小鼠饮酒模型在酒精中毒药物治疗发展中的应用: 黑暗中的饮酒和两瓶选择
Summary
酒精使用障碍 (aud) 是一个主要的国家健康问题, 需要开发更有效的治疗方法来抵消这些病人群体的需求。为此, 下面的协议使用两个简单的啮齿类动物饮酒模型来评估铅抗酒精化合物的临床前疗效。
Abstract
酒精使用障碍 (aud) 是一个主要问题, 估计全世界有超过7600万人符合诊断标准。目前的治疗方法仅限于三种 fda 批准的药物, 即使与心理社会干预相结合, 这些药物也基本无效, 高复发率就证明了这一点。因此, 寻找更多的新治疗方法是一个重要的公共卫生目标。为此, 下面的协议使用两个简单的啮齿类动物饮酒模型来评估铅抗酒精化合物的临床前疗效: 两瓶选择 (tbc) 和在黑暗中饮酒 (did)。前者允许小鼠适度自愿饮酒, 而后者则诱导小鼠在短时间内自愿饮酒, 模仿暴饮暴食。这两种模式的简单和高吞吐量特性允许快速筛选药理制剂或识别表现出某些自愿饮酒行为的小鼠菌株。
Introduction
在过去的25年多时间里, 在开发治疗酒精使用障碍 (aud)1的药物方面做出了巨大的努力。尽管取得了许多进展, 但澳元仍然是一个重大的公共卫生问题, 影响到 1 800多万美国人, 每年花费 2, 200多亿美元。目前只有三种 fda 批准的药物, 二硫酮、纳曲酮和阿卡莫酸, 所有这些药物在临床试验中都产生了不一致的结果, 即使与临床环境中的心理社会干预相结合, 也取得了有限的成功4 个,5,6,7。
目前 aud 治疗失败的一个主要原因与 aud8的异质性有关。虽然环境和遗传因素都有助于澳元的发展, 但遗传力估计占发病风险的 50-60%.与抑郁症的治疗类似, 人们普遍认为, 患有澳元的患者将需要各种量身定制的药物, 以满足每个患者的需求 1 0.
显然, 迫切需要更有效的治疗方法, 如果简化本已艰巨和耗时的药物发现过程3, 将有助于这种治疗。为此, 下面的协议证明了两种广泛用于检测 aud 11 的神经生物学基础的啮齿类动物饮酒模型的临床前适用性.更具体地说, 本文介绍的方法可以评估候选化合物在减少酒精消费的有效性, 在 “适度” 和 “暴饮暴食” 的情况下, 利用两瓶选择 (tbc) 和在黑暗中饮酒 (did) 的范式,分别。这两种范式都考察了非操作乙醇自我管理, 即小鼠口头和随意摄入乙醇, 从而说明了高脸和构建有效性作为人类酒精中毒的模型11。
在 tbc 饮酒, 也被称为免费饮酒、偏好饮酒或社交饮酒, 两瓶溶液在家里的笼子里不断提供。一个瓶子含有水, 另一个含有稀释的乙醇溶液, 因此乙醇的浓度可以改变 (例如, 5-30% v/v)11,12。老鼠可以不断地接触到这两个瓶子, 因此, 可以选择从每个瓶子里喝多少酒。
该模型评估每只小鼠的乙醇消费量 (g/kg), 以及乙醇偏好比 (乙醇消费量; 总液体消费量)。它通常被用来比较不同品种小鼠的饮酒水平, 或者在特定的基因操纵 (如基因敲除或敲除) 之后, 导致血液乙醇浓度 (bec) 类似于在人类身上发现的水平。适量、14岁。
在 did 过程中, 在黑暗循环开始3小时后, 用 20% (v/v) 的乙醇溶液交换家庭笼瓶, 以进行有限的饮用。饮酒是连续4天的周期, 在4-day 持续 2小时, 第4天持续4小时。第4天作为第4天测试前的酒精适应期。因此, 小鼠将可靠地消耗足够的乙醇, 以达到 bec & gt;100 mg/dl, 因此, 表现出中毒的行为影响, 在人类中发现的暴饮暴食 13,14,15.除饮酒场所之外, 任何时候都可以获得水。
有限的获取饮料有几种变化。例如, 在间歇性访问模型中, 老鼠仅在周一、周三和周五收到两瓶 (一瓶含有20% 的水, 另一瓶含有20% 乙醇), 平日和周末分别有24小时和48小时的戒断期, 为 16.经过几个星期的间歇性访问, 小鼠将逐渐和自愿上升的饮酒水平, 最终达到相关的 dod 模型中观察到的 beck。然而, did 似乎是评估暴饮暴食行为最常用的模型。其他间歇性饮酒的模式也存在, 但它们依赖于限制获得食物或蒸汽室导致自愿自我管理增加的情况, 这使得它们对自愿饮酒的代表性降低了16。
Protocol
Representative Results
Discussion
全世界的估计表明, 多达7600万人符合酒精使用障碍 (aud) 诊断的标准。不幸的是, 目前可用的药物治疗基本无效, 需要进一步发展, 以抵消这一临床人群的需求20。为此, 以下协议旨在通过举例说明两种最基本的啮齿类动物饮酒模式来促进这一努力: 双瓶选择 (tbc) 和黑暗中饮酒 (did)。这两种模型都测量乙醇的非操作性自管理, 即小鼠口服乙醇。在 tbc 范式中, 乙醇 (10% vv) 和水都是连续?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 ctsi nhcrr/ncat驻 ul1tr000130 (d. l. d.)、aa022448 (d. l. d.) 和 usc 药学学校研究赠款的部分支持。
Materials
| 1 L graduated cylinder | VWR | https://us.vwr.com/store/product/20935285/marisco-single-scale-cylinder-graduates-john-m-maris-co | To prepare ethanol solution. |
