Mild Traumatic Brain Injury and Opiate Exposure Crosstalk: Neuropathological, Neurobehavioral, and Neuroproteomic Assessments

创伤性脑损伤（TBI）是退伍军人人群发病的主要原因。轻度TBI（mTBI） 与药物滥用有关的共病，阻碍了康复和治疗工作 任何一种情况。特别令人感兴趣的是退伍军人中阿片类药物滥用的上升趋势。现有证据 表明TBI和阿片类药物滥用的重叠分子和神经通路;例如，炎症 介质和增加的神经变性和小胶质细胞活化在阿片滥用和 mTBI。据我们所知，目前还没有临床前研究的报道， mTBI和随后的阿片类药物暴露在神经行为、神经病理 和神经生物化学后果。本研究建议使用一种经过验证的啮齿动物模型， 头部损伤（rCHI）与长期暴露于芬太尼药物配对，以密切模拟mTBI的真实世界条件 和鸦片的使用芬太尼是一种非常有效但容易上瘾的止痛药，常用于治疗慢性疼痛。 该提议的中心假设是，mTBI后持续的阿片剂暴露将诱导特征性的 神经功能和神经病理学变化不同于单独暴露于mTBI或阿片类药物时观察到的变化， 这些变化可以通过神经行为学、生物化学和神经蛋白质组学来识别和评估， 接近。这一提议是PI实验室的一个新方向，可能会改变范式。要求1 将确定1个月芬太尼阿片类药物暴露，随后在r-CHI后停药1个月， 认知、疼痛感觉、运动活动和自发性阿片戒断症状。雄性小鼠（C57 BL 6）将 将有四个实验组：（a） 假手术+生理盐水，（B）rCHI+生理盐水，（c）假手术+芬太尼，（d）rCHI-随后暴露于芬太尼。功能 评估测试包括：TBI相关认知（空间记忆表现;莫里斯水迷宫）和焦虑- 行为（高架迷宫）或阿片类药物使用/戒断试验;运动相关疼痛敏感性试验 （温水尾部戒断和口面福尔马林试验）（药物暴露和戒断阶段），以及药物 戒断依赖症状（在芬太尼戒断阶段）。结果将与 单独的rCHI或芬太尼暴露的观察。目标2将评估关键的神经病理学、神经细胞 三个脑区（皮质、海马和腹侧被盖区）的标记物和阿片受体水平 涉及TBI和/或阿片受体介导的奖励回路。将在2个终点分析脑样本 - 在芬太尼给药结束时（第1个月）和停药1个月后（第2个月）， 没有rCHI。神经元、轴突、星形胶质细胞、小胶质细胞、突触和白色物质标记物，沿着和 通过免疫印迹/ELISA试验， 并通过组织化学-组织病理学分析。目标3将确定长期mTBI后果， 阿片类药物滥用共病相关的病理通路/关键驱动因素作为假定的神经治疗靶点， 神经蛋白质组学和系统生物学分析。总的来说，这一新的调查路线旨在提供见解 mTBI和损伤后阿片类药物滥用之间的神经生物学和神经病理学相互作用。结果 可能指导可能的新治疗策略，以治疗退伍军人TBI和共病阿片类药物依赖。

Traumatic brain injury (TBI) is a major cause of morbidity among the Veteran population. Mild TBI (mTBI) has been associated with substance abuse as a comorbid condition, hampering rehabilitation and treatment efforts of either condition. Of particular interest is the rising trend of opiate abuse among Veterans. Existing evidence indicates overlapping molecular and neural pathways for TBI and opiate abuse; for example, inflammatory mediators and increased neurodegeneration and microglial activation are observed in both opiate abuse and mTBI. To the best of our knowledge, there are no reported preclinical studies that have systematically examined the crosstalk between mTBI and subsequent opiate exposure in terms of neurobehavioral, neuropathological and neurobiochemical consequences. This study proposes to use a validated rodent model of repeated closed head injury (rCHI) paired with chronic exposure to fentanyl drug to closely simulate real-world conditions of mTBI and opiate usage. Fentanyl is a highly effective but addictive analgesic commonly used for treating chronic pain. The central hypothesis of this proposal is that sustained opiate exposure post-mTBI will induce characteristic neurofunctional and neuropathological changes that differ from those seen with mTBI or opiate exposure alone, and that these changes can be identified and evaluated via neurobehavioral, biochemical and neuroproteomic approaches. This proposal is a new direction for the PI’s laboratory and is potentially paradigm-shifting. Aim 1 will determine the effects of 1 month fentanyl opiate exposure followed by 1 month withdrawal after r-CHI on cognition, pain sensation, motor activity and spontaneous opiate withdrawal symptoms. Male mice (C57BL6) will be used and subjected to rCHI or sham procedure (non-injury).There will be four experimental groups: (a) Sham+saline, (b) rCHI+saline, (c) sham+fentanyl, (d) rCHI-followed by Fentanyl exposure. Functional assessment tests include: TBI-related cognition (spatial memory performance; Morris water maze) and anxiety- behavior (elevated plus maze), or opiate-use/withdrawal tests; locomotive activity-related pain sensitivity tests (warm-water tail withdrawal and Orofacial formalin test) (during drug exposure and withdrawal phases), and drug withdrawal-dependence symptoms (during the fentanyl withdrawal phase). The results will be compared with the observations from rCHI or fentanyl exposure alone. Aim 2 will assess key neuropathological, neural cellular markers and opiate receptor levels in three brain regions (cortex, hippocampus and ventral tegmental area) implicated in TBI and/or opioid receptor-mediated reward circuits. Brain samples will be analyzed at 2 endpoints - at the end of fentanyl administration (month 1), and after 1 month of withdrawal (month 2), in animals with or without rCHI. Neuronal, axonal, astroglial, microglial, synaptic and white matter markers, along with and the three subtypes of opioid receptors will be assessed in separate sets of animals by immunoblotting/ELISA essays and by immunohistochemical-histopathological analyses. Aim 3 will identify long-term mTBI consequences and opiate abuse comorbidity-related pathological pathways/key drivers as putative neurotherapeutic targets using neuroproteomic and System Biology analysis. Collectively, this new line of investigation aims to provide insights into the neurobiological and neuropathological interplay between mTBI and post injury opioid abuse. The results may guide possible new therapeutic strategies to treat TBI and comorbid opioid dependence in Veterans.