多亏了 Rassam 的样本收集, 伊迪丝 Klobetz--对 FCM 的分析, PD 博士. 兽医。habil。萝丝维塔梅尔协助统计分析, 并 Wiebke Gentner 校对手稿。这项研究是柏林-勃兰登堡研究平台 BB3R (www.bb3r.de) 的一部分, 由德国联邦教育部和研究部资助 (赠款号: 031A262A) (www.bmbf.de/en/index.html)。

这项研究是根据《德国动物福利法》规定的准则进行的, 并得到了柏林国家当局 ("Landesamt 并且一厢情愿和 Soziales") 的批准, 允许号码: G0053/15)。
注: 本议定书的主要目的是研究反复麻醉对糖皮质激素代谢物的影响。为了确定要使用的动物数量, 进行了一个样本大小计算: n ≥ 2 x (s/µ1-µ2)2 x (zα + zβ)2。µ1-µ2是指在其中进行功率和样本大小计算 (α = 5%, β = 80%) 的填充方式之间的差异; zα = 1.96 和 zβ = 0.84 是标准正态分布的分位数。图 1说明了此协议的时间线。如果协议的参数显示与控制级别的差异, 应密切监视动物, 并在适当的时间后再测量参数。例如, 如果增加了特质焦虑相关行为, 则应在一周后再次测试此行为, 以帮助确定期间直到完全恢复。本协议中定义的时间点和周期可用于其他过程。在更改时间点时, 应按照协议中的说明来保留适应期。为了减少可能影响小鼠行为的因素, 需要进行更多操作的测试应在不干扰小鼠正常行为的试验后进行。图 2使用摘要计分单汇总了协议的所有测试。图 3提供了福利等级的简化刻度, 它概述了如何解释测试结果。
1. Habituating 老鼠处理的实验者
<ol><li>允许老鼠习惯到动物设施至少2周后, 他们已经从其他设施或供应商获得。</li>
	<li>小鼠在小组和维护他们在标准条件下 (室温 22 2 °c; 相对湿度 55 * 10%) 在光: 黑暗的周期 12:12 h。</li>
	<li>为所有组提供隧道和棉花 nestlets 作为标准浓缩, 并提供食物和水的ad 随意。</li>
	<li>在测试12之前, 将所有鼠标习惯到隧道和/或杯处理至少一周。 注意: 通过尾部拾取鼠标可以诱发压力或焦虑, 进而影响福祉, 也会对此协议12的结果产生影响。</li>
</ol>2. 准备行为测试室和仪器
注意: 提供一个单独的测试空间, 最好是在保存动物的房间附近。在手术前至少60分钟将老鼠送到试验室。如果可能, 在执行该过程的同一测试室中进行本协议的所有测试。
<ol><li>准备一个观察笼来测试穴居行为8 , 并拍摄照片, 以用于在 mg13 (图 4)。<ol>
<li>使用一个玻璃盒, 建筑面积约220毫米 x 290 毫米, 高度为390毫米。</li>
		<li>用大约0.5 厘米的寝具材料盖住这个盒子的地板。</li>
		<li>在新的床上用品材料的顶部, 将一小部分使用过的床上用品从家庭笼子里撒出来, 以减少新环境造成的痛苦。</li>
		<li>提供食物, 通常作为饮食和水供应的同类食品。 注: 如有可能, 使用水瓶, 因为老鼠可以用床上用品填充水碗。</li>
	</ol>
</li>
	<li>为24小时观察期间准备一个保持架 (III 型: 420 毫米 x 260 毫米 x 150 毫米), 为其单独存放鼠标 (图 5)。 注: 为了最大限度地减少个体住房的持续时间, 在这一时期收集巢楼行为、家庭笼活动、食物摄取和粪便皮质酮代谢物 (FCM) 的数据。<ol>
<li>将新的床上用品材料放在笼中 (大约0.5 厘米深), 并在新材料的顶部分散一小部分使用过的寝具材料, 而不粪便从主笼上, 以减少遇险。</li>
		<li>提供标准的方形棉 nestlet 的定义的重量, 作为环境浓缩仅 (参见材料表)14。 注意: 商业 nestlets 的重量可能不同。因此, 我们修改了执事描述的 nestlet 的重量, 使用了 2.0 g 而不是 2.7 g14。</li>
		<li>在保持架顶部安装红外线传感器时, 使用红外线传感器测量主笼活动 (请参阅材料表)。</li>
		<li>提供食物, 通常作为饮食供应的同类, 和水ad 随意。</li>
	</ol>
</li>
</ol>3. 鼠标鬼脸秤
注: 在观察笼中拍摄的三个时间点的照片: (i) 在程序前2天记录基线的标准, (ii) 30 分钟后的程序, 和 (iii) 150 分钟后的程序。当福利被削弱时, 他的分数就会增加。如果在150分钟后仍能观察到增加的。
<ol><li>使用高清相机拍摄照片。</li>
	<li>将鼠标轻轻地转移到观察笼中, 让鼠标习惯到新的环境中至少30分钟。</li>
	<li>在 1-2 分钟内连续拍摄每段时间的 30-40 张照片。</li>
	<li>对所有照片进行排序, 选择尖锐的正面或侧面照片, 丢弃模糊的照片或照片, 显示鼠标面对从其他角度比正面或侧面的看法。</li>
	<li>从每个时间点随机选择一张照片, (即过程前2天, 程序后30分钟, 以及过程后150分钟) 为每个鼠标。</li>
	<li>裁剪照片以仅显示鼠标的头部, 以使正文位置不可见13。</li>
	<li>为每张照片创建一个电子表格文件, 并在每张纸上添加一个表, 包括五面部动作单元。 注: 该文件包含基线照片和照片后的过程。</li>
	<li>随机化工作表的顺序。</li>
	<li>将计算机屏幕上的文件显示给三名独立人员, 这些人以前受过训练, 使用由兰福德et . 开发的 "特里", 并让他们用3点的比例 (0 = 不存在, 1 = 适度存在, 2 = 明显) 得分面部动作单位。目前)。 注: 评分依据的是以下参数13: 轨道收紧 ("眶面积缩小, 眼睑紧密闭合或眼挤压");鼻子隆起 ("在鼻子的桥梁可看见的被圆的延伸皮肤);面颊凸起 ("面颊肌肉的凸的出现);耳朵位置 ("耳朵从基线位置分离和向后, 或具有垂直脊, 形成由于耳朵被拉回的提示");晶须变化 ("晶须从基线位置向后、正面或向前移动, 仿佛站在末端;胡须也可能聚集在一起 ")。</li>
	<li>分析分数, 如下所示 (从兰福德et 。13)。<ol>
<li>平均所有的面部动作单位为每张照片, 以产生的 "中" 得分。 注: 如果一个面部动作单位不能得分, 平均其余的面部动作单位。</li>
		<li>减去平均值的基线照片从平均值的照片后的过程, 以获得一个不同的分数为每一个鼠标。</li>
		<li>测试不同的人之间的差异评分 (非参数测试的相关样本)。 注: 如果有显著差异 (p < 0.05), 确定是否所有照片的分数或只有几张照片的分数不同的人之间。如果后者是真的, 重复评分这些照片。否则, 这些人应该重复训练, 然后再拍照片。</li>
