Establishing the Molecular Mechanisms of Reduced Ethanol Drinking in Calcineurin-Mediated Immunosuppression Treated Rodents

建立钙调神经磷酸酶介导的免疫抑制治疗的啮齿动物减少乙醇饮酒的分子机制

• 批准号: 10847322
• 负责人: THOMAS P BERESFORD
• 金额: --
• 依托单位: VA EASTERN COLORADO HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10847322
• 关键词: Abstinence; Address; Admission activity; Affect; Alcohol consumption; Alcohol dependence; Alcoholism; Alcohols; American; Anatomy; Brain; Calcineurin; Calcineurin inhibitor; Caring; Chemicals; Consumption; Cyclosporine; DSM-V; Darkness; Data; Diagnosis; Enzyme-Linked Immunosorbent Assay; Ethanol; FK506; Family; Flow Cytometry; Frequencies; Functional Magnetic Resonance Imaging; Future; General Population; Genetic; Glutamates; Goals; Health; Health care facility; Human; Immune; Immune system; Immunohistochemistry; Immunosuppression; Immunosuppressive Agents; Injections; Inpatients; International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10); Intervention; Knock-out; Knockout Mice; Knowledge; LoxP-flanked allele; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Mediating; Medical; Medicine; Mental Depression; Metabolic; Metabolism; Microglia; Microinjections; Mitochondria; Modeling; Molecular; Morbidity - disease rate; Mus; Neuroglia; Neuroimmune; Neurons; Neurotransmitters; Organ Transplantation; Pathway interactions; Patient Admission; Patients; Peripheral; Persons; Play; Prevention; Procedures; Productivity; Protein Inhibition; Protein phosphatase; Protocols documentation; Quantitative Reverse Transcriptase PCR; Rattus; Recovery; Regulation; Research; Research Personnel; Resolution; Rewards; Rodent; Rodent Model; Role; Self Administration; Signal Pathway; Signal Transduction Pathway; Signaling Molecule; Sirolimus; T-Lymphocyte; Tacrolimus; Testing; Time; Transplantation; Ventricular; addiction; alcohol use disorder; analog; brain metabolism; clinical application; consumption measures; cost; design; drinking; experimental study; gamma-Aminobutyric Acid; innovation; inpatient service; liver transplantation; military veteran; mortality; mouse model; neurochemistry; neuroinflammation; neuromechanism; neurotransmission; novel strategies; pandemic disease; preference; preservation; prevent; problem drinker; prolonged abstinence; psychosocial; translational study

Veteran populations present alcoholism rates that are far higher than general population frequencies. Despite this, the treatment options for alcoholism are very few and much in need of new approaches for medical interventions. This project grows from the observation that alcoholic persons receiving liver transplant evidence very high rates of sustained abstinence after the liver grafting procedure that we could not explain by psychosocial or selection factors. We asked whether this might be due to a common factor shared by all of these patients: immunosuppressant medicines given for very long periods after transplant to keep the grafted organ in a state of health. In two separate rodent experiments we found that 1) cyclosporine (CSA) had an antidipsic effect in an open choice model of ethanol (alternatively alcohol, or ethyl alcohol) drinking, and 2) that CSA and tacrolimus (TRL, alternately FK506) had similar effects in a drinking-in-the-dark (DID) ethanol drinking model while sirolimus (SRL, alternately rapamycin) had no effect but had lower concentrations in the brain. These findings implicate the immunosuppressants blocking brain calcineurin (CLN) activity that may enhance the rodent's choice against drinking ethanol through 1) direct CLN modulation in the brain or 2) through the systemic immunosuppressive and neuroimmune pathways. This study asks whether further evidence supports one or the other of these possible mechanisms of action. Following the overall goal of this project--to elucidate the mechanism(s) of reduction of alcohol preference by CLN directly or by CLN-mediated immunosuppression, the study's primary hypothesis asserts that inhibition limiting CLN activity in the brain itself will decrease rodent ethanol choice. An alternative hypothesis states that the peripheral effects of CLN inhibition in the immune system will result in a decrease in ethanol preference. The overall aim of the project is therefore to establish which of these two possible mechanisms mediates the effect of immunosuppressant agents on rodent's choice not to drink ethanol. Our experimental approaches address the primary hypothesis using a genetic knockout approach. In Specific Aim 1, we will compare immunosuppressants in brain-specific CLN knock-out mice that include a) pan-brain knockout (CamKIIα Cre x floxed CLN) rodents, b) CRF neuron specific calcineurin knockout (CRHCrex floxed CLN), and c) focal CLN knockouts in extended reward regions (VTA, NAc, CeA) utilizing AAV-Cre microinjections in floxed CLN mice. In Specific Aim 2, we will characterize the possible mechanisms of central effects of CLN inhibition on alcohol consumption by assessing 1) the brain's metabolic protection by CLN inhibitors, and 2) addiction related downstream signal molecules in extended brain reward networks. In Specific Aim 3, we test whether drinking causes neuroinflammation that drives subsequent drinking. We will use a combination of approaches (flow cytometry, immunohistochemistry, qRT-PCR, ELISA) to determine the effects of CLN inhibition on ethanol induced neuroinflammation. And in Specific Aim 4 we will non-invasively assess anatomical, metabolic and functional changes in the mouse brain using multi-parametric magnetic resonance imaging (MRI). We will use non-invasive 9.4 Tesla MRI protocols longitudinally on our mouse models of drinking preference treated with CsA, TRL and SRL. We expect the data from these procedures to answer the study questions in a definitive manner and to point the path to new answers in the neurochemistry of alcohol choice. This in turn can lead to better understanding of the mechanisms involved in stopping or limiting drinking and alcohol addiction in veteran populations. The overall goal is to find new medicinal agents that can treat alcoholism a condition that affects up to one in every two patients admitted to VA inpatient services with an estimated half of those actively drinking.

