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

描述（由申请人提供）：脊髓损伤（SCI）导致称为“进行性出血性坏死”（PHN）的自毁过程，导致脊髓组织的破坏性损失。PHN的两个关键组成部分是：1）进行性继发性出血; 2）坏死性细胞死亡。我们最近发现，新表达的SUR 1调节的NC（Ca-ATP）通道与坏死细胞死亡和SCI后继发性出血密切相关。在这里，我们将进一步描述SUR 1调节的NC（Ca-ATP）通道在SCI中的作用。我们的首要假设是NF-κ B信号传导的激活在内皮细胞、神经元和少突胶质细胞中的SUR 1调节的NC（Ca-ATP）通道的从头表达中起关键作用，并且随后通过ATP耗竭打开通道导致毛细血管的灾难性衰竭、瘀点形成以及神经元和少突胶质细胞的坏死性死亡，这又引起氧化应激和炎症，它们一起为PHN提供燃料。我们在小鼠和大鼠挫伤SCI模型中的数据表明，通过使用格列本脲对SUR 1进行药理学阻断，通过使用反义寡脱氧核苷酸（AS-ODN）对SUR 1进行基因抑制（其优先靶向半暗带毛细血管），以及通过转基因SUR 1-KO（SUR 1-/-）小鼠的基因抑制，并且这3种治疗或病症与短期神经行为功能的显著改善相关。在特定目标（SA）1中，使用针对SCI小鼠和大鼠模型中SUR 1的基因抑制策略，我们将评估进行性继发性出血对短期后遗症（包括炎症和氧化应激）和长期后遗症（包括组织病理学和神经行为功能）的作用。其他初步数据表明，PHN中最关键的细胞是毛细血管内皮细胞，神经元和少突胶质细胞。在SA 2中，使用野生型（WT）与SUR 1-KO小鼠的小鼠脊髓微血管内皮细胞、神经元和少突胶质细胞的原代培养物，我们将证实每种细胞类型均可上调SUR 1调节的NC（Ca-ATP）通道，我们将表征新诱导的通道，确定其通过pH的生理调节及其在细胞死亡中的作用。其他初步数据表明，NF κ B，这是已知的突出参与SCI，可能作为一个重要的转录激活NC（Ca-ATP）通道的从头表达。在SA 3中，使用SCI后培养的细胞和脊髓组织，我们将确定NF κ B刺激的转录在SUR 1的从头表达和功能性SUR 1调节的NC（Ca-ATP）通道的从头表达中的作用。这些研究将产生一个更彻底的了解NC（钙ATP）通道在SCI中的作用，将导致新的分子见解和重要的新治疗方法，这种毁灭性的人类条件。公共卫生相关性使用大鼠和小鼠脊髓损伤模型，我们发现药物抑制以及基因抑制SUR 1调节的NC（Ca-ATP）通道导致进行性继发性出血和出血性坏死显著减少，并与短期神经功能的显着改善相关。在这个提议中，我们将在大鼠和小鼠脊髓损伤模型中使用基因抑制策略来确定SUR 1抑制的短期和长期后果，并更全面地表征SCI中控制NC（Ca-ATP）通道表达和功能的基本分子原理。这些研究将为未来脊髓损伤的新疗法奠定基础。

DESCRIPTION (provided by applicant): 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 NF-kappa B 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. PUBLIC HEALTH RELEVANCE Using rat and mouse models of spinal cord injury, we discovered that pharmacological inhibition as well as gene suppression of SUR1-regulated NC (Ca-ATP) channels cause a striking reduction in progressive secondary hemorrhage and in hemorrhagic necrosis, and are associated with dramatic improvements in short-term neurological function. In this proposal, we will use gene suppression strategies in rat and mouse models of spinal cord injury to determine short and long-term consequences of SUR1 inhibition and to more fully characterize essential molecular principles governing NC (Ca-ATP) channel expression and function in SCI. These studies will form the basis for novel future therapies for spinal cord injury.