兽研摘要
近年来，在人类和动物中的研究探索了替代的药物给药途径，例如药针疗法，这是一种在穴位处皮下注射低剂量药物的技术。在这些穴位中，督脉20（GV20）因其在人类和犬中报道的镇静和安定作用而在兽医学中引起了特别关注。有研究提出，在该部位给予镇静药物可能增强其效果，从而在保持疗效和减少不良反应的同时降低剂量。本研究旨在比较成年豚鼠（Cavia porcellus）在GV20穴位给予美托咪定与肌肉注射途径的镇静效果。二十只动物采用交叉设计，通过两种途径接受美托咪定（100 µg/kg），洗脱期为2周。使用先前发表的量表（0-19）评估镇静程度。镇静起效定义为达到19分中>3分的时间，持续时间定义为恢复到19分中≤3分的时间。评估在给药前和给药后进行，前10分钟每隔1分钟评估一次，之后每2分钟评估一次直至30分钟，此后每5分钟评估一次直至恢复。两种途径之间最大镇静评分、起效时间和持续时间的比较使用Wilcoxon符号秩检验分析。GV20途径与肌肉注射途径在最大评分、起效时间或镇静持续时间方面未发现显著差异。这些结果表明，在本研究条件下，与肌肉注射途径相比，在GV20给予美托咪定并未增强镇静效果或延长作用持续时间。
缩写和首字母缩略词：GV20，督脉20
引言
实验室动物经常需要使用镇静剂，以尽量减少操作或实验模型中进行的操作所产生的焦虑和应激。因此，开发有效的镇静策略以减少应激、便于操作并促进动物福利具有特别的意义。豚鼠（Cavia porcellus）越来越多地被用作实验室动物和伴侣动物，它们经常需要可靠和安全的镇静来进行影像学检查、采血和小型干预等操作。
目前，有多种药物和组合可用于实验室动物的镇静，其中α2受体激动剂如赛拉嗪、美托咪定和右美托咪定以其镇静、肌肉松弛和镇痛特性而著称。实验室动物麻醉指南和比较麻醉综述报告，在小哺乳动物中美托咪定皮下或肌肉注射剂量为30-100 µg/kg，右美托咪定剂量为15-50 µg/kg，并指出α2-肾上腺素能激动剂常与氯胺酮或阿片类药物联合使用以增强镇静或诱导麻醉。在包括大鼠和小鼠在内的某些啮齿类动物中，注射麻醉方案通常依赖氯胺酮-赛拉嗪或氯胺酮-美托咪定组合，通常通过腹腔或皮下途径给药，α2-肾上腺素能激动剂的剂量也在上述范围内。
美托咪定及相关α2受体激动剂在小哺乳动物和其他物种中已知的不良反应包括剂量依赖性心动过缓、心输出量减少、外周血管收缩伴体循环血管阻力增加，以及潜在的呼吸抑制，特别是在较高剂量或与其他药物联合使用时。在犬和其他接受α2受体激动剂的物种中，也有胃肠道副作用如呕吐、流涎或运动性改变的报道。
犬中α2受体激动剂的给药途径可调节这些心血管、呼吸和胃肠道效应的起效、程度和临床表现。静脉给药与快速起效和更急剧的血流动力学变化相关，而肌肉注射和皮下途径通常产生较慢的起效和更平稳的心血管特征，但可能因血管收缩而引起局部疼痛或组织刺激。例如，肌肉注射右美托咪定显示吸收可变，峰值效应延迟出现在给药后约10-20分钟，而静脉注射后起效约1分钟，这一差异归因于局部血管收缩和组织灌注效应。
皮下、肌肉和腹腔途径是实验室动物给予镇静剂最常用的途径。然而，近年来在人类和动物中的研究探索了药物给药的替代方法。在这一意义上，“药针疗法”——一种在穴位处皮下给予低剂量药物的技术——已逐渐获得认可。据推测，使用药针疗法可能允许减少通过其他途径通常所需的剂量，同时保持疗效并尽量减少副作用的发生。在兽医学中最受关注的穴位之一是督脉20（GV20）穴，传统东方医学已对其在人类和动物中的治疗特性进行了研究。
GV20穴位位于颅骨背侧中线与连接头顶耳基部前缘的连线的交点处。人类和犬的研究表明，当该穴位通过针灸、电针或激光刺激时，可产生镇静和安定作用。其作用机制尚不清楚；然而，一些作者提出穴位可能作为药物的入口，促进其吸收并增强其治疗效果。
