MECHANISMS AND PREVENTION OF HEMOGLOBIN NEUROTOXICITY

以血红蛋白为基础的化合物正在被开发为血液替代品 用于外科手术或急救医学，但放置的后果 直接接触中枢神经系统组织的以血红蛋白为基础的化合物是未知的。 这些研究将继续开发一种细胞培养模型，可以 用于评估血红蛋白或基于血红蛋白的化合物对人体的毒性 中枢神经系统。此外，该模型还可以用于识别和测试 治疗血红蛋白介导的可能的治疗方法 细胞损伤。 此前的研究结果表明，血红蛋白具有神经毒性。氧合血红蛋白 会导致脑血管痉挛，皮质内注射血红蛋白有 被用来制作癫痫的动物模型。初步实验 已经证明了血红蛋白和以血红蛋白为基础的氧气 载体对培养的小鼠神经细胞的毒性作用呈浓度依赖性 时尚。这种毒性随着时间的推移而发展，甚至在 去除了血红蛋白。所测试的血红蛋白溶液中没有一种 似乎对小鼠神经胶质细胞有毒性。 目前的提案将解决培养物中血红蛋白介导的损伤问题。 大鼠小脑神经元、皮质神经元、纯星形胶质细胞和联合 星形胶质细胞和小胶质细胞的培养。浓度依赖关系和 将描绘出血红蛋白介导的损伤的时间进程。特定的 血浆蛋白将被评估为保护剂，重点是蛋白质 结合并促进血红蛋白或血红素清除的物质--结合珠蛋白 或者是血液凝集素。其他可能对癌症有治疗潜力的化合物 将对血红蛋白介导的损伤的治疗进行评估。这些化合物 将包括抗氧化剂(如曲洛克斯、α-生育酚和21- 氨基类固醇)、螯合剂(如去铁胺和一种新的去铁胺 衍生物)，以及阻止钙进入神经元的药物 (NMDA、海人藻酸/AMPA、代谢性受体拮抗剂和钙 通道阻滞剂)。将进行更多的实验来测试 抗坏血酸和血红蛋白依赖性神经毒性的假说是 通过类似的机制，抗坏血酸可能会加剧 血红蛋白介导的损伤。最后，给出了热冲击的表达式 蛋白质HSP70和血红素加氧酶-L将在神经元和 血红蛋白和抗坏血酸依赖过程中的星形胶质细胞 受伤。这些被认为具有保护性的蛋白质可能会被诱导 不同细胞的差异，部分解释了这种差异 神经元和星形胶质细胞对血红蛋白的敏感性。

Hemoglobin-based compounds are being developed as blood substitutes to be used in surgery or emergency medicine, yet the consequences of placing hemoglobin-based compounds in direct contact with CNS tissue are unknown. These studies will continue to develop a cell culture model that can be used to assess the toxicity of hemoglobin or hemoglobin-based compounds to the CNS. Additionally, this model can be utilized to identify and test possible therapeutic approaches for the treatment of hemoglobin-mediated cellular injury. Previous results suggest that hemoglobin is neurotoxic. Oxyhemoglobin causes cerebral vasospasm, and intracortical injections of hemoglobin have been used to generate animal models of epilepsy. Preliminary experiments have demonstrated that both hemoglobin and a hemoglobin-based oxygen carrier are toxic to cultured murine neurons in a concentration-dependent fashion. This toxicity developed over time and progressed even after hemoglobin was removed. None of the hemoglobin solutions tested have appeared to be toxic to murine glial cells. The current proposal will address hemoglobin-mediated injury in cultures of rat cerebellar neurons, cortical neurons, pure astrocytes, and co- cultures of astrocytes and microglia. The concentration dependence and time course of hemoglobin-mediated injury will be delineated. Specific plasma proteins will be evaluated as protectants, emphasizing proteins that bind and facilitate the clearance of hemoglobin or heme--haptoglobin or hemopexin. Other compounds that may have therapeutic potential for the treatment of hemoglobin-mediated injury will be evaluated. These compounds will include antioxidants (such as Trolox, alpha tocopherol, and the 21- aminosteroids), chelators (such as deferoxamine and a novel deferoxamine derivative), as well as drugs that block the calcium entry into neurons (NMDA, kainate/AMPA, and metabotropic receptor antagonists and calcium channel blockers). Additional experiments will be conducted to test the hypotheses that ascorbate- and hemoglobin-dependent neurotoxicity are mediated through similar mechanisms and that ascorbate may exacerbate hemoglobin-mediated injury. Finally, the expression the of heat shock proteins HSP7O and heme oxygenase- l will be examined in neurons and astrocytes during the course of hemoglobin- and ascorbate-dependent injury. These proteins, which are thought to be protective, may be induced differently in different cells, partially explaining the differential sensitivity of neurons and astrocytes to hemoglobin.