Role And Intervention Of Non-selective Cation Channel TRPA1 In White Matter Damage After Intracerebral Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)is a devastating disease that is characterized by high morbidity and high mortality.ICH has an annual incidence of 10–30/100 000 people and accounts for approximately 10% to 30% of all types of stroke.ICH mostly occurs at the basal ganglia,which is rich in nerve fibres;thus,hemiplegia is quite common in ICH patients with partial sensory disturbance and ectopic blindness.In the clinic,those symptoms are considered to originate from the white matter inju ry in the area,but the exact mechanisms are unknown,and currently,no effective drug treatments are available to improve the prognosis.Clarifying the mechanisms will contribute to the development of new treatment methods for patients.The transient receptor potential ankyrin 1(TRPA1)channel is a non-selective cation channel that plays key roles in inflammatory pain sensation,nociception,emotion,cognition and social behaviour.Here,we report that TRPA1 is involved in myelin damage and oxidative stress injury in a mouse ICH model.Intervention with the TRPA1 channel may be a new method to improve the motor function of patients in the early stage of ICH.Part? Neuronal tract damage and motor dysfunction in the ICH miceObjectiveTo explore the condition of neuronal tract damage and motor dysfunction in the ICH mice.MethodsAdult male C57BL/6 mice(aged 8–10 weeks old,22–30 g)were addressed ICH in the brain using the autologous blood injection model.The mice were randomly divided into two groups: Sham and ICH.L-?-lysophosphatidylcholine was used to construct a demyelinating model.Several indexes including Immunofluorescence,Western Blot Analysis,Transmission Electron Microscopy,Electrophysiologic Recording,Weight,Behavioural assessments were assessed comprehensively.Results1.Compared with the sham group mice,the ICH mice showed a lower score in the BMS and beam balance tests on D0-D3.The forelimb muscle strength of the ICH mice was lower than that of the sham group mice on D0-D3.No significant differences were observed in the BMS score,the beam balance test score and forelimb muscle strength between the two groups on D7 and D14.No significant difference in weight was noted between the two groups throughout the experimental period.2.In contrast to that of the sham group mice,the latency of MEPs of the ICH mice was significantly extended on D1 and D3,and returned to the sham group level on D7 and D14.3.The coronal sections showed a progressive,overt decrease based on staining for normal MBP expression in the peri-haematoma region on D1 and D3 after ICH,and the immunofluorescence gradually recovered on D7 and D14.MBP protein expression showed a similar trend.Simultaneously,the DMBP immunofluorescence was enhanced after ICH,which indicated that myelin degradation was increased.Moreover,the decrease in NF200 was parallel with that of MBP.4.We noted oedema of the axons and disintegration of myelin in the peri-haematoma white matter,and an increased G-ratio indicated thinning of the myelin sheath under TEM.The myelin sheath of the ICH mice was compared to that of the demyelinating model,and the pathological changes of the white matter were similar.ConclusionThese results indicated severe neuronal tract damage and motor dysfunction in the acute phase of ICH mice.Part ? Study on the effect and mechanism of TRPA1 blockade on white matter damage of ICH miceObjectiveTo explore the effect and mechanism of TRPA1 blockade on white matter damage of ICH mice.MethodsAdult male C57BL/6 mice(aged 8–10 weeks old,22–30 g)were addressed ICH in the brain using the autologous blood injection model.The mice were randomly divided into the following groups: Sham,ICH,ICH + HC-030031,ICH + A-967079,and ICH + polygodial.Several indexes including Immunofluorescence,Western Blot Analysis,Transmission Electron Microscopy,Electrophysiologic Recording,Behavioural assessments,Kaplan-Meier survival curves were assessed comprehensively.Results1.The immunofluorescence and western blot analyses demonstrated that TRPA1 was expressed in the white matter and was co-labelled with the MBP.2.HC-030031 and A-967079 shortened the MEPs latency of the ICH mice on D1 and D3.In contrast,the TRPA1 agonist polygodial application in the ICH mice could not induce MEPs.3.HC-030031 and A-967079 obviously improved the BMS,beam balance test and the forelimb muscle strength scores in the first three days of ICH.4.The NF200 and MBP immunofluorescence was significantly enhanced on D1 and D3 after HC-030031 and A-967079 application in the ICH mice.The improved MBP protein expression indicated that HC-030031 and A-967079 reduced the degradation of myelin skeletal proteins.The reduced fluorescence intensity of DMBP showed that HC-030031 and A-967079 also reduced MBP degradation.Conversely,we found that polygodial deteriorated myelin damage with a decreased fluorescence intensity and reduced MBP expression on D1.5.TEM showed that HC-030031 and A-967079 reduced the degree of demyelination in the acute phase of ICH,leading to a decline in the G-ratio and the improvement of the myelin injury score.Additionally,HC-030031 could ameliorate the axonal oedema,whereas application of polygodial showed a large number of demyelinated axons.6.NOX1 expression was increased after ICH.A significant decrease of NOX1 was observed after application of HC-030031 and A-967079.Calpain1 expression was increased in the ICH mice and significantly down-regulated after administration of HC-030031 and A-967079.ConclusionThe above findings suggested that 1)TRPA1 blockers could significantly ameliorate nerve conduction and motor dysfunction during the acute phase in ICH mice;2)TRPA1 played a vital role in myelin damage after ICH;3)Up-regulation of NOX1 and Calpain1 mediated the myelin degradation induced by TRPA1 activation.