Plasmalemma permeability and necroptosis: New targets for intracerebral hemo

描述(申请人提供)：非创伤性脑出血(ICH)是一个主要的公共卫生问题，缺乏专门的治疗方法。我们提出了几条证据，表明胶原酶诱导的小鼠脑出血后，质膜通透性和坏死参与了细胞死亡。脑出血后，质膜对碘化丙啶(PI)的通透性是细胞损伤和死亡的一个关键特征；在肿瘤坏死因子α和Fas受体缺乏的小鼠中，脑出血后的质膜通透性降低了50%；通过EM、组织学和生化指标评估，脑出血后出现坏死样细胞死亡；通过RIPK3基因敲除(一种控制程序性坏死的关键分子)，脑出血后PI+细胞减少。这些初步发现有力地表明，坏死性下垂(由肿瘤坏死因子/Fas启动，由RIPK1和RIPK3介导)会导致脑出血后细胞死亡，可能还会导致神经功能障碍。Necrostatins是RIPK1的小分子抑制剂，可以抑制细胞内的坏死性下垂，保护脑创伤和缺血性中风模型中的细胞死亡和功能缺陷。Kollidon VA64是FDA批准的用于药丸生产的配方成分，用于在体内重新密封CCI和ICH后的受损细胞。VA64可减轻CCI后小鼠血脑屏障损伤、脑水肿、细胞死亡和运动功能障碍，减少胶原酶脑出血后损伤细胞和BBB损伤。利用小鼠胶原酶和自体血液脑出血模型，我们提出了3个特定的目标来检验以下中心假设：(1)质膜损伤是脑出血后细胞死亡的标志和中介；(2)程序性坏死对脑出血后细胞死亡和功能结局有贡献。目的1将利用脉冲标记实验追踪脑出血后受损细胞的体内命运，并验证质膜完整性丧失是脑出血后致死性细胞损伤的生物标志物的假说；目的2将验证VA64恢复质膜完整性，减少继发性损伤并拯救脑出血后受损脑细胞免于死亡的假说；目的3将使用RIPK3基因敲除小鼠和Necrostatins，验证坏死下垂导致脑出血后质膜完整性丧失和细胞死亡的假说，以及使用Necrostatins+VA64的翻译方法。这些目标的完成将使坏死成为脑出血的核心，并可能使VA64和坏死抑素成为治疗脑出血的新疗法。

DESCRIPTION (provided by applicant): Non-traumatic intracerebral hemorrhage (ICH) is a major public health problem that lacks specific therapy. We present several lines of evidence suggesting that plasmalemma permeability and necrosis contribute to cell death after collagenase induced ICH in mice. Following ICH, plasmalemma permeability to propidium iodide (PI) is a key feature of cellular injury and death; plasmalemma permeability after ICH is reduced by 50% in mice deficient in TNF alpha and Fas receptor; necrotic-like cell death, assessed by EM, histological, and biochemical criteria occurs after ICH; and PI+ cells are reduced after ICH by RIPK3 knockout (a key molecule governing programmed necrosis). These preliminary findings strongly suggest that necroptosis (initiated by TNF/Fas and mediated by RIPK1 and RIPK3) contribute to cell death, and perhaps neurological dysfunction, after ICH. Necrostatins are small molecule inhibitors of RIPK1 that inhibit necroptosis in cells and protect against cell death and functional deficits in brain trauma and ischemic stroke models. Kollidon VA64 is a FDA approved formulary component in pill manufacture that reseals injured cells after CCI and ICH in vivo. VA64 administration reduces blood brain barrier damage, brain edema, cell death, and motor dysfunction after CCI, and reseals injured cells and reduces BBB damage after collagenase ICH in mice. Using mouse collagenase and autologous blood ICH models, we propose 3 specific aims to test the central hypotheses that (1) plasmalemma damage is a marker and mediator of cell death after ICH, and (2) that programmed necrosis contributes to cell death and functional outcome after ICH. Aim 1 will use pulse labeling experiments to follow the fate of injured cells in vivo and test the hypothesis that loss of plasmalemma integrity is a biomarker of fatal cellular injury after ICH; Aim 2 will test the hypothesis that VA64 restores plasmalemma integrity, reduces secondary injury and rescues injured brain cells from death after ICH; Aim 3 will test the hypothesis that necroptosis induces loss of plasmalemma integrity and cell death after ICH, using RIPK3 knockout mice and necrostatins, and a translational approach using necrostatins plus VA64. Completion of these Aims will establish necrosis as central to ICH, and may establish VA64 and necrostatins as novel therapies for ICH.