退伍军人人口的酗酒率远高于一般人口频率。 尽管如此，酒精中毒的治疗选择很少，而且需要新方法 医疗干预措施。该项目从接受肝移植的酗酒者的观察结果发展 肝移植程序后，我们无法解释的证据很高的持续节制率很高 社会心理或选择因素。我们问这是否可能是由于所有人共享的一个共同因素 这些患者：移植后长期服用的免疫抑制药物以保持移植 身体状况良好。在两个独立的啮齿动物实验中，我们发现1）环孢素（CSA）具有 在开放选择乙醇（替代酒精或乙醇）饮用的开放选择模型中的抗替代作用，2） CSA和他克莫司（TRL，FK506）在黑暗中饮酒（DID）乙醇也有类似的作用 饮酒模型虽然西罗莫司（SRL，雷帕霉素）无效，但浓度较低 脑。这些发现的免疫抑制剂阻断了脑钙调神经酶（CLN）活性，可能 通过1）大脑中的直接CLN调制或2 通过系统性免疫抑制和神经免疫性途径。这项研究询问是否进一步 证据支持这些可能的作用机制中的一种或另一种。 遵循该项目的总体目标 - 阐明酒精的减少机制 直接或CLN介导的免疫抑制的CLN偏爱，该研究的主要假设断言 抑制限制大脑本身的CLN活性会减少啮齿动物乙醇的选择。另一种 假设指出，CLN抑制在免疫系统中的外围作用将导致减少 乙醇偏好。因此，该项目的总体目的是确定这两个可能的哪个 机制介导了免疫抑制剂对啮齿动物选择不喝乙醇的影响。 我们的实验方法使用遗传基因敲除方法解决了主要假设。在 特定的目标1，我们将比较包括a在内的脑特异性CLN敲除小鼠中的免疫抑制剂 泛脑敲除（CamkiiαCrex Floxed CLN）啮齿动物，b）CRF神经元特异性钙调神经蛋白敲除 （CRHCREX FLOXED CLN）和C）使用扩展奖励区域（VTA，NAC，CEA）的焦点CLN敲除 AAV-CRE微型注射在Floxed CLN小鼠中。在特定目标2中，我们将表征可能的机制 通过评估1）通过评估CLN抑制对酒精消耗的核心影响 CLN抑制剂和2）与成瘾相关的大脑奖励网络中相关的下游信号分子。在 特定目标3，我们测试饮酒是否引起神经炎症，导致随后的饮酒。我们将 结合方法（流式细胞仪，免疫组织化学，QRT-PCR，ELISA）来确定 CLN抑制对乙醇诱导神经炎症的影响。在特定的目标4中，我们将不可侵入 使用多参数磁性评估小鼠大脑中的解剖学，代谢和功能变化 共振成像（MRI）。我们将在鼠标上纵向使用无创的9.4 Tesla MRI协议 用CSA，TRL和SRL处理的饮酒偏好模型。 我们期望这些程序中的数据能以明确的方式回答研究问题 指向酒精选择神经化学的新答案的道路。反过来可能会导致更好 了解停止或限制退伍军人饮酒和饮酒的机制 人群。总体目标是找到可以治疗酒精中毒的新医疗特工 每两名患者中，多达一名患者接受VA住院服务，估计有一半的患者积极地 喝。