		<li>如果所有的人的结果没有显著的差异, 平均每只老鼠的不同得分者的差异评分。</li>
		<li>使用非参数统计检验, 比较研究组之间平均差异评分。</li>
	</ol>
</li>
</ol>4. 穴居行为8,15,16
<ol><li>在标准不透明的塑料水瓶 (250 毫升, 150 毫米长度, 55 毫米直径, 45 毫米直径的瓶子颈) 上放置 140 "2 克" 的食物颗粒, 以此来准备洞穴8。 注意: 小鼠喜欢宽管, 有68毫米直径的洞穴可以按照执事16的描述使用。</li>
	<li>在驯化的过程前5天, 将洞穴填满食物颗粒在笼子里。 注意: 笼子里的常规食品分配单位不应该被清空, 但也应该留满食物颗粒, 因为老鼠已经习惯了。</li>
	<li>进行两次测试, 2 天前的程序 (基线);执行最后30分钟的开机自检程序。<ol>
<li>让鼠标习惯至少30分钟的观察笼, 在那里拍摄的照片为的。</li>
		<li>将塑料水壶装满与观察笼的后壁平行的食物小球。</li>
		<li>在2小时后的洞穴中, 称食物颗粒 (g)。</li>
	</ol>
</li>
	<li>计算相对于初始重量的小鼠从洞穴中取出的食物颗粒的重量 (%)。</li>
</ol>5. 24 小时观察期
注意: 老鼠单独安置, 如2.2 所述。(图 5), 为期24小时, 以测量食物摄取量、家庭笼活动、巢建筑行为和 FCM 水平。24 h 的观察发生两次: (一) 在程序开始之日前2天 (二)。
<ol><li>
食物摄取量
	<ol>
<li>定期对老鼠进行称量 (例如麻醉前2天, 麻醉前、麻醉后2天、麻醉后的每周), 以评估体重的变化 (评分表的一部分)。 注意: 体重需要计算每克体重的食物摄取量。在24小时观察期间也可以测量进水量。如果食物摄入量减少, 福利可能会受损。</li>
		<li>确定笼中食物单位 (约100克) 所提供的标准食物饮食 (克) 的初始重量。</li>
		<li>在24小时的观察期结束时确定标准饮食的重量。</li>
		<li>仔细扫描食物单位下方的笼侧, 以便食物溢出, 并添加任何额外的食物颗粒, 发现食品颗粒剩余的食物单位的重量。</li>
		<li>计算每单位体重的食物摄取量。</li>
	</ol>
</li>
	<li>
家庭笼活动 注: 以下说明是指使用红外线传感器 (请参阅材料表), 但家庭笼子活动也可以用其他程序评估。家庭笼活动从控制级别的偏差 (例如hypoactivity、多动症) 可能是幸福度受损的标志。<ol>
<li>启动程序。</li>
		<li>选择一个采样间隔为1分钟和一个采集时间为24小时, 这意味着脉冲记录每分钟24小时。 注: 如果实验者在录制开始后几次进入房间, 则只使用句点中的数据, 当鼠标未受到干扰时 (即在黑暗期间)。</li>
		<li>总结10分钟的脉冲间隔。</li>
		<li>计算时间曲线下的区域 (脉冲 x min)。</li>
	</ol>
</li>
	<li>
巢大厦行为 注意: 复杂和高巢可以作为福祉的指标。<ol>
<li>放置一个方形棉花 nestlet (参见材料表), 其定义的重量 (例如2.0 g) 在笼子中间。</li>
		<li>根据执事14在第二天早晨, 大约2小时在光打开以后, 在5点刻度上评分巢 (如下所示)。权衡任何 untorn nestlet 件, 至少是最初 nestlet 重量的5%。将巢分如下14<ol>
<li>如果90% 的 nestlet 完好无损, 则分配 "1" 分。</li>
			<li>指定分数为 "2", 如果它是 50-90% 完好无损。</li>
			<li>分配分数 "3", 如果 50-90% 的 nestlet 被粉碎。</li>
			<li>分配评分 "4", 如果超过90% 被粉碎, 但巢是平的, 少于50% 的周长比鼠标身高时, 蜷缩起来。</li>
			<li>分配评分 "5", 如果超过 90% nestlet 被撕碎和巢是高, 超过50% 的周长高于身高的一个卷曲的鼠标。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
粪皮质酮代谢物 注意: 在控制水平以上的 FCM 的增加反映了24小时 postanesthetic 期间的急性应激水平。<ol>
<li>在24小时观察期结束时使用镊子从笼中收集所有干粪丸, 并清除尿液中的湿颗粒。</li>
		<li>提取 FCM 根据棕榈, et 等。17, 如下所示。<ol>
<li>干燥粪样品在温度 60-70 °c。</li>
			<li>用灰浆融汇粪便样品。</li>
			<li>将0.05 克的整除与1毫升80% 甲醇在离心管中摇动, 在多涡上进行30分钟。</li>
			<li>离心机样品在 2500 x g 15 分钟。</li>
			<li>将0.5 毫升的上清液注入另一离心管。</li>
			<li>储存粪便样品 (和提取物) 在至少-18 摄氏度。</li>
			<li>用 5α-pregnane-3 b、11 b、21-三元醇-20-一酶免疫分析法 (EIA) 18、19或另一次完全验证的环境影响评估 FCM.</li>
		</ol>
</li>
		<li>计算 fcm 浓度相对于基线 fcm 浓度的百分比变化。</li>
	</ol>
</li>
</ol>6. 自由探索范式
<ol><li>把笼子从架子上拿出来, 在24小时观察期结束时把它放在桌面上。</li>
	<li>将网格化笼顶 (不带食物或水瓶) 放在位于45°角的笼中, 置于笼的较长一侧。 注意: 不要破坏巢, 它充当老鼠的藏身之处, 但将笼顶对角置于巢之上。</li>
	<li>显示器或视频-记录小鼠10分钟从距离约1.5 米。<ol>
<li>启动计时器。</li>
		<li>注意当鼠标爬到笼顶 (所有四只爪子放在笼顶) 或离开笼顶 (用一个或更多的爪子在笼底) 的所有时间。 注意: 一些老鼠可能爬上笼顶, 离开它, 以便沿着笼子的边缘走。一些老鼠也在笼子顶上。把这些病例当作老鼠仍然放在笼子顶上。</li>
	</ol>
</li>
	<li>根据伯特et计算参数。20。<ol>
<li>分析第一次探测的滞后时间 (秒)。</li>
		<li>分析探索的次数。</li>
		<li>分析勘探的总持续时间 (秒)。 注意: 第一次勘探的潜伏期很高, 探索的次数少, 总的勘探时间也很低, 这表明有较高的特质焦虑水平。</li>
	</ol>
</li>
</ol>