犬的研究表明，在GV20穴位皮下注射右美托咪定优于静脉注射，可最大限度减少不良反应并提高安全性。此外，与肌肉注射途径相比，观察到镇静效果持续时间更长、疗效更高，表明在GV20穴位给药可作为犬镇静的有效选择。这些研究使用了临床标准剂量的右美托咪定而非减量，以初步确立该技术的可行性，将潜在剂量节省效果的评估留待后续研究。近期鸟类研究显示，与肌肉注射途径相比，在GV20穴位应用镇静药物需要更低剂量、缩短起效时间并提高镇静程度。这些发现表明，在该穴位给予镇静药物可应用于日常实践，便于管理、减少焦虑和应激并改善动物福利。
在豚鼠中，皮下（GV20穴位）与肌肉注射α2受体激动剂的镇静效果比较尚未被研究；因此，尚不清楚该途径是否具有与其他物种报道的相似优势。选择豚鼠作为实验动物是基于与其他实验啮齿动物相比，该物种镇静和麻醉方面的现有文献相对有限。此外，大多数已发表的关于GV20穴位药物注射的研究是在犬、猫、马和鸟类中进行的。本研究假设，与肌肉注射相比，在GV20穴位皮下注射美托咪定可缩短起效时间并提高镇静程度和镇静效果持续时间。
本研究旨在比较成年豚鼠在GV20穴位给予美托咪定（100 µg/kg）与肌肉注射给药所达到的最大镇静评分、起效时间和作用持续时间。
材料与方法
伦理审查。
本研究经布宜诺斯艾利斯大学兽医学院IACUC批准（编号2024/29）。
研究设计。
在20只动物中进行了一项前瞻性、盲法、随机、交叉实验研究。每只动物以随机分配的顺序接受两种处理（GV20穴位皮下注射和肌肉注射），间隔2周洗脱期。
样本量。
基于对10只动物进行的预试验（未发表数据）收集的数据，使用G*Power软件估算样本量为20只豚鼠。用于计算所需样本量的主要结局指标为作用持续时间（肌肉注射组和GV20组分别为均值79.7和68.7分钟，标准差13.99和14.41分钟），检验效能为90%，α值为0.05。
纳入和排除标准。
仅纳入经繁殖设施证明健康的动物。如果怀疑注射位置错误（即在GV20穴位区域之外给药），该动物的数据将从分析中排除。本研究中无动物被排除（n = 20）。
随机化。
处理根据区组随机化方法分配，使用免费Randomizer工具（https://www.randomized.org/）。为尽量减少潜在混杂变量，动物在研究期间单独饲养于独立笼中，并使用无毒永久标记笔清晰标识。
盲法。
给药始终由同一研究人员进行，由2名负责保定和束缚的研究人员协助。一名对处理不知情的独立研究人员进行镇静评分评估，由另一名研究人员协助记录数据。此外，所有参与分组分配和药物给药的研究人员在评估阶段均不在场。
结局指标。
在注射美托咪定前后，使用先前发表的量表评估镇静程度，该量表评估5个特定方面：运动、肌张力、对操作的 реакция、姿势和翻正反射。通过将这些变量的值相加，得出0至19分的评分，其中0表示无镇静或正常行为，19反映最大镇静水平。此外，根据同一评估工具，记录起效时间（预先定义为达到19分中>3分所需的时间）以及作用持续时间（定义为恢复到19分中≤3分所需的时间）。该阈值是基于该量表此前的应用以及初步预试验观察结果而确定的。
统计学方法。
数据使用 GraphPad Prism 软件（版本 10.1.1）在 macOS 系统（GraphPad Software，圣迭戈，加利福尼亚州）上进行分析。由于所分析的变量为离散型数据，数据按非正态分布处理。结果以中位数和四分位距（Q3 − Q1）表示。最大镇静程度、起效时间和作用持续时间在不同给药途径之间的比较采用配对样本的 Wilcoxon 符号秩检验。统计显著性设定为 P < 0.05。
实验动物。
