The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia–reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death.

在缺血性中风中看到的主要细胞死亡形式有两种主要类型：坏死/坏死性凋亡形式，以及在损伤半影区常见的凋亡形式。通常，凋亡与坏死性程序性细胞死亡在本质上被描述为竞争性的，其中坏死性凋亡通常被描述为对凋亡起辅助作用。在本研究中，我们在添加和不添加药理学 RIPK1 磷酸化抑制剂 necrostatin-1 的野生型和 caspase-3 缺失小鼠的缺血再灌注损伤小鼠 endothelin-1 模型中检查了这两种形式的细胞死亡之间的关系。通过细胞和体积评估、电子显微镜、TUNEL 染色、活化的 caspase-3 和 caspase-7 染色以及 CD11b 和 F4/80 染色对缺血性脑损伤进行分析。抑制 caspase-3 或 RIPK1 磷酸化显示出显着的神经保护作用，这些作用是非累加的，并且在受保护区域中表现出显着的重叠。有趣的是，皮质的形态分析表明RIPK1 抑制后细胞凋亡减少。与此一致，RIPK1 抑制降低了 caspase-3 和 caspase-7 的激活水平。此外，这种保护似乎独立于继发性炎症介质。总之，这些观察结果表明，坏死性凋亡蛋白 RIPK1 改变了 caspase-3/-7 的活性，最终导致神经元凋亡减少。因此，这些发现修改了凋亡/坏死性凋亡信号传导的传统排斥观点，揭示了这些主要的细胞死亡形式之间的一种新的相互作用形式。