<ol>
	<li>2010 EU. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directive+2010/63/EU.">Directive 2010/63/EU.</a> Official Journal of the European Union. , (2010).</li><li>Russell, W. M. S., Burch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The principles of humane experimental technique. , (1959).</li><li>Morton, D. B., Griffiths, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+the+recognition+of+pain,+distress+and+discomfort+in+experimental+animals+and+an+hypothesis+for+assessment.">Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment.</a> Vet Rec. 116 (16), 431-436 (1985).</li><li>Bugnon, P., Heimann, M., Thallmair, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+the+literature+tells+us+about+score+sheet+design.">What the literature tells us about score sheet design.</a> Lab Anim. 50 (6), 414-417 (2016).</li><li>Boissy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+positive+emotions+in+animals+to+improve+their+welfare.">Assessment of positive emotions in animals to improve their welfare.</a> Physiol Behav. 92 (3), 375-397 (2007).</li><li>Arras, M., Rettich, A., Cinelli, P., Kasermann, H. P., Burki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+post-laparotomy+pain+in+laboratory+mice+by+telemetric+recording+of+heart+rate+and+heart+rate+variability.">Assessment of post-laparotomy pain in laboratory mice by telemetric recording of heart rate and heart rate variability.</a> BMC Vet Res. 3, 16 (2007).</li><li>Jirkof, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing+and+nest+building+behavior+as+indicators+of+well-being+in+mice.">Burrowing and nest building behavior as indicators of well-being in mice.</a> J Neurosci Methods. 234, 139-146 (2014).</li><li>Jirkof, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing+behavior+as+an+indicator+of+post-laparotomy+pain+in+mice.">Burrowing behavior as an indicator of post-laparotomy pain in mice.</a> Front Behav Neurosci. 4, 165 (2010).</li><li>Hawkins, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+defining+and+implementing+protocols+for+the+welfare+assessment+of+laboratory+animals:+eleventh+report+of+the+BVAAWF/FRAME/RSPCA/UFAW+Joint+Working+Group+on+Refinement.">A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement.</a> Lab Anim. 45 (1), 1-13 (2011).</li><li>Hohlbaum, K., Bert, B., Dietze, S., Palme, R., Fink, H., Th&#246;ne-Reineke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severity+classification+of+repeated+isoflurane+anesthesia+in+C57BL/6JRj+mice-Assessing+the+degree+of+distress.">Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice-Assessing the degree of distress.</a> PLoS ONE. 12 (6), e0179588 (2017).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+the+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.</a> PLoS Biol. 8 (6), e1000412 (2010).</li><li>Hurst, J. L., West, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+anxiety+in+laboratory+mice.">Taming anxiety in laboratory mice.</a> Nat Methods. 7 (10), 825-826 (2010).</li><li>Langford, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nat Methods. 7 (6), 447-449 (2010).</li><li>Deacon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+nest+building+in+mice.">Assessing nest building in mice.</a> Nat Protoc. 1 (3), 1117-1119 (2006).</li><li>Deacon, R. M., Raley, J. M., Perry, V. H., Rawlins, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing+into+prion+disease.">Burrowing into prion disease.</a> Neuroreport. 12 (9), 2053-2057 (2001).</li><li>Deacon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burrowing+in+rodents:+a+sensitive+method+for+detecting+behavioral+dysfunction.">Burrowing in rodents: a sensitive method for detecting behavioral dysfunction.</a> Nat Protoc. 1 (1), 118-121 (2006).</li><li>Palme, R., Touma, C., Arias, N., Dominchin, M. F., Lepschy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+extraction:+get+the+best+out+of+faecal+samples.">Steroid extraction: get the best out of faecal samples.</a> Wien Tierarz Monats. 100 (9-10), 238-246 (2013).</li><li>Touma, C., Palme, R., Sachser, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+corticosterone+metabolites+in+fecal+samples+of+mice:+a+noninvasive+technique+to+monitor+stress+hormones.">Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones.</a> Horm Behav. 45 (1), 10-22 (2004).</li><li>Touma, C., Sachser, N., Mostl, E., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sex+and+time+of+day+on+metabolism+and+excretion+of+corticosterone+in+urine+and+feces+of+mice.">Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice.</a> Gen Comp Endocrinol. 130 (3), 267-278 (2003).</li><li>Bert, B., Schmidt, N., Voigt, J. P., Fink, H., Rex, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cage+leaving+behaviour+in+rats+as+a+free+choice+paradigm.">Evaluation of cage leaving behaviour in rats as a free choice paradigm.</a> J Pharmacol Toxicol Methods. 68 (2), 240-249 (2013).</li><li>Lister, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethologically-based+animal+models+of+anxiety+disorders.">Ethologically-based animal models of anxiety disorders.</a> Pharmacol Ther. 46 (3), 321-340 (1990).</li><li>Belzung, C., Berton, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+pharmacological+validation+of+the+BALB/c+neophobia+in+the+free+exploratory+paradigm+as+an+animal+model+of+trait+anxiety.">Further pharmacological validation of the BALB/c neophobia in the free exploratory paradigm as an animal model of trait anxiety.</a> Behav Pharmacol. 8 (6-7), 541-548 (1997).</li><li>Finlayson, K., Lampe, J. F., Hintze, S., Wurbel, H., Melotti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+indicators+of+positive+emotions+in+rats.">Facial indicators of positive emotions in rats.</a> PLoS ONE. 11 (11), e0166446 (2016).</li><li>Miller, A., Kitson, G., Skalkoyannis, B., Leach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+isoflurane+anaesthesia+and+buprenorphine+on+the+mouse+grimace+scale+and+behaviour+in+CBA+and+DBA/2+mice.">The effect of isoflurane anaesthesia and buprenorphine on the mouse grimace scale and behaviour in CBA and DBA/2 mice.</a> Appl Anim Behav Sci. 172, 58-62 (2015).</li><li>Miller, A. L., Golledge, H. D., Leach, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+isoflurane+anaesthesia+on+the+rat+grimace+scale.">The influence of isoflurane anaesthesia on the rat grimace scale.</a> PLoS ONE. 11 (11), e0166652 (2016).</li><li>Deacon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+burrowing,+nest+construction,+and+hoarding+in+mice.">Assessing burrowing, nest construction, and hoarding in mice.</a> J Vis Exp. (59), e2607 (2012).</li><li>Felton, L. M., Cunningham, C., Rankine, E. L., Waters, S., Boche, D., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCP-1+and+murine+prion+disease:+separation+of+early+behavioural+dysfunction+from+overt+clinical+disease.">MCP-1 and murine prion disease: separation of early behavioural dysfunction from overt clinical disease.</a> Neurobiol Dis. 20 (2), 283-295 (2005).</li><li>Deacon, R. M., Croucher, A., Rawlins, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+cytotoxic+lesion+effects+on+species-typical+behaviours+in+mice.">Hippocampal cytotoxic lesion effects on species-typical behaviours in mice.</a> Behav Brain Res. 132 (2), 203-213 (2002).</li><li>Filali, M., Lalonde, R., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subchronic+memantine+administration+on+spatial+learning,+exploratory+activity,+and+nest-building+in+an+APP/PS1+mouse+model+of+Alzheimer's+disease.">Subchronic memantine administration on spatial learning, exploratory activity, and nest-building in an APP/PS1 mouse model of Alzheimer's disease.</a> Neuropharmacology. 60 (6), 930-936 (2011).</li><li>Guenther, K., Deacon, R. M., Perry, V. H., Rawlins, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+behavioural+changes+in+scrapie-affected+mice+and+the+influence+of+dapsone.">Early behavioural changes in scrapie-affected mice and the influence of dapsone.</a> Eur J Neurosci. 14 (2), 401-409 (2001).</li><li>Deacon, R. M., Reisel, D., Perry, V. H., Nicholas, J., Rawlins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+scrapie+infection+impairs+operant+DRL+performance+in+mice.">Hippocampal scrapie infection impairs operant DRL performance in mice.</a> Behav Brain Res. 157 (1), 99-105 (2005).</li><li>Jirkof, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+postsurgical+distress+and+pain+in+laboratory+mice+by+nest+complexity+scoring.">Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring.</a> Lab Anim. 47 (3), 153-161 (2013).</li><li>Atanasov, N. A., Sargent, J. L., Parmigiani, J. P., Palme, R., Diggs, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+train-induced+vibration+and+its+effect+on+fecal+corticosterone+metabolites+in+mice.">Characterization of train-induced vibration and its effect on fecal corticosterone metabolites in mice.</a> J Am Assoc Lab Anim Sci. 54 (6), 737-744 (2015).</li><li>Voigt, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+stress+response+of+laboratory+mice+to+conventional+and+minimally+invasive+bleeding+techniques.">Hormonal stress response of laboratory mice to conventional and minimally invasive bleeding techniques.</a> Anim Welf. 22 (4), 449-455 (2013).</li><li>Walker, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+less+stressful+alternative+to+oral+gavage+for+pharmacological+and+toxicological+studies+in+mice.">A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice.</a> Toxicol Appl Pharmacol. 260 (1), 65-69 (2012).</li><li>Miyashita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+stress+increases+biopyrrins,+oxidative+metabolites+of+bilirubin,+in+mouse+urine.">Social stress increases biopyrrins, oxidative metabolites of bilirubin, in mouse urine.</a> Biochem Biophys Res Commun. 349 (2), 775-780 (2006).</li><li>Bains, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+individual+mouse+activity+in+group+housed+animals+of+different+inbred+strains+using+a+novel+automated+home+cage+analysis+system.">Analysis of individual mouse activity in group housed animals of different inbred strains using a novel automated home cage analysis system.</a> Front Behav Neurosci. 10 (106), (2016).</li><li>Saibaba, P., Sales, G. D., Stodulski, G., Hau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behaviour+of+rats+in+their+home+cages:+daytime+variations+and+effects+of+routine+husbandry+procedures+analysed+by+time+sampling+techniques.">Behaviour of rats in their home cages: daytime variations and effects of routine husbandry procedures analysed by time sampling techniques.</a> Lab Anim. 30 (1), 13-21 (1996).</li></ol>

