Calcium-related Neurotoxicity of Cocaine

描述（由申请人提供）：可卡因引起的中风和脑出血是最严重的医疗并发症之一。这些作用的机制尚不清楚，但可能部分反映了其血管活性作用，这种作用可能会随着重复药物暴露而加剧。我们对可卡因的脑血管效应的了解不多，部分原因是我们同时评估并区分可卡因的神经元效应和血管效应的能力存在技术限制。在这里，我们的目标是应用我们新开发的光学/荧光成像技术（OFIs），分别研究急性和慢性可卡因暴露的神经和脑血管的影响。OFI集成了（1）双波长激光散斑成像，能够在大视场内以高时空分辨率同时检测脑血流量（CBF）、血容量（CBV）和组织血红蛋白氧合（StO 2），（2）数字频率斜坡多普勒光学相干断层扫描，用于神经血管网络的体内3D定量成像，（3）Rhod 2荧光成像以测量细胞内钙（[Ca 2 +]i），和（4）微导管探针（mOFI）以在真实的时间和高时空分辨率下评估可卡因对皮层下脑区域的作用。 我们的初步研究表明，急性可卡因降低CBF，同时增加脱氧血红蛋白含量和增加细胞内钙含量，并有敏感性，这些影响与重复曝光。由于细胞内[Ca 2 +]i的增加与缺血后的神经元死亡相关，我们假设这些慢性可卡因暴露的致敏反应增加了神经元组织对缺氧损伤的脆弱性。为了验证这些假设，我们将使用OFI同时测量急性和慢性可卡因对脑血管反应（CBF，CBV），组织氧合（StO 2）和皮质和皮质下脑区神经元[Ca 2 +]i变化的影响。我们将使用一个动物模型的强迫性可卡因自我管理（短期和长期访问），模仿人类成瘾的关键方面。此外，我们将研究钙通道阻滞剂在预防可卡因诱导的细胞内Ca 2+含量增加中的功效，作为降低可卡因脑血管毒性的潜在治疗策略。我们提出以下具体目的：评估急性和慢性可卡因对皮质神经元和血管的影响，（使用OFI，Aim 1）和皮质下区域（使用mOFI，目标2）;描述慢性可卡因暴露导致的“缺血样”神经血管功能障碍（目标3），并检查钙通道阻滞剂改善可卡因诱导的脑内[Ca 2 +]i和血流动力学变化的功效（目的4）。 拟议中的研究将更好地了解可卡因脑血管毒性的机制，这可能有助于开发治疗干预措施来抵消这种毒性。此外，拟议的研究将引入一种成像能力，这种能力可用于研究其他滥用药物的影响和研究有相关动物模型的其他脑部疾病。

DESCRIPTION (provided by applicant): Cocaine-induced stroke and hemorrhage in the brain are among the most serious medical complications. The mechanisms underlying these effects are poorly understood but are likely to reflect in part its vasoactive effects that may be exacerbated with repeated drug exposures. Our poor understanding of the cerebrovascular effects of cocaine are due in part to technological limitations in our ability to concurrently assess and thus distinguish cocaine's neuronal effects from its vascular effects. Here we aim to apply our newly developed optical/ fluorescence imaging techniques (OFIs), to study separately the neuronal and cerebrovascular effects of acute and chronic cocaine exposures. OFIs integrate (1) dual-wavelength laser speckle imaging to enable concurrent detection of cerebral blood flow (CBF), blood volume (CBV), and tissue hemoglobin oxygenation (StO2) at high spatiotemporal resolutions across a large field of view, (2) digital-frequency-ramping Doppler optical coherence tomography for in vivo 3D quantitative imaging of the neurovascular network, (3) Rhod2 fluorescence imaging to measure intracellular calcium ([Ca2+]i), and (4) a micro-catheter probe (mOFI) to assess cocaine's effects on subcortical brain regions in real time and at high spatiotemporal resolution. Our pilot study has shown that acute cocaine decreases CBF while increasing deoxyhemoglobin content and increasing intracellular Ca content and that there is sensitization to these effects with repeated exposures. Since increases in intracellular [Ca2+]i are associated with neuronal death following ischemia, we hypothesize that these sensitized responses with chronic cocaine exposures increase the vulnerability of neuronal tissue to damage from hypoxia. To test these hypotheses, we will use OFIs to concurrently measure the effects of acute and chronic cocaine on cerebrovascular responses (CBF, CBV), tissue oxygenation (StO2) and neuronal [Ca2+]i changes in cortical and subcortical brain regions. We will use an animal model of compulsive cocaine self- administration (short and long access) that mimics key aspects of human addiction. In addition, we will examine the efficacy of calcium channel blockers in preventing cocaine-induced increases in intracellular Ca2+ content, as a potential therapeutic strategy to reduce cocaine's cerebrovascular toxicity. We propose the following Specific Aims: Assess the neuronal and vascular effects of acute and chronic cocaine on cortical (using OFI, Aim 1) and subcortical regions (using mOFI, Aim 2); Characterize the 'ischemia-like' neurovascular dysfunction resulting from chronic cocaine exposure (Aim 3), and examine the efficacy of calcium channel blockers to ameliorate cocaine-induced intracellular [Ca2+]i and hemodynamic changes in brain (Aim 4). The proposed studies will provide a better understanding of the mechanisms that underlie cocaine's cerebrovascular toxicity, which could help develop therapeutic interventions to counteract this toxicity. In addition, the proposed studies will introduce an imaging capability that could be applied to studies of the effects of other drugs of abuse and to studies of other brain disorders for which there are pertinent animal models.