload model through Co-culture of FeCl₃ with oligodendrocytes,DFO was used for intervention of cells.Cells were divided into 4 groups including Control group,FeCl₃ group,FeCl₃+vehicle group,and FeCl₃+DFO group.Intracellular iron overload was detected by Perl's staining.Oligodendrocytes activity was evaluated with CCK-8 assay and LDH releasing method.The expression of Tf R and DMT1,at gene and protein level was detected by RT-PCR and Western blotting respectively.The production of ROS in oligodendrocytes was measured by using flow cytometry.Ferroptosis of oligodendrocytes was examined via transmission electron microscopy.Results1.Oligodendrocytes iron overload model was successfully established.Perl,s staining showed obvious iron overload in oligodendrocytes after co-culture of FeCl₃ and oligodendrocytes.2.CCK-8 assay showed that the viability of oligodendrocytes decreased significantly after intracellular iron overload.The LDH method revealed that the cells were obviously damaged in the FeCl₃ group and FeCl₃+vehicle group compared with the Control group.However,after DFO was administered,intracellular iron overload was significantly reduced,which was accompanied by increased cells viability and decreased cells damage.3.In the FeCl₃ group and FeCl₃+vehicle group,the expression of Tf R and DMT1 at gene and protein level were both increased compared to Control group.However,the expression of Tf R and DMT1 was much less in the FeCl₃+DFO group than that in the FeCl₃ group and FeCl₃+vehicle group.Then the iron overload in the cells reduced.4.The production of ROS increased significantly in the cells after iron overload.Ferroptosis was observed obviously in the FeCl₃ group and FeCl₃+vehicle group,which was manifested by condensed mitochondria and increased mitochondrial membrane density.But when DFO was applied to treat the cells,ROS production decreased and ferroptosis was less observed.Conclusion1.Iron treatment of oligodendrocytes could lead to intracellular iron overload and iron overload could result in oligodendrocyte injury;2.Increased expression of Tf R anf DMT1 may be related to the intracellular iron overload of oligodendrocytes;3.Iron overload leads to the continuous production of ROS,which may cause ferroptosis of oligodendrocytes,suggesting that ferroptosis may be an important mechanism of white matter injury.