作者没有什么可透露的。

该议定书最初是为了评估接受单一麻醉或重复异氟醚麻醉的 C57BL/6JRj 小鼠的福祉而制定的。研究结果证实, 对奢侈品行为的测试以及其他措施 (例如自由探索范式、mg、穴居食物摄取) 都是评估幸福感的敏感方法。反复异氟醚麻醉对特质焦虑相关行为、特征和穴居行为产生短期影响。此外, 反复异氟醚麻醉似乎影响食物摄取量10。
自由探索范式表明, 减少了探索行为, 因此, 在反复麻醉的小鼠中, 与仅麻醉一次的小鼠相比, 较高的特质焦虑水平和控制。但是, 所有组的鼠标都探索了网格化笼顶部10, 20.在调查与焦虑相关的行为时, 区分特征和状态焦虑是很重要的21,22。将鼠标放置在不熟悉的环境中会诱发状态焦虑。相比之下, 自由探索范式允许动物呆在自己的笼子里, 从而调查特质焦虑。
反复麻醉后小鼠探索性行为的减少不能通过运动活动的减少来解释。在黑暗时期, 家庭笼活动在研究组之间没有显著差异。这表明在进行自由探索范式试验时, 老鼠已经从麻醉中恢复了体力。
该人实际上是开发来评估疼痛, 但有证据表明, 面部表情也改变了压力和积极情绪13,23。小鼠脸上的照片表明, 单次和多次异氟醚麻醉在近期麻醉后的短期内增加了对该药的评分。由于该差异评分保持在1以下, 压力的水平似乎是温和的10。结果与米勒et . 的最近观察一致。24,25并显示鼠标返回控制级别150分钟后麻醉10。
奢侈行为, 如穴居和巢建筑行为是物种特有的, 可以作为健康的指标7和良好的一般健康的小鼠26。只有当所有的基本需求都满足时, 老鼠才会表现出奢侈的行为。如果福利减少, 奢侈行为将首先被削弱5,7。以前的研究表明, 挖洞和鸟巢建筑物都可以受到疼痛和窘迫的损害6,8, 但也有证据表明, 海马病变可能会影响这两个行为15,27,28,29,30,31。
奢侈行为被调查了在早期麻醉后时期 (挖洞行为) 和由次日早晨 (巢大厦行为)。对执事et等) 开发的穴居行为进行了试验, 并对 Jirkof et 等人所采用的方法进行了修改, 以适应 (小组住房而不是单独住房) 和行为测量的持续时间 (仅2小时而不是24小时)。8,15,16。
值得注意的是, 反复麻醉减少了穴居行为, 表明有一个损害的福祉立即后麻醉10, 这也是报告的 Jirkof et 等。8. 但是在第二天早上, 当老鼠有更多的时间从麻醉中恢复时, 他们所建造的高而复杂的巢表明他们正在经历良好的10。这些发现与以前在32早期取得巢的报告不同。因此, 在较早时间内调整协议和评分巢可能是有用的。然而, 巢建筑行为的昼夜节律仍然必须考虑, 因为老鼠倾向于准备他们的巢在黑暗的阶段的末端32。
反复麻醉后1天, 食物摄入量略有下降, 但一周后增加。因为老鼠并没有失去体重 (见 Hohlbaum et 等。10) 可能会发生某种补偿机制, 而食物摄入方面的福利损害应归类为轻度10。食物摄取提供了对小鼠术后窘迫、幸福感和食欲的洞察力, 可通过术后恶心26和术后应激27来损害。
FCM 可靠地指示小鼠的压力33,34,35。峰值 FCM 浓度通常发生在压力源后8-10 小时, 但取决于肠道转运时间18。因此, 也必须监测家庭笼活动, 如本议定书所载。由于生理节律对排泄的 FCM18的影响, 最好在24小时内收集粪便样本, 在24小时的麻醉后的时间内反映急性压力。由于 fcm 水平因人而异, 计算了相对基线的 fcm 水平的百分比变化。本研究的 FCM 结果表明, 无论是麻醉还是反复麻醉的经验都没有显著增加下丘脑-垂体-肾上腺 (HPA) 轴活动10。
总而言之, 这一发现表明, 反复异氟醚麻醉引起的短期轻度窘迫和受损的福祉在早期的 postanesthetic 期略多于单一经验的异氟醚麻醉10。
评估福利的议定书不应对动物造成额外的痛苦。本议定书的一个限制是, 它包括在24小时观察期内的个别住房, 这是已知的增加血浆皮质酮水平36。然而, 个人住房是必要的收集有效的数据, 个人的家庭笼活动, 食物摄取, 巢建设行为, 和 FCM 水平。通过调查四个参数同时 (即在24小时观察期间), 减少了单个住房的持续时间。为了防止单个住房对结果产生偏见, 控制小鼠进行了同样的试验, 并考虑了其结果。如果方法可用于度量组壳环境中的这些参数, 则应使用它们 (例如家庭笼活动的自动主笼分析系统37和食物摄取量)。然而, 自动化的家庭笼分析系统需要额外的设备, 这可能是不可用的。在研究集中在动物群而不是个体, 家庭笼子活动, 食物摄取, 巢大厦行为和 FCM 水平可能也被评估为一组老鼠。这同样适用于自由探索范式, 它只要求老鼠明显地被标记, 以便他们可以被区分。如果一组患者的福利减少, 则应仔细检查组中的所有小鼠 (使用评分表和临床检查) 来确定有关的老鼠或老鼠。为了确定组值的灵敏度, 需要在组级别进行进一步的观察。
关于改进, 这项议定书可以适应并纳入正在进行的研究, 以评估特定程序对小鼠福祉的影响。根据具体程序和研究设计, 应选择该协议中最适合的测试。在这里, 穴居和鸟巢的建筑试验, 对食物摄取的自由探索范式和测量似乎是特别有益的。当老鼠的行为遵循昼夜节律38时, 最好在定义的时间点对所有组的小鼠进行行为测试。老鼠在早晨比在下午38更活跃。但是, 如果研究设计不允许在上午测试, 测试可能会在另一个时间完成。同样重要的是要确保每一个测试都在同一时间点进行所有组。否则, 参数的昼夜节律的影响可能导致内部或团体的差异。此外, 在研究中应纳入控制小鼠, 以便对小鼠和对照鼠的结果进行比较。控制小鼠必须在同一时间点进行检测, 以治疗小鼠。如果议定书表明福利受到损害, 应改进程序, 并重复该议定书。这种方法可以说明改进程序的努力是否有效。
本议定书可作为一般援助, 以估计程序造成的严重程度。因此, 它有助于将程序的严重性归类为以科学和动物为中心的水平。然而, 该议定书并没有提供将程序分类为轻度、中等或严重的规模。为了对程序的严重性进行分类, 有必要参考《欧盟指令2010/63 》附件八中的例子。
最后, 本议定书支持以科学的、以动物为中心的方式对小鼠的福祉进行系统评估, 并遵循使用全麻的程序。

