Pathological mechanisms of the ischemic white matter injury in animal model of lacunar infarction

Abstract

White matter is composed of axons and axon-wrapping myelin. Physiological role of white matter is rapid signal transduction with saltatory conduction. The humansare 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 postischemic inflammatory responses. There was a significantly increaseof 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 proinflammatory 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 vivowithout 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 contentssuch 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 vivoin 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 effectson 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 areasin vivowithout 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.

뇌백질은 축삭과 수초로 이루어져 있으며, 수초는 희소돌기교세포에서 생성된다. 허혈성뇌졸중에서 뇌백질은 흔하게 침범되는 부위이며, 뇌백질의 허혈성손상은 궁극적으로 혈관성인지장애를 유발하는 것으로 알려져 있다. 허혈성백질손상으로 인한 신경학적 손상은 허혈성손상 후 탈수초화가 일어나며 이 결과 신경세포들의 신호전달이 느려짐으로 인하여 발생하게 된다. 허혈성백질손상의 주된 병리소견은 탈수초화와 수초를 생성하는 희소돌기교세포의사멸로 알려져있다. 본 연구에서는 백질부위에 국한된 열공성 뇌경색동물모델을 이용하여 허혈성백질손상의 기전을 규명하고자하였다. Endothelin-1 (ET-1)이라는 혈관수축제를 생쥐의속막에 주입하여 백질에 국한된 열공성 뇌경색 모델을 확립하였다. 허혈성백질손상부위에서 허혈성탈수초화와 희소돌기교세포의세포소실이 관찰되었으며, 동시에 염증세포의 침윤과 Toll-like receptor 2(TLR2)의 발현이 백질열공성뇌경색 부위에서 증가함을 확인 하였다. 이는 허혈성백질손상의 인체부검 소견과도 일치하였다. TLR2 결핍생쥐를 이용하여 백질내열공성뇌경색에서 TLR2의 역할을 규명하고자 하였으며, 그 결과 TLR2 결핍생쥐가 대조군생쥐보다 ET-1에 의한 백질내열공성뇌경색의 크기가 유의하게 커짐을 확인하였다. TLR2결핍생쥐에서의 병변크기 증가는 허혈성 손상 후 일어나는 염증반응과는 무관하게 발생하였다. 열공성뇌경색 후 희소돌기교세포에서 TLR2의 발현이 증가함을 확인 할 수 있었고, TLR2 결핍생쥐에서 대조군보다 희소돌기교세포의 사멸이 증가함을 확인하였다. 특히 TLR2 결핍생쥐에서는 희소돌기교세포 생존에 중요한 ERK1/2의인산화가 일어나지 않았다. 희소돌기교세포의 세포실험에서도 생체내실험과 동일한 결과를 확인하였으며, TLR2 자극제인 Pam3CSK4를 처리하였을 때, 세포실험 및 생체내실험 모두에서 희소돌기교세포의 세포사멸이 감소하거나 병변크기의 감소를 확인하였다. 희소돌기교세포는 허혈성자극 후에 TLR2의 작용제로 알려진 HMGB1이라는 핵단백질을 방출함이 관찰되었다. 방출된 HMGB1은 TLR2를 통하여 희소돌기교세포의 사멸을 허혈성자극으로부터 억제하였고, 방출된HMGB1를 억제 하였을경우, 희소돌기교세포의 세포사멸이 증가하였다. 세포실험을 통한 HMGB1의 역할을 생체내에서 규명하기 위하여 ET-1과 HMGB1의 억제제인 glycyrrhizin을 함께 속막에 주사하였다. Glycyrrhizin을 함께 주사한 생쥐는 대조군 생쥐에 비하여 백질내열공성병변의 크기가 증가하였을 뿐 아니라, 심한 신경학적 결손을 보였다. 본 연구는 백질에 국한된 허혈성병변모델을 이용하여, 허혈성백질병변에서 TLR2와 TLR2의 작용제를 통한 조절이 향후허혈성백질병변 및 희소돌기교세포사멸의 새로운 치료타겟이될 수 있음을 시사한다.