Neuronal Somatic Response to Traumatic Brain Injury

描述（由申请人提供）：本次竞争性更新旨在继续探索由未合并挫伤或肿块病变形成的弥漫性创伤性脑损伤（TBI）引发的神经元躯体损伤和死亡。虽然以前的研究认为TBI后细胞凋亡和坏死的细胞死亡集中在受体介导的，氧自由基诱导和/或缺血引发的细胞变化，在以前的资助期间进行的工作并不支持这些假设。相反，我们最近发表的研究表明，TBI导致立即创伤后膜扰动，然后设置后续的细胞损伤或坏死细胞死亡的基础上，神经元的能力，重新密封最初的膜损伤的阶段。这些现象在整个新皮层的弥漫性损伤的神经元胞体中观察到，散布着其他完整和未改变的神经元细胞体。与这种弥漫性神经元体细胞膜扰动相一致，还观察到分散的体周轴突切断，但需要注意的是，这种损伤不会导致神经元细胞死亡。在这次竞争性更新中，我们利用上一个资助期开发的技术来扩展这些发现，以检测创伤诱导的神经元膜穿孔，其随时间的持续性及其对坏死细胞死亡的总体影响。在这些死亡途径中，将在24小时内对特定神经元进行随访，这些神经元显示通过鞘内给予的荧光葡聚糖的摄取而发生的初始和持续体细胞膜变化的证据。将通过共聚焦显微镜和电子显微镜在同一神经元中评价钙蛋白酶介导的血影蛋白水解、5-钙蛋白酶活化、溶酶体相关膜蛋白释放和/或组织蛋白酶B活化的伴随免疫细胞化学检测。与这些描述性和机制研究一致，将评估颅内压升高的调节作用，因为它是人类TBI的重要混淆因素。最后，由于膜穿孔很可能不适用于用于减轻TBI损伤后果的传统疗法，我们将使用新型泊洛沙姆密封受损的膜并提供保护。拟议的研究也将集中在弥漫性周围轴突切断术，以更好地了解其关系，或缺乏，上述现象。我们相信，这些研究是新颖和创新的，如果成功的话，它们将要求重新思考弥漫性TBI后神经元是如何受伤和死亡的。公共卫生相关性：我们相信，提出的项目是翻译和直接相关的，我们更好地了解创伤性脑损伤的病理生物学及其治疗管理。探索的主题是非常新颖的，并将当代细胞生物学方法与临床相关参数相结合，以解决以前未被充分认识的创伤性脑病理学领域及其通过颅内压和全身生理学变化进行的潜在调节。

DESCRIPTION (provided by applicant): This competitive renewal seeks to continue its exploration of that neuronal somatic damage and death triggered by a diffuse traumatic brain injury (TBI) uncomplicated by contusion or mass lesion formation. Although previous studies considering apoptotic and necrotic cell death following TBI have focused on receptor-mediated, oxygen radical-induced and/or ischemic-initiated cell change, work conducted in the previous funding period did not support these assumptions. Rather, our recently published studies have demonstrated that TBI causes immediate posttraumatic membrane perturbation that then sets the stage for subsequent cell damage or necrotic cell death based upon the neuron's ability to reseal the initial membrane damage. These phenomena were observed in diffusely injured neuronal somata throughout the neocortex, interspersed with other intact and unaltered neuronal cell bodies. In concert with this diffuse neuronal somatic membrane perturbation, scattered perisomatic axotomy was also observed, with the caveat that such injury did not result in neuronal cell death. In this competitive renewal, we expand upon these findings utilizing technologies developed in the last funding period to detect traumatically induced neuronal membrane poration, its persistence over time, and its overall implications for necrotic cell death. In these death pathways, specific neurons, showing evidence of initial and continued somatic membrane change via the uptake of intrathecally administered fluorescent dextrans, will be followed over a 24h period. Concomitant immunocytochemical detection of calpain-mediated spectrin proteolysis, 5-calpain activation, lysosomal associated membrane protein release, and/or cathepsin B activation will be evaluated in the same neuron by confocal microscopy and electron microscopy. In concert with these descriptive and mechanistic studies, the modulatory effect of elevated intracranial pressure will be assessed as it is an important confound of human TBI. Lastly, in that membrane poration is most likely non amenable to traditional therapies used to attenuate the damaging consequences of TBI, we will use a novel poloxamer to seal damaged membranes and provide protection. The proposed studies will also focus on diffuse perisomatic axotomy, to better understand its relation, or lack thereof, to the above described phenomena. We believe that the proposed studies are novel and innovative and if successful, they will mandate a rethinking on how neurons are injured and die following diffuse TBI. PUBLIC HEALTH RELEVANCE: We believe that the project proposed is translational and of direct relevance to our better understanding of the pathobiology of traumatic brain injury and its therapeutic management. The themes explored are extremely novel and integrate contemporary cell biological approaches with clinically relevant parameters to address previously underappreciated areas of traumatically-induced brain pathology and its potential modulation by changes in intracranial pressure and systemic physiology.