欧盟指令 2010/631规定, 每当需要动物试验时, 由罗素和2开发的3R 原理 (替换、还原、细化) 将被应用.欧盟指令的最终目标是逐步淘汰所有动物试验, 但该指令承认, 目前还需要一些动物实验来进行研究, 以保护人类和动物的健康。因此, 如果动物实验不能被任何替代方法所取代, 只有最小数量的实验动物被用来获得可靠的结果。此外, 使用适当的细化措施, 应尽量减少伴随实验程序的疼痛、痛苦和苦恼的数量。欧盟指令2010/63 规定, 过程的严重性必须前瞻性归类为非恢复、温和、适度或严重的1。由于严重性分类是根据个案来决定的, 因此有科学的声音工具来估计给定过程的严重性是很重要的。
根据莫顿和格里菲斯的建议, 评分表3是检测任何偏离正常状态的重要工具, 包括对福祉4的负面影响。评分表用于回顾性地确定由实验引起的疼痛、痛苦和苦恼, 并着重于个体动物 (例如、体重、皮毛、步态) 的物理状态的可见变化。虽然, 欧盟指令2010/63 的附件八提供了每个严重性类别的例子, 但研究人员仍然缺乏利用科学基础的数据来估计给定程序严重性程度的工具。
缺乏显示消极福祉的指标并不是确定动物状况的唯一方法;指向正福利的指示器的存在也很重要5、6、7、8。例如, 动物只在满足所有基本需求时, 才会表现出诸如挖洞和筑巢等奢侈行为。如果减少了福利, 奢侈品行为首先会下降5,7。用于评估福祉的议定书应包括指示动物生理、生理/生物化学和心理状态的指标, 以便以详细和全面的方式对其福祉进行评估9。
在改进的范围内, 制定了一项议定书, 以满足这些要求, 并评估一项涉及全麻对小鼠福祉的程序的影响10。同时, 目标是尽量减少任何额外的压力, 使协议易于集成到给定的实验中。该协议考虑穴居行为, 家庭笼行为, 如活动, 食物摄取, 和巢, 和特质焦虑相关的行为。此外, 还包括小鼠的鬼脸量表和粪便中皮质酮代谢物的无创性分析。该议定书的目的是促进以科学和动物为中心的方式评估福祉, 并提供支持严重程度分类的福祉信息。除了评分表之外, 它还可以为过程的严重性分类提供有用的信息。由于该协议易于执行且不需要广泛的设备, 因此可以将其集成到一个正在进行的实验中, 而不影响研究结果。应该指出的是, 动物研究: 报告的活体实验 (到达) 指南11将在所有涉及动物实验的研究中观察到, 目的是改进设计、分析和报告。

<table><tbody><tr><td>Isofluran</td><td>CP-Pharma Handelsgesellschaft mbH</td><td>1214</td><td></td></tr><tr><td>InfraMot - Sensore Units</td><td>TSE Systems</td><td>302015-SENS</td><td></td></tr><tr><td>InfraMot - Control Units</td><td>TSE Systems</td><td>302015-C/16</td><td></td></tr><tr><td>InfraMot - Software</td><td>TSE Systems</td><td>302015-S</td><td></td></tr><tr><td>Nestlet N</td><td>Ancare - Plexx</td><td>NES3600</td><td></td></tr><tr><td>Camera EOS 350D</td><td>Canon</td><td></td><td></td></tr></tbody></table>

systematic assessment of well-being in mice for procedures using general anesthesia

根据罗素和科里开发的3R 原理 (替换、还原、细化), 科学研究应尽可能使用动物实验的替代品。如果不能替代动物实验, 那么所使用的实验动物总数应该是获取有价值数据所需的最低数量。此外, 应采用适当的细化措施, 以尽量减少伴随实验程序的痛苦、痛苦和痛苦。用于分类疼痛、痛苦和痛苦程度的类别是不恢复的、轻度的、中等的或严重的 (欧盟指令 2010/63)。要确定哪些类别适用于个别案例, 使用科学的声音工具是至关重要的。
这里提出的幸福评估协议是为使用全麻的程序设计的。该协议的重点是家庭笼活动, 鼠标鬼脸规模, 和奢侈行为, 如穴居和巢建筑行为作为福祉的指标。它也使用自由探索范式的特质焦虑相关行为。粪便皮质酮代谢物作为急性应激指标的测量在24小时后麻醉期间。
该议定书提供了有关全麻后小鼠的福祉的科学可靠的信息。由于其简单性, 该协议可以很容易地被修改和集成在一个计划的研究。虽然它没有提供一个规模, 根据欧盟指令2010/63 分类的苦恼, 它可以帮助研究人员估计一个程序的严重性程度, 使用科学的声音数据。它提供了一种以科学、动物为中心的方式来改进对幸福的评估的方法。