共使用 20 只成年雌性 Hartley 豚鼠，体重 952 ± 149 g（平均值 ± 标准差），年龄 1 至 2 岁，原 enrolled 于一项无关研究。繁育设施（阿根廷布宜诺斯艾利斯国家实验室与卫生研究所“Dr. Carlos G. Malbrán”）对动物的健康状况进行了认证。作者不了解繁育设施常规开展哪些检测来评估动物健康状况。实验对象饲养于布宜诺斯艾利斯大学兽医学院麻醉科实验室，使用尺寸为 80 × 50 × 43 cm 的塑料笼具（Savic，科特赖克，比利时），配备栅栏顶盖。在适应期及操作之间的常规饲养期间，豚鼠按照其社交需求和机构指南以每笼最多 3 只的方式群养。每次镇静会话时，为便于交叉设计中的个体识别、操作和数据采集，动物在给予镇静剂前后立即暂时单笼饲养于其原笼中。该暂时单笼饲养时间不超过每次会话中镇静、评分和恢复所需的时间。一旦达到恢复标准且动物恢复至基线行为，即将其放回群养笼中。垫料为高压灭菌松木刨花。室温维持在 19 至 23 °C，采用 12 小时光照/12 小时黑暗周期，标准日间工作时间内开灯，相对湿度保持在约 40%-60%。饮食包括专为豚鼠配制的颗粒饲料（Ganave，Grupo Pilar，布宜诺斯艾利斯，阿根廷）、苜蓿干草和蔬菜，并提供添加抗坏血酸的过滤水自由饮用。
实验操作。
研究在 2 周适应期后开始进行。操作室保持安静条件，限制人员流动，且不同时进行其他操作。通过保持门关闭并在评分期间限制走动和交谈，最大限度减少突发的听觉和视觉干扰。
动物在研究开始前禁食 2 小时，并在镇静前数小时移除新鲜蔬菜。该方法符合豚鼠及其他小型食草动物的实验动物麻醉建议，即通常不鼓励长时间禁食以降低肠梗阻风险。
每只豚鼠接受 0.05% 右美托咪定溶液（Detor，Richmond Vet Pharma，布宜诺斯艾利斯，阿根廷），剂量为 100 µg/kg，总注射体积为 0.2 mL/kg，使用 1 mL 低死腔注射器和 25 号、⅝ 英寸针头给药，或经半膜肌肌肉注射（肌肉注射组），或于 GV20 穴位皮下注射（GV20 组）（图 1）。选择 100 µg/kg 的右美托咪定剂量是基于现有实验动物中 α2 受体激动剂使用建议，该建议指出此范围可产生可靠的中度镇静。尽管有建议豚鼠可使用高达 500 µg/kg 的剂量，但通用实验动物麻醉教材列出的右美托咪定剂量在小哺乳动物（包括啮齿类及其他小型物种）中为 30 至 100 µg/kg 皮下注射，更高剂量主要延长镇静时间而非显著增加镇静深度。因此，选择 100 µg/kg 剂量以确保两种途径下所有动物均达到中度镇静，并为比较 GV20 与肌肉注射之间的起效时间和持续时间提供 adequate 范围。
[图表内容请查看原文]
两次给药之间间隔 2 周洗脱期。所有操作均在光照期进行，处于相对狭窄且一致的日间工作窗口内（上午 8：00 至下午 1：00）。每只豚鼠的两次镇静给药均安排在可比的时间范围内，以尽量减少昼夜节律变异。由 3 名研究者组成的团队负责保定和注射操作，而由一名盲法评估者评估研究结果。动物由 2 名熟悉豚鼠操作的研究人员轻柔手动保定，采用胸部和骨盆支撑相结合的方式，以尽量减少挣扎并防止过度颈部或脊柱屈曲。对于肌肉注射，目标为半膜肌，需要适度后肢伸展并短暂稳定骨盆肢。对于 GV20 注射，按照其他物种中 GV20 的解剖定位描述，将动物头部稳定于中立位置，同时触诊颅骨背中线及连接耳基前缘的连线以确定穴位。所有注射均由同一名经验丰富的操作者完成，由 2 名助手协助，以尽量减少保定的和给药的变异性。
在每次给药前，使用先前建立的量表评估镇静程度。给药后，在给药后的前10分钟内以1分钟间隔应用该量表，随后以2分钟间隔直至30分钟，最后以5分钟间隔直至动物的评分恢复至19分中的3分或更低（终点）。记录每种给药途径的以下参数：镇静深度、起效时间和作用持续时间。研究期间连续观察动物的呼吸模式和黏膜颜色。