| 1 L glass bottle | Pyrex (Fisher Scientific) | https://www.fishersci.com/shop/products/pyrex-reusable-media-storage-bottles-12/p-42752 | To prepare ethanol solution. |
| 100 mL graduated cylinder | Fisher Scientific | https://www.fishersci.com/shop/products/kimble-chase-kimax-class-a-to-contain-graduated-cylinders-8/p-4369311 | To prepare ethanol solution. |
| Analox | One potential method of analyzing DID blood samples is by using the analox machine | ||
| ball-bearing sipper tubes | Ancare Corp. | http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point | Length: 2.5 inches, Diameter: 5/16 inches, Model: TD100 |
| C57BL/6J Mice | Jackson lab | https://www.jax.org/strain/000664 | May also come from internal breeding colony |
| disposable serological pipets | VWR International (VWR) | https://us.vwr.com/store/product/4760455/vwr-disposable-serological-pipets-polystyrene-sterile-plugged | 10 mL, 18 mL, or 25 mL |
| ethanol, pure, 190 proof (95%), USP, KOPTEC | Decon Labs (VWR) | https://us.vwr.com/store/product/4542412/ethanol-pure-190-proof-95-usp-koptec | — |
| heat gun | Master Appliances Corp. | http://www.masterappliance.com/master-heat-guns-kits/ | |
| heat shrink tubing | — | — | Diameter: 3/8 inches |
| industrial knife/blade | — | — | — |
| metal cage plate | — | — | Should be available through the university/institutional vivarium |
| mouse RO water | — | — | Should be available through the university/institutional vivarium |
| portable electronic scale | Ohaus (VWR) | https://us.vwr.com/store/product/4789377/portable-electronic-cs-series-scales-ohaus | — |
| red light headlamp | nyteBright (Amazon) | https://www.amazon.com/LED-Headlamp-Flashlight-Red-Light/dp/B00R0LMMF8/ref=sr_1_1?ie=UTF8&qid=1499591137&sr=8-1-spons&keywords=red+lamp+headlamp&psc=1 | — |
| silicone stoppers | Fisher | — | — |
| thermometer | Fisher Scientific | https://www.fishersci.com/shop/products/fisher-scientific-hygro-thermometer-clock-large-display-2/p-4077232 | — |
| weigh boat | VWR International (VWR) | https://us.vwr.com/store/product/16773534/vwr-pour-boat-weighing-dishes | The lid from a pipete tip box is an appropriate alternative |
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