本议定书最初是为了评估 C57BL/6JRj 小鼠在单异氟醚麻醉 (一次45分钟麻醉疗程, n = 13 女性) 或重复异氟醚麻醉 (六45分钟麻醉疗程 3-4 天) 后的福祉而制定的。在麻醉疗程之间, n = 13 女性) 与控制小鼠 (n = 6 位女性) 的福利相比,10, 没有接受麻醉, 但根据相同的措施测试。我们评估了单一经验的异氟醚麻醉和重复异氟醚麻醉对 C57BL/6JRj 小鼠的幸福与未经治疗的控制小鼠的影响。这里, 女性 C57BL/6JRj 小鼠的代表性结果, 包括以前在 Hohlbaum et . 中发布的一些数据。显示10以及以前未发布的结果。
统计分析
对每个参数进行了探索性数据分析和常态测试。如图传说中所述, 研究组 (即对照、单异氟醚麻醉、重复异氟醚麻醉等) 之间的差异进行了分析。当数据符合正态分布假设时, 进行1路方差分析。用克鲁斯卡尔-沃利斯试验对非正常分布数据进行了分析. 在 p < 0.05 中, 差异被认为是显著的。
基线值
在执行过程之前收集的基线值对于确定治疗组在各自参数中是否不同是至关重要的。如图 6所示,图 7,图 8,图 9和图 10, 克鲁斯卡尔评分的基线级别 (p = 0.762, 克鲁斯卡尔-沃利斯测试), 奢侈行为, 如穴居 (p = 0.896,-沃利斯测试) 和嵌套 (p = 0.723, 克鲁斯卡尔-沃利斯试验), 食物摄取量 (p = 0.398, 1 方式方差分析), 和家庭笼活动 (p = 0.208, 克鲁斯卡尔-沃利斯试验) 没有显著差异的组。此外, 没有发现基线 FCM 浓度的显著差异 (中位, 四分位范围在方括号 [ng/50 毫克]: 控制: 123.01 (82.70-193.46); 单次麻醉: 118.31 (101.73-153.54); 反复麻醉: 129.55 (92.58-139.48) (p= 0.904, 克鲁斯卡尔-沃利斯测试)。如果基线级别发生差异, 则可以计算增量值。
鼠标鬼脸秤
当对照组的平均成绩进行比较时, 在麻醉的单次经历 (p = 0.001) 和最后一次麻醉之后 (p = 0.021) 后, 在最后一次麻醉剂后30分钟后发现了显著较高的得分与对照 (图 6A).在上一次麻醉后150分钟, 组间不再有差异 (p = 0.910)。
为了考虑到在所有情况下, 基线的方法分数不等于 0, 根据协议中的描述, 计算出了不同评分。麻醉 (p = 0.002) 和反复麻醉 (p = 0.008) 两个单一的经验增加了在麻醉后30分钟的药敏差异评分与对照。在最后一次麻醉后的150分钟, 所有的老鼠都返回到控制级别 (p = 0.617) (图 6B)10。
穴居行为
反复麻醉大大减少了从洞穴中移除的食物小球的重量百分比与控制 (p = 0.036, Kruksal-沃利斯测试) (图 7)10。
巢大厦行为
单次麻醉经验、反复麻醉和控制 (p = 0.240、Kruksal-沃利斯测试) (图 8)10之间的巢得分没有显著差异。
食物摄取量
在上一次麻醉后的1天, 反复麻醉的老鼠与经历过单次麻醉的小鼠相比明显减少了食物摄入量 (p = 0.047, 1 路方差分析)。相比之下, 一周后, 接受过反复麻醉的老鼠消耗的食物比对照组 (p = 0.012, 1 路方差分析) 或小鼠接受了单一麻醉 (p = 0.001, 1 方式方差分析) (图 9)10。
家庭笼活动
在上一次麻醉后1天, 在活动曲线下的区域显示的在黑暗期内的家庭笼活动, 在接受过单一麻醉、反复麻醉或控制治疗的小鼠之间没有显著差异 (p = 0.498,克鲁斯卡尔-沃利斯测试) (图 10)10。
自由探索范式
当试验进行时, 所有的老鼠都在笼子顶上摸索。然而, 在上一次麻醉后1天, 反复麻醉的老鼠 (中位, 四分位范围在括号内 [s]: 78.00 (55.00-89.00)) 比对照组更迟地探索笼顶部 (31.00 (18.25-42.75); p = 0.009,克鲁斯卡尔-沃利斯试验) 和小鼠接受了单一麻醉 (27.00 (21.00-45.50); p = 0.001, 克鲁斯卡尔-沃利斯测试), 如以前发布的10。探查的参数总持续时间 (图 11A) 和探索次数 (图 11B) 类似于第一次勘探的滞后时间。反复麻醉大大减少了探索与控制的次数 (p = 0.023, 克鲁斯卡尔-沃利斯试验) 和总勘探时间 (p = 0.032, 克鲁斯卡尔-沃利斯试验) 与单一麻醉在1天后最后一次麻醉。在上次麻醉8天后所有参数,即到第一次探测的滞后时间 (控制: 27.50 (13.50-47.25); 单次麻醉: 18.00 (9.50-38.50); 反复麻醉: 20.00 (12.50-42.00); p = 0.722, 克鲁斯卡尔-沃利斯试验), 数量探索 (p = 0.057) 和总勘探持续时间 (p = 0.579), 研究组之间不再有差异 (图 11)。
粪皮质酮代谢物
为了考虑获得的基线值, 计算了相对于基线的百分比变化, 发现组之间没有显著差异 (p = 0.119, 克鲁斯卡尔-沃利斯测试) (图 12)10。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57046/57046fig1.jpg"> 图 1: 协议的时间行.灰色和白色的颜色场分别象征着一天的黑暗和光明时期。根据程序的不同, 此协议可能会被修改。"对洞穴的适应" 意味着, 在进行穴居试验之前, 老鼠必须适应使用塑料水瓶作为他们家笼中的洞穴。B、基线值;FCM, 粪便皮质酮代谢物。此数字已从 Hohlbaum et . 中进行了修改。10.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57046/57046fig2.jpg"> 图 2: 摘要评分表.此工作表包括所有测试, 并且可以为每个单独的鼠标填写。在进行测试时应添加日期和时间。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57046/57046fig3.jpg"> 图 3: 福利等级的刻度.