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

描述(申请人提供)：脊髓损伤(SCI)导致称为进行性出血性坏死(PHN)的自杀性过程，导致脊髓组织的毁灭性损失。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SUR1调节的NC(Ca-ATP)通道与坏死性细胞死亡和脊髓损伤后继发性出血密切相关。在这里，我们将进一步研究SUR1调节的NC(钙-ATP)通道在脊髓损伤中的作用。我们的总体假设是，核因子-kappa B信号的激活在内皮、神经元和少突胶质细胞中SUR1调节的NC(Ca-ATP)通道的从头表达中起关键作用，随后ATP耗竭导致通道的开放导致毛细血管灾难性衰竭、点状出血的形成以及神经元和少突胶质细胞的坏死性死亡，这反过来又引发氧化应激和炎症，共同促进PHN。我们在小鼠和大鼠脊髓挫伤模型上的数据表明，使用格列本脲的SUR1药物阻断，通过使用优先靶向于半暗带毛细血管的反义寡核苷酸(AS-ODN)抑制SUR1的基因，以及通过转基因SUR1-KO(SUR1-/-)小鼠的基因抑制，可以显著减少出血，这三种治疗或条件与短期神经行为功能的显著改善有关。在特定目的(SA)1中，利用针对SUR1的基因抑制策略在小鼠和大鼠脊髓损伤模型中，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，参与PHN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SA2中，使用来自野生型(WT)和SUR1-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，我们将证实每种细胞类型都能上调SUR1调节的NC(Ca-ATP)通道，我们将表征新诱导的通道，确定它们的生理调节，以及它们在细胞死亡中的作用。其他初步数据表明，NFkappaB可能是NC(Ca-ATP)通道从头表达的重要转录激活因子，它与脊髓损伤密切相关。在SA3中，利用脊髓损伤后的培养细胞和脊髓组织，我们将确定NFkappaB刺激的转录在SUR1从头表达和SUR1调节的NC(Ca-ATP)功能通道从头表达中的作用。这些研究将对NC(钙-ATP)通道在脊髓损伤中的作用产生更彻底的理解，将导致对这种毁灭性的人类疾病的新的分子见解和重要的新治疗方法。公共卫生相关性利用大鼠和小鼠脊髓损伤模型，我们发现，药物抑制以及SUR1调节的NC(钙-ATP)通道的基因抑制可以显著减少进行性继发性出血和出血性坏死，并与短期神经功能的显著改善有关。在这个方案中，我们将在大鼠和小鼠的脊髓损伤模型中使用基因抑制策略来确定SUR1抑制的短期和长期后果，并更全面地描述调控脊髓损伤中NC(钙-ATP)通道表达和功能的基本分子原理。这些研究将为未来治疗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.