White matter is composed of axons and axon-wrapping myelin. Physiological role of white matter is rapid signal transduction with saltatory conduction. The humans are a unique species of mammals that has larger white matter than gray matter in their brain. As a consequence of larger volume of white matter, many neurological diseases affect white matter. White matter is also frequently involved in ischemic stroke, and progressive white matter injuries are associated with vascular cognitive impairment. Ischemic vascular cognitive impairment is caused by reduced signal conduction across white matter because of decreased signal conduction speed. Decreased signal conduction in ischemic white matter lesion results from the loss of myelin which wraps axon to provide saltatory conduction for enhancing signal transduction from neuron to neurons. Myelin is produced by oligodendrocytes (OLs) in central nervous system. Demyelination and oligodendrocyte loss are prominent features of the white matter stroke.
To elucidate the pathomechanism of ischemic white matter lesion, I used focal white matter infarction model, which resembled acute human subcortical white matter lacunar infarction, by using endothelin-1 (ET-1), a potent vasoconstrictor to cause localized ischemia. Stereotaxic endothelin-1 injection into the internal capsule resulted in a localized demyelinating lesion in mice, where loss of OL lineage cells and inflammatory cells infiltration were observed. Because of inflammatory cells infiltration, I focused on the toll-like receptor (TLR) in ET-1 induced ischemic demyelinating lesion because TLRs are main mediator of post-ischemic inflammatory responses. There was a significantly increase of TLR2 in the ET-1 induced ischemic white matter lesion. Increased expression of TLR2 in ischemic white matter lesion was also observed in human sample. Intriguingly, the extent of demyelinating pathology was markedly larger in TLR2 deficient mice than that of wild type (WT) mice. The aggravation of demyelinating lesions was not associated with increased microglial activation or up-regulation of pro-inflammatory cytokines like TNF-α and IL-1β. Instead, TLR2 deficient mice showed enhanced OL death and decreased phosphorylation of ERK1/2 compared to WT animals. Cultured OLs from TLR2 deficient mice were more vulnerable to oxygen-glucose deprivation (OGD) than WT OLs. Applying TLR2 agonists after OGD substantially rescued WT OL death with augmentation of ERK1/2 phosphorylation. Treatment with TLR2 agonists also reduced the extent of ET-1 induced ischemic demyelination in vivo without any increase of inflammatory response in non-lesional area.
To modulate or activate TLR2 on OLs, endogenous ligands of TLR2 must be present and bind to the cognate receptor. Several endogenous ligands from intracellular contents such as heat shock protein family or high mobility group box 1 (HMGB1).have been identified. I found substantial accumulation of HMGB1, but not members of heat shock protein family, in OL-bathing media after OGD. OLs expressed HMGB1 in nucleus in vitro and in vivo in uninjured condition. After OGD, HMGB1 in OLs was translocated from nucleus to cytoplasm and eventually released into the extracellular space. To determine role of released HMGB1 on OGD induced OLs death, HMGB1 inhibitor or HMGB1 depleted media were used. After application of HMGB1 inhibitor or HMGB1 depleted media, OGD induced OLs death was augmented. HMGB1 containing conditioned media could not show any protective effects on TLR2 (-/-) OLs. Exogenous application of HMGB1 also showed rescued OLs from OGD induced OLs death. HMGB1 application increased ERK1/2 and CREB phosphorylations. In addition to the direct effects on OLs, HMGB1 application also increased IGF-1 in primary microglia in a TLR2 dependent manner. Finally, application of HMGB1 inhibitor, glycyrrhizin, to the ischemic white matter lesion aggravated ET-1 induced ischemic demyelinating areas in vivo without changing post-ischemic inflammatory response. HMGB1 inhibitor applied mice showed worsening neurobehavioral outcomes than vehicle applied mice, showing a strong correlation between the neurobehavioral outcomes and with the extent of ET-1 induced demyelinating lesion.
The present study showed demyelinations with oligodendrocyte loss were prominent features of ET-1 induced localized white matter lacunar infarction in mice resembling human pathology of ischemic white matter injury. Furthermore, this study suggests TLR2 activation on oligodendrocytes with its endogenous ligand may be a novel therapeutic target to treat ischemic white matter injuries and related neurological deficits.