鳞片范围从 "福利未受损伤的" (绿色) 到 "好被削弱的" (红色) 并且表达测试结果的意思用一个简化的方式。在这一阶段, 关于福利指标的知识, 我们不能就每项考试的福利等级作出明确的说明, 只作一般性发言。老鼠鬼脸秤。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57046/57046fig4.jpg"> 图 4: 观察笼.该笼用于穴居行为的测试, 并根据鼠标的鬼脸量表对照片进行分析。笼子的前面是清楚的, 并且, 根据小鼠的毛皮颜色, 其他三个墙壁应该被上色黑白或白色与老鼠对比。玻璃盒的地板上覆盖着大约0.5 厘米的床上用品材料, 包括从主笼中使用的床上用品材料。提供标准的食物和水。如果可能的话, 应该使用水瓶而不是碗, 因为老鼠可能会用床上用品填充碗。在至少30分钟的适应期之后, 增加洞穴。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57046/57046fig5.jpg"> 图 5:24 小时观察期间.为了评估巢的建筑行为, 家庭笼活动, 食物摄取和收集粪便样本, 以测量粪便皮质酮代谢物 (FCM), 小鼠单独安置24小时 (笼型 III: 420 毫米260毫米150毫米; 床上用品材料大约0.5 厘米深与使用的床上用品材料从家庭笼散落在顶部, 棉花 nestlet, 自来水和标准饮食饮食ad 随意)。红外传感器安装在笼顶部。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57046/57046fig6.jpg"> 图 6: 鼠标鬼脸缩放比例.该框表示四分位范围 (IQR), 方框边缘为25 th 和 75个th 四位。胡须代表的值不大于 1.5 x IQR。点是介于 1.5-3.0 x IQR 之间的值的异常。彩色星号是值大于 3.0 x IQR 的异常。(A) 表示。(B) 表示差异评分。p 值的计算使用克鲁斯卡尔-沃利斯试验: * p < 0.05;** < 0.01。由于技术故障 (照相机, 鳞片), 四只老鼠在唯一麻醉小组必须被排除从统计。此数字已从 Hohlbaum et . 中进行了修改。10.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig6large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57046/57046fig7.jpg"> 图 7: 挖洞行为.该框表示四分位范围 (IQR), 方框边缘为25 th 和 75个th 四位。胡须代表的值不大于 1.5 x IQR。彩色星号是值大于 3.0 x IQR 的异常。p 值的计算使用克鲁斯卡尔-沃利斯测试: * p < 0.05。由于技术故障 (照相机, 鳞片), 四只老鼠在单一麻醉组必须排除在统计分析。此数字已从 Hohlbaum et . 中进行了修改。10.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig7large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57046/57046fig8.jpg"> 图 8: 嵌套生成行为.该框表示四分位范围 (IQR), 方框边缘为25 th 和 75个th 四位。胡须代表的值不大于 1.5 x IQR。点是介于 1.5-3.0 x IQR 之间的值的异常。用克鲁斯卡尔-沃利斯试验计算 p 值;d, 天。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57046/57046fig9.jpg"> 图 9: 食物摄入量.数据均为标准偏差。p 值的计算使用1路方差分析 (后 Tukey-HSD): * p < 0.05;** < 0.01。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/57046/57046fig10.jpg"> 图 10: 家庭笼活动.该框表示四分位范围 (IQR), 方框边缘为25 th 和 75个th 四位。胡须代表的值不大于 1.5 x IQR。点是介于 1.5-3.0 x IQR 之间的值的异常。用克鲁斯卡尔-沃利斯试验计算 p 值.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/57046/57046fig11v2.jpg"> 图 11: 自由探索范式.该框表示四分位范围 (IQR), 方框边缘为25 th 和 75个th 四位。胡须代表的值不大于 1.5 x IQR。点是介于 1.5-3.0 x IQR 之间的值的异常。彩色星号是值大于 3.0 x IQR 的异常。(A) 勘探总时间。(B) 探索的次数。p 值的计算使用克鲁斯卡尔-沃利斯测试: * p < 0.05。此数字已从 Hohlbaum et . 中进行了修改。10.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig11v2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/57046/57046fig12.jpg"> 图 12: 粪皮质酮代谢物 (FCM).该框表示四分位范围 (IQR), 方框边缘为25 th 和 75个th 四位。胡须代表的值不大于 1.5 x IQR。彩色星号是值大于 3.0 x IQR 的异常。FCM 水平测量2天之前和1天后程序。计算了相对于各自基线值的 FCM 浓度的百分比变化 [%]。用克鲁斯卡尔-沃利斯试验对数据进行了分析.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57046/57046fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia at JoVE.com

