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

摘要 脊髓损伤(SCI)导致称为“进行性”的自我破坏过程。 出血性坏死(PHN)，导致脊髓组织的毁灭性损失。 PHN的关键成分是：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) 通道在坏死性细胞死亡和继发性出血中起关键作用。 SCI。在这里，我们将进一步表征SUR1调节的NC(钙-ATP)通道的作用 在SCI中。我们的主要假设是NFkappaB信号的激活起着关键作用 SUR1调节的内皮细胞NC(Ca-ATP)通道从头表达的作用 神经元和少突胶质细胞，以及随后由ATP开放的通道- 衰竭导致毛细血管灾难性的衰竭，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，共同助长了PHN。我们在小鼠和大鼠模型中的数据 挫伤脊髓损伤显示药物治疗可显著减少出血。 用格列本脲阻断SUR1，反义抑制SUR1基因 寡核苷酸(AS-ODN)，优先针对半暗带毛细血管；以及 在转基因SUR1-KO(SUR1-/-)小鼠中通过基因抑制，这3种 治疗或情况与短期内的显著改善有关。 神经行为功能。在特定目标(SA)1中，使用基因抑制策略 以SUR1为靶点，在小鼠和大鼠脊髓损伤模型中，我们将评估进行性的作用 短期后遗症继发性出血，包括炎症和氧化 压力，以及包括组织病理学和神经行为在内的长期后遗症 功能。其他初步数据表明，在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SA2中，使用主要培养物 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)和SUR1-KO小鼠中，我们将证实每种细胞类型都可以 上调SUR1调节的NC(钙-ATP)通道，我们将表征新诱导的 通道，通过pH决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，这是已知的显著 参与脊髓损伤，可能在新生中扮演重要的转录激活因子的角色 NC(Ca-ATP)通道的表达。在SA3中，使用培养的细胞和脐带组织后 我们将确定NFkappaB刺激的转录在从头开始中的作用 SUR1的表达及SUR1调节的NC(Ca-ATP)从头表达 频道。这些研究将使我们更深入地了解NC(钙-钙)的作用 在脊髓损伤中的ATP)通道将导致新的分子洞察力和重要的新治疗 对这种毁灭性的人类状况负责。

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.