The Promoting Effects Of Exogenous Hydrogen Sulfide On White Matter Repair Following Subcortical White Matter Stroke And Underlying Mechanisms

Research Purposes:Ischemic stroke is a major health problem globally.Subcortical white matter stroke is a very unique subtype of stroke,accounting for 25%of strokes.It is the second leading cause of dementia and is very common in the elderly.At present,there is no effective treatment for subcortical white matter stroke.Previous research from our group showed that the exogenous slow-releasing hydrogen sulfide donor anethole trithione(5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione,ADT)conferred therapeutic effects on ischemic stroke in animal models,yet the underlying mechanisms remain unclear yet.Here,I investigated whether ADT also conferred therapeutic effects following subcortical white matter stroke.Moreover,we recently reported that sulfide:quinone oxidoreductase(SQR)is the key mediator of H2S signaling.Oxidation of H2S by SQR drove reverse electron transfer at mitochondrial complex I and consequently induced mitochondrial production of reactive oxygen radicals(ROS).Mitochondrial ROS,in turn,activated uncoupling protein 2 and subsequently leads to the activation of AMP-activated protein kinase(AMPK).Furthermore,microglia/macrophage SQR mediated the therapeutic effects of ADT in an animal model of intracerebral hemorrhage(Sci Adv,2020).Therefore,I further investigated whether ADT acted through the signaling cascade mediated by SQR to promote white matter repair following subcortical white matter stroke.Methods:(1)For induction of subcortical white matter stroke in mice,N5-(1-iminoethyl)-L-ornithine dihydrochloride(L-NIO)was stereotactically injected into the corpus callosum of mice.The sham-operated mice were injected with an equal volume of saline.Then,the mice subjected to subcortical white matter stroke were randomly divided to receive intraperitoneal injection of ADT or vehicle(Veh 10%dimethyl sulfoxide in corn oil)daily,starting at 24 h after L-NIO injection.The mice were subjected to the novel object recognition test at the 2,4,9 weeks after L-NIO injection.Collect brain tissues and frozen sections at the 9th week after surgery,and use immunohistochemical methods to detect the recovery of white matter ischemic area damage in each group of mice to verify that ADT promotes long-term repair of ischemic white matter effect.(2)Brains were collected,and sectioned cronally on a cryostat cronal at the 9 weeks after L-NIO injection.To determine whether ADT promoted repair following subcortical white matter injury,Immunohistochemical methods were used to detect white matter injury volumes in each group of mice.(3)Genetically engineered mice with soecific deletion of SQR in microglia and macrophages(Cx3cr1Cre:Sqrfl/fl)were used to determine whether ADT acted through microglial SQR to promote repair following subcortical white matter stroke.As described above,we injected L-NIO into the corpus callosum of knockout and control to induce subcortical white matter stroke.Then,the mice were randomly divided into to receive daily inection of ADT or vehicle,starting at 24 hours after L-NIO,for 9 weeks.At 2,4,and 9 weeks after L-NIO injection,the mice were subjected to the novel object recognition test.The brains were harvested and coronally sectioned on a cryostat at 9 weeks after L-NIO injection,and the sections were used for the assay of lesion volumes and evaluation of the integrity of nodes of Ranvier using immunohistochemistry approaches.(4)Brains were collected,and sectioned cronally on a cryostat cronal at the 2 weeks after L-NIO injection.To determine whether ADT promoted microglia to remove myelin debris following subcortical white matter injury by Immunohistochemical methods.(5)Genetically engineered mice(NG2CreERT+/-:RosatdT/+)were prepared for lineage tracing of the maturation of oligodendrocyte precursor cells(OPC).The lineage mapping mice(OPC reporter mouse)that is used to label OPC lineage cells.To specifically label OPC with the red fluorescent protein(tdT),tamoxifen(100 mg/kg)was daily injected into NG2CreERT+/-:RosatdT/+ mice via the intraperitoneal route for 5 days.Ten days after tamoxifen injection,L-NIO was stereotactically injected into the corpus callosum.Sham-operated mice received was injection of the same amount of saline.The mice subjected to L-NIO injection were randomly divided to receive daily injection of ADT or Vehicel,starting at 24 h after surgery.The brains were collected and sectioned cronally on a cryostat.Antibodies against CC1 was used to label mature oligodendrocytes,and Ng2 was used to label inmature OPCs.To determine whether ADT promoted the proliferation of OPCs and the differentiation of OPCs into mature OLs,the number of OPCs(tdT+),that of inmature OPCs(Ng2+tdT+)and that of mature oligodendrocytes from OPCs(CC1+tdT+)were examined in the lesion areas following subcortical white matter stroke using immunohistochemistry approaches.(6)To investigate whether ADT acted through SQR-mediated signaling pathway to promote repair following subcortical white matter injury,endogenous expression of complex I subunit NDUFS3,UCP2 or AMPK in the corpus callosum was knocked down by injection of the lentiviruses expressing NDUFS3-shRNA,UCP2-shRNA or AMPK-shRNA into the corpus callosum beofore the induction of white matter stroke.Then,L-NIO was injected to induce subcortical white matter stroke.Mice received daily injection of ADT or vehicle via the intraperitoneal route for 9 weeks,starting at 24 h after L-NIO injection.Mice were subjected to the novel object recognition test at 2,4,and 9 weeks after L-NIO injection.At 9 weeks after L-NIO injection,brains were harvested and sectioned coronally on a cryostat and frozen sections were used the assay of lesion volumes in each group of mice.Results:(1)Compared with vehicle administration,the administration of the exogenous slow-releasing hydrogen sulfide donor ADT promoted the remyelination of mice and facilitated the long-term recovery after subcortical white matter injury.(2)Deletion of SQR in microglia macrophages abrogated the promoting effects of ADT on white matter repair and functional recovery following subcortical white matter injury.(3)The hydrogen sulfide donor ADT promoted microglia to remove myelin debris following subcortical white matter injury.(4)The hydrogen sulfide donor ADT promoted the proliferation of OPCs and the differentiation of OPCs into mature oligodendrocytes.(5)Knockdown of Complex I in the corpus callosum abolished the promoting effects of ADT on white matter repair and functional recovery following subcortical white matter injury.Knockdown of UCP2 in the corpus callosum abolished the promoting effects of ADT on white matter repair and functional recovery following subcortical white matter injury.Knockdown of AMPK in the corpus callosum abolished the promoting effects of ADT on white matter repair and functional recovery following subcortical white matter injury.Conclusion:The hydrogen sulfide donor ADT plays a role in promoting long-term repair after ischemic white matter damage through the SQR signaling pathway.