结果
未观察到临床明显的局部或全身不良反应，包括组织反应，如肿胀、红斑或溃疡。未观察到可见的发绀、呼吸暂停或持续性异常呼吸模式发作，豚鼠均顺利恢复，无明显并发症。
所有动物及其完整的个体数据集均纳入分析，每组共20个数据集。最大镇静评分不服从正态分布。关于观察到的镇静效果，肌肉注射组最大镇静评分中位数为11.0，四分位距为3.75（Q3 − Q1 = 13.0-9.25），而GV20组中位数为12.0，四分位距为3.75（Q3 − Q1 = 13.0-9.25）（图2）。肌肉注射组起效时间中位数为7.5分钟，四分位距为7分钟（Q3 − Q1 = 10-3），GV20组为5.5分钟（四分位距 = 5.5分钟，Q3 − Q1 = 9.5-4.0）（图3）。关于作用持续时间，肌肉注射组中位数为86分钟（四分位距 = 23.5分钟；Q3 − Q1 = 95.75-72.25），而GV20组中位数为82.5分钟（四分位距 = 26.75分钟；Q3 − Q1 = 89.0-62.25）（图4）。Wilcoxon检验结果显示，两种给药途径在最大评分（P = 0.3237）、起效时间（P = 0.5072）和作用持续时间（P = 0.0648）方面均无显著差异。
[图表内容请查看原文]
[图表内容请查看原文]
[图表内容请查看原文]
讨论
本研究旨在比较美托咪定（100 μg/kg）在豚鼠GV20穴位皮下注射与肌肉注射的最大镇静评分、起效时间和作用持续时间。结果表明，豚鼠GV20穴位给予美托咪定的镇静特征与肌肉注射途径无显著差异。
这一结果与其他物种先前发表的结果形成对比，后者提示采用药物针刺时可改善镇静评分、起效时间、作用持续时间或减少副作用。药物针刺已被提出作为兽医学中增强疗效和/或减少镇静药物剂量的替代方法，可能有助于减少副作用。在本研究中，两种途径刻意使用相同的美托咪定剂量（100 µg/kg），以便对由途径本身引起的镇静特征进行受控比较，而不受剂量影响。这一设计选择遵循了其他物种多项药物针刺研究的方法，即初步比较时在不同途径使用等效剂量以建立基线等效性或优越性，然后再测试减量方案。在豚鼠中，GV20穴位给药可能还具有比静脉给药更易操作的额外优势。
犬类研究表明，与等量肌肉注射相比，GV20穴位皮下注射右美托咪定可提高镇静和镇痛评分并延长作用持续时间，且起效更快。此外，与静脉途径相比，GV20给药后观察到的不良事件更少。同样，在鸟类（蓝额亚马逊鹦鹉）中，GV20穴位半剂量氯胺酮-咪达唑仑药物针刺达到了与全剂量肌肉注射相同的镇静程度，且恢复更快、更平稳。作者认为，针刺与镇静剂的结合可能是设计鸟类更安全麻醉方案的有用工具。这些在不同物种和药物（α2受体激动剂及其他药物）中的一致发现凸显了药物针刺增强药物疗效或减少副作用的潜力。
另一方面，某些研究报告的发现与本 study 结果一致。在猫中，GV1穴位右美托咪定药物针刺增加了热痛觉缺失，但与相同剂量的肌肉注射途径相比，在镇静或肌肉松弛评分方面未产生显著差异。同样，最近一项在猫中比较GV20穴位邻近部位皮下注射右美托咪定与肌肉注射的研究报告了相当的镇静水平。
在最近一项在小鼠中进行的研究中，比较了皮下肩胛间途径、GV20穴位和腹腔注射氯胺酮/赛拉嗪镇静-麻醉复合制剂的效果。结果显示，与其他2种评估途径相比，在GV20部位给予该药物组合并无显著优势。这些发现与本研究的结果一致。基于现有证据，如其他物种所报道，在啮齿动物中GV20部位与其他给药途径之间未显示出显著差异。
