| BackgroundIntracerebral hemorrhage(ICH),a subtype of stroke with high morbidity and mortality,occurs mainly in the basal ganglia and causes white matter injury(WMI),resulting in severe motor dysfunction and poor prognosis in patients.The preservation of the white matter around the hematoma is crucial to motor function recovery,but there is no effective treatment for WMI following ICH until now.Lithium has been widely used for the treatment of bipolar disorder for decades.The protective effects of lithium on neurodegenerative diseases and cerebral trauma have been studied in recent years,but whether it can be used to alleviate the WMI after ICH remains to be researched.In this study,after lithium chloride(Li Cl)administration in mice with ICH,behavioral performance and electrophysiologic functions were improved,and ICH-induced white matter pathological injury including myelin sheath and axon degeneration,was ameliorated.Furthermore,LiCl treatment decreased death of mature oligodendrocytes(OLGs)in ICH mice,which may be attributed to the enhanced expression of brain derived neurotrophic factor(BDNF)regulated by Li Cl-induced inhibition of glycogen synthase kinase-3?(GSK-3?).The decreased death of OLGs was closely associated with decreased destruction of myelin sheath and then alleviated degradation of axons.In summary,this study suggested that the protective effect of lithium on WMI after ICH might be related to the increased level of BDNF and that LiCl treatment may be a potential therapeutic method to palliate the WMI after ICH.Part? Pathological damage of white matter and motor dysfunction in mice after intracerebral hemorrhageObjectiveThe research aimed to explore the motor dysfunction and the pathological changes of white matter in mice after intracerebral hemorrhage.MethodsThe model of intracerebral hemorrhage was established in adult male C57/ BL6 mice(8-10 weeks old,23-30 g).Mice were randomly divided into two groups: sham group and ICH group.The scores of behavioral evaluation,latency of motor evoked potential,fluorescence density,gray value analysis and myelin sheath injury scores of electron microscopy were analyzed by means of BMS,beam balance test and forelimb muscle force test(n = 6),electrophysiological motor evoked potential record(n = 6),immunofluorescence(n = 5),immunoblotting staining(n = 5)and transmission electron microscopy(TEM)(n = 6).Results1.The results of BMS scores,beam balance test and forelimb muscle force test revealed that the motor functions of mice were significantly lower from day1 to day14 after ICH than those of sham group.2.The results of MEPs indicated that the latent period of left limb(corresponding to right intracerebral hemorrhage)was significantly prolonged from the day1 to the day14 after ICH compared with sham group.3.The results of immunofluorescence showed that,compared with sham group,the fluorescence densities of NF200 and MBP was significantly reduced,and the fluorescence densities of SMI-32 and DMBP were significantly enhanced in ICH mice.4.Western blotting analysis showed that MBP and NF200 protein level in white matter around hematoma decreased significantly compared with sham group,while SMI-32 protein level increased significantly.5.The results of TEM showed that the white matter around the hematoma was drastically degenerated and destroyed on day3 and day7 after ICH.At the same time,the score of myelin sheath injury showed that compared with sham group the relatively intact sheath ratio was significantly lower on the day3 and day7 after ICH,and the G-ratio of myelin indicated that the myelin sheaths around the hematoma thinned and disintegrated after ICH.ConclusionIn summary,the white matter fibers around the hematoma were obviously degraded,and abnormal nerve conduction and obvious motor dysfunction also existed after ICH in mice.Part II The effect and mechanism of lithium on white matter injury in ICH miceObjectiveThe purposes of this research were verifying the toxicity and relative safe dose of lithium chloride in mice,and exploring whether the intervention of lithium can improve the white matter injury around hematoma and its specific mechanism.Methods1.Wild type adult male C57/BL6 mice(8-10 weeks old,23-30g)were intraperitoneally injected with lithium chloride at different doses and divided into the following four groups: 42.4 mg/kg group,84.8 mg/kg group,169.6 mg/kg group and 339.2 mg/kg group.The short-term changes of motor function of mice were evaluated at different time points after single Li Cl treatment,and the motor function and weight of mice were analyzed after long-term continuous drug treatment for 7 day.2.The model of intracerebral hemorrhage was established in mice by injection of autologous blood.The mice were allocated into four groups randomly: ICH group,sham group,ICH+42.4 mg/kg group and ICH+84.8 mg/kg group.The scores of behavior evaluation,latent period of MEPs,immunofluorescence density,gray value of western blotting bands,scores of myelin sheath injury and G-ratio were statistically analyzed by means of BMS,beam balance test(n = 6),motor evoked potentials(MEPS)(n = 6),immunofluorescence(n = 5),immunoblotting(n = 5)and TEM(n = 6).3.The methods of immunofluorescence,western blotting,TUNEL staining and PI cell death detection method were used to detect the protein expression and cell location of related pathways,such as BDNF and GSK-3 ?,and to detect the effect of lithium on the death of mature oligodendrocytes(OLGs).Results1.High dose treatment of Li Cl(169.6 mg/kg and 339.2 mg/kg)brought about different degrees of reduction in motor behavior scores and weight loss caused by ICH.However,42.4 mg/kg and 84.8 mg/kg Li Cl intervention did not show significant changes in motor behavior.2.The 42.4 mg/kg Li Cl treatment could restore the motor function scores and MEPs latency to some extent in mice with ICH,while 84.8 mg/kg Li Cl could not significantly improve the motor dysfunction and MEPs latency.3.Mice were treated with 42.4 mg/kg Li Cl on the day 3 after ICH.The results indicated that the immunofluorescence densities and protein levels of NF200 and MBP were significantly increased,whereas immunofluorescence densities of SMI-32 and DMBP and protein level of SMI-32 were decreased significantly.4.The results of TEM showed that 42.4 mg/kg Li Cl treatment reduced the degree of demyelination on day 3 after ICH.Moreover,the decrease of G-ratio,the improvement of myelin injury score indicated that 42.4 mg/kg Li Cl treatment decreased the myelin damage level on day 3 after ICH.5.Li Cl intervention reduced the death of mature oligodendrocytes induced by ICH.Besides,Li Cl treatment inhibited GSK-3 ? and increased the protein expression of BDNF.ConclusionThese results suggest that: 1)the relative safe doses of Li Cl to treat wild-type male mice are 42.4 mg/kg and 84.8 mg/kg;2)the treatment of 42.4 mg/kg Li Cl can significantly improve the white matter injury of mice after ICH;3)the improvement of white matter injury after ICH treatment with Li Cl may be related to the increase of BDNF result from GSK-3 ? inhibition and the decrease of death of mature oligodendrocytes. |
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