Spinal cord injury, progressive hemorrhagic necrosis and the NC(Ca-ATP) channel

摘要 脊髓损伤 (SCI) 导致自毁过程，称为“渐进性” 出血性坏死”（PHN），导致脊髓组织毁灭性损失。两个 PHN 的关键组成部分是：1）进行性继发性出血； 2）坏死细胞 死亡。我们最近发现新表达的 SUR1 调节的 NC(Ca-ATP) 通道在坏死细胞死亡和术后继发性出血中发挥着重要作用。 SCI。在这里，我们将进一步表征 SUR1 调节的 NC(Ca-ATP) 通道的作用 在SCI中。我们的首要假设是 NFkappaB 信号传导的激活起着关键作用 在内皮细胞中 SUR1 调节的 NC(Ca-ATP) 通道从头表达中的作用， 神经元和少突胶质细胞，以及随后 ATP- 通道的打开 耗尽会导致毛细血管灾难性衰竭，形成点状出血 以及神经元和少突胶质细胞的坏死性死亡，进而引发氧化 压力和炎症共同加剧 PHN。我们在小鼠和大鼠模型中的数据 SCI 挫伤表明，药物可显着减少出血 使用格列本脲阻断 SUR1，通过使用反义基因抑制 SUR1 寡脱氧核苷酸（AS-ODN），优先靶向半影毛细血管，以及 通过转基因 SUR1-KO (SUR1-/-) 小鼠中的基因抑制，这 3 治疗或状况与短期内的显着改善有关 神经行为功能。在具体目标（SA）1中，使用基因抑制策略 针对小鼠和大鼠 SCI 模型中的 SUR1，我们将评估进行性进展的作用 继发性出血的短期后遗症，包括炎症和氧化 压力以及长期后遗症，包括组织病理学和神经行为学 功能。其他初步数据表明，与 PHN 关系最密切的细胞是 毛细血管内皮细胞、神经元和少突胶质细胞。在 SA2 中，使用原代培养物 小鼠脊髓微血管内皮细胞、神经元和少突胶质细胞 从野生型 (WT) 与 SUR1-KO 小鼠的比较中，我们将确认每种细胞类型都可以 上调 SUR1 调节的 NC(Ca-ATP) 通道，我们将表征新诱导的 通道，通过 pH 值确定其生理调节及其在细胞死亡中的作用。 其他初步数据表明 NFkappaB，众所周知， 参与 SCI，可能在 de novo 中充当重要的转录激活剂 NC(Ca-ATP) 通道的表达。在 SA3 中，使用培养细胞和脐带组织后 SCI，我们将确定 NFkappaB 刺激转录在从头的作用 SUR1 的表达和功能性 SUR1 调节的 NC(Ca-ATP) 的从头表达 渠道。这些研究将使人们更深入地了解 NC(Ca- SCI 中的 ATP）通道将带来新颖的分子见解和重要的新疗法 对于这种毁灭性的人类状况。

ABSTRACT Spinal cord injury (SCI) results in the autodestructive process termed "progressive hemorrhagic necrosis" (PHN), which leads to devastating loss of spinal cord tissue. Two critical components of PHN are: 1) progressive secondary hemorrhage; 2) necrotic cell death. We recently discovered that newly expressed SUR1-regulated NC(Ca-ATP) channels are critically involved in necrotic cell death and in secondary hemorrhage post- SCI. Here, we will further characterize the role of SUR1-regulated NC(Ca-ATP) channels in SCI. Our overarching hypothesis is that activation of NFkappaB signaling plays a key role in de novo expression of SUR1-regulated NC(Ca-ATP) channels in endothelium, neurons and oligodendrocytes, and that subsequent opening of the channels by ATP- depletion results in catastrophic failure of capillaries, formation of petechial hemorrhages and necrotic death of neurons and oligodendrocytes, which in turn provokes oxidative stress and inflammation, which together fuel PHN. Our data in mouse and rat models of contusion SCI demonstrate that hemorrhage is dramatically reduced by pharmacological block of SUR1 using glibenclamide, by gene suppression of SUR1 using antisense oligodeoxynucleotide (AS-ODN), which preferentially targets penumbral capillaries, and by gene suppression in transgenic SUR1-KO (SUR1-/-) mice, and that these 3 treatments or conditions are associated with dramatic improvements in short-term neurobehavioral function. In specific aim (SA) 1, using gene suppression strategies targeting SUR1 in mouse and rat models of SCI, we will assess the role of progressive secondary hemorrhage on short-term sequelae, including inflammation and oxidative stress, and on long-term sequelae, including histopathology and neurobehavioral function. Other Preliminary Data indicate that the cells most critically involved in PHN are capillary endothelial cells, neurons and oligodendrocytes. In SA2, using primary cultures of murine spinal cord microvascular endothelial cells, neurons and oligodendrocytes from wild-type (WT) vs. SUR1-KO mice, we will confirm that each cell type can upregulate SUR1-regulated NC(Ca-ATP) channels, we will characterize newly induced channels, determine their physiological regulation by pH and their role in cell death. Other Preliminary Data demonstrate that NFkappaB, which is known to be prominently involved in SCI, is likely to act as an important transcriptional activator in de novo expression of NC(Ca-ATP) channels. In SA3, using cultured cells and cord tissues post- SCI, we will determine the role of NFkappaB-stimulated transcription in de novo expression of SUR1 and de novo expression of functional SUR1-regulated NC(Ca-ATP) channels. These studies will yield a more thorough understanding of the role of NC(Ca- ATP) channels in SCI will lead to novel molecular insights and significant new treatments for this devastating human condition.