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

我们制定了一个协议, 以评估小鼠的福祉在程序中使用全麻。分析了一系列的行为参数, 说明了幸福水平和糖皮质激素代谢产物。该议定书可作为一般援助, 以科学的、以动物为中心的方式估计严重程度。

全身麻醉下小鼠幸福度的系统评价

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

systematic-assessment-well-being-mice-for-procedures-using-general

Nanyang Technological University

Research

JoVE Journal

Behavior

12.1K 次观看.  Freie Universität Berlin. 我们制定了一个协议, 以评估小鼠的福祉在程序中使用全麻。分析了一系列的行为参数, 说明了幸福水平和糖皮质激素代谢产物。该议定书可作为一般援助, 以科学的、以动物为中心的方式估计严重程度。

视频: Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

科研

行为学

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

使用开放式现场迷宫来衡量小鼠的运动能力和焦虑样行为

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

植入的无线电遥测发射器产生心电图，心率，身体核心温度和自由移动的实验室小鼠的活动上的数据

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

医学

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

对小鼠高架十字迷宫

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

生物学

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

诱导小鼠抑郁行为的慢性固定性压力协议

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

通过嗅觉挖掘任务评估小鼠判断偏差

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

针灸慢性缺氧小鼠的认知改善

Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice rooted in traditional Chinese medicine, entails the insertion of fine needles into precise points on the body and is commonly employed for the management of diverse conditions. Recently, acupuncture has emerged as a promising therapeutic intervention for a range of neurological diseases, including anxiety and respiratory disorders. However, the potential of acupuncture in treating cognitive dysfunction induced by chronic hypoxia has not yet been explored. This paper presents a comprehensive protocol for establishing a mouse model of chronic hypoxia-induced cognitive impairment, administering mild anesthesia, performing acupuncture treatment, and assessing behavioral changes and memory abilities using open field tests and water mazes. The step-by-step protocol provides detailed instructions on accurately locating and positioning acupoints and needles for cognitive improvement. By employing this protocol, researchers can conduct systematic studies to thoroughly evaluate the therapeutic potential of acupuncture for cognitive dysfunction.

作者聚焦:改进增强小鼠针刺研究的麻醉方案

Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment.

针灸治疗慢性缺氧诱导认知功能障碍小鼠模型

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice ...

全身麻醉下小鼠幸福度的系统评价

摘要

探索更多视频

此视频中的章节

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

使用开放式现场迷宫来衡量小鼠的运动能力和焦虑样行为

应用立体定向注射技术在研究动物行为的遗传效应

植入的无线电遥测发射器产生心电图，心率，身体核心温度和自由移动的实验室小鼠的活动上的数据

对小鼠高架十字迷宫

诱导小鼠抑郁行为的慢性固定性压力协议

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

通过嗅觉挖掘任务评估小鼠判断偏差

针灸治疗慢性缺氧诱导认知功能障碍小鼠模型

针灸治疗慢性缺氧诱导认知功能障碍小鼠模型