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

脊髓损伤、进行性出血性坏死和 NC(Ca-ATP) 通道

• 批准号: 8013900
• 负责人: J. Marc Simard
• 金额: $ 32.16万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013900
• 关键词: Blood capillaries; Capillary Endothelial Cell; Cell Death; Cell Survival; Cells; Cessation of life; Characteristics; Contusions; Cultured Cells; Data; Electrophoretic Mobility Shift Assay; Employee Strikes; Endothelial Cells; Endothelium; Exhibits; Failure; Future; Genes; Genetic Transcription; Glyburide; Health; Hemorrhage; Histopathology; Human; In Vitro; Inflammation; Knock-out; Lead; Mediating; Modeling; Molecular; Mus; NF-kappa B; Necrosis; Nervous System Physiology; Neurons; Oligodendroglia; Outcome Measure; Oxidative Stress; Physiological; Play; Process; Property; Rattus; Regulation; Reporter Genes; Role; Signal Transduction; Spinal Cord; Spinal cord injury; Tissues; Transcription Coactivator; Transcriptional Regulation; Transgenic Organisms; base; capillary; cell type; chromatin immunoprecipitation; improved; in vivo; insight; knockout gene; mouse model; neurobehavioral; novel; patch clamp; research study; response

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.

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)坏死细胞死亡。我们最近发现新表达的sur1调控的NC （Ca-ATP）通道在坏死细胞死亡和脊髓损伤后继发性出血中起关键作用。在这里，我们将进一步表征sur1调节的NC （Ca-ATP）通道在SCI中的作用。我们的总体假设是，nf - κ B信号的激活在内皮细胞、神经元和少突胶质细胞中sur1调节的NC （Ca-ATP）通道的新生表达中起关键作用，随后ATP耗竭导致通道的开放导致毛细血管的灾难性衰竭、点状出血的形成和神经元和少突胶质细胞的坏死死亡，这反过来又引发氧化应激和炎症，共同加剧PHN。我们在小鼠和大鼠挫伤性脊髓损伤模型中的数据表明，使用格列本脲药物阻断SUR1，使用优先靶向半暗毛细血管的反义寡脱氧核苷酸（AS-ODN）基因抑制SUR1，以及通过转基因SUR1- ko （SUR1-/-）小鼠基因抑制SUR1，可以显著减少出血，并且这3种治疗或条件与短期神经行为功能的显着改善有关。在特定目的（SA） 1中，我们将在小鼠和大鼠脊髓损伤模型中使用靶向SUR1的基因抑制策略，评估进行性继发性出血对包括炎症和氧化应激在内的短期后遗症以及包括组织病理学和神经行为功能在内的长期后遗症的作用。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，使用野生型（WT）小鼠与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可以上调sur1调节的NC（Ca-ATP）通道，我们将表征新诱导的通道，确定pH对它们的生理调节及其在细胞死亡中的作用。其他初步数据表明，NFkappaB可能在NC （Ca-ATP）通道的从头表达中扮演重要的转录激活因子的角色。在SA3中，我们将利用培养的细胞和脊髓损伤后的组织，确定nfkappab刺激的转录在SUR1的从头表达和SUR1调节的功能性NC （Ca-ATP）通道的从头表达中的作用。这些研究将更深入地了解NC （Ca- ATP）通道在脊髓损伤中的作用，并为这种毁灭性的人类疾病带来新的分子见解和重要的新治疗方法。利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sur1调节的NC （Ca-ATP）通道可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着改善相关。在本研究中，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SUR1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。