有几个因素可以解释为何在豚鼠中于GV20穴位给予美托咪定未显示出优于肌内注射的显著优势。物种间的解剖和生理差异可能影响药物吸收和分布。通过红外成像评估，在猫中观察到GV20穴位处皮下组织的灌注高于肩胛间皮下区域。作者提出，这种灌注增加可能是猫在GV20部位给予镇静剂时药物吸收增强的原因。据我们所知，尚无研究评估豚鼠皮下血管分布的区域差异，可能在该物种中GV20处的灌注模式与其他皮下区域无显著差异，从而限制了任何穴位特异性的药代动力学优势。此外，吸收速率或局部血流的细微差异可能不会转化为镇静评分上有临床意义的变化。在小鼠中的发现——GV20皮下注射氯胺酮-赛拉嗪并未较肩胛间皮下或腹腔途径带来明显优势——支持了GV20药物穴位注射的潜在益处可能具有物种或剂量依赖性的观点。此外，所使用的镇静量表虽然此前曾应用于豚鼠，但尚未经过正式验证，对于检测不同途径之间在起效或峰值效应上的细微差异可能敏感性有限，尤其是在较高镇静水平时。缺乏额外的行为学测量（例如特异性反射测试）以及缺乏生理监测可能降低了检测微小途径依赖性差异的能力。
药物穴位注射的确切机制仍不清楚。一些作者提出，穴位可能作为药物的“门户”，促进其吸收并增强其治疗效果。已显示GV20穴位刺激可影响人类的大脑活动，在大鼠中也已证明可降低大脑皮质的兴奋性。GV20穴位刺激可通过增加α波段功率和降低β波段功率来影响大脑活动，提示具有“情绪调节性放松效应”。α2肾上腺素能激动剂，如美托咪定，通过激活脑桥蓝斑中的α2a受体、阻断去甲肾上腺素释放并调节自主神经系统而产生镇静作用。有研究提出，在犬GV20穴位施针可能至少部分通过激活α2肾上腺素能系统而诱导镇静效应，支持证据显示镇静反应可被阿替美唑拮抗，而纳洛酮未能逆转该效应。
药物穴位注射已被提出作为增强兽医学中镇静药物吸收和疗效的可行替代方案。然而，在豚鼠给予美托咪定的具体案例中，结果表明肌内注射产生的镇静特征与GV20穴位皮下注射所观察到的相当。这些发现强调了根据所研究物种的具体生理和行为特征来验证每种给药途径的重要性。
本研究存在一定局限性。首先，未评估非GV20皮下注射部位，这限制了对任何差异究竟源于穴位特异性特性还是更普遍地源于皮下给药这一问题的结论。未来研究可增加另一皮下给药部位（即肩胛间皮下）以确定任何获益是与穴位相关还是与皮下血流或组织结构的差异相关。此外，尽管所用镇静评分量表此前已有描述，但尚未经过正式验证。而且，该镇静量表未包含特定的面部或口腔参数。啮齿类动物面部表情量表及基于面部的疼痛评分提示面部和口腔特征可能是感觉与情感状态改变的敏感指标。因此，未来在豚鼠中进行的研究可纳入更多项目，包括对口腔撤回反射或面部反应的标准化评估，以增强该物种镇静评估工具的敏感性和结构效度。本研究仅纳入雌性受试动物，且未监测血流动力学和呼吸变量等生理参数，因而无法评估治疗相关的不良反应发生率差异。此外，尽管穴位注射通常与不良副作用减少和总体药物用量降低相关，但本研究未专门评估这些方面，主要关注镇静疗效、起效时间和持续时间。在犬中，某些评估GV20穴位注射的研究在首次确立该技术可行性时也使用了临床标准剂量的右美托咪定而非减量，将剂量缩减评估留待后续研究。本研究中缺乏显著差异并不能排除剂量缩减获益的可能性，但在本研究特定条件（剂量和量表）下未证实此类获益。可开展进一步研究，探索美托咪定的较低（亚临床）剂量，或评估生理参数和副作用，以阐明GV20穴位注射在豚鼠中的潜在获益。
未对心血管和呼吸参数进行定量监测，因此无法评估不同给药途径之间心肺抑制的潜在差异。未观察到明显的心肺功能受损或恢复延迟的临床体征，但这不能排除轻度或亚临床效应，且缺乏定量心肺数据无法对细微的心血管或呼吸抑制做出明确评估。未来研究可纳入基本生理监测，并考虑供氧的可能性以提高安全性。
总体而言，两种给药途径下动物在进针时均表现出轻微活动。尽管研究者的主观印象认为GV20穴位给药更容易，且注射后可观察到的反应相对较低，但未测量应激的客观指标（例如皮质酮浓度或心率变异性）以评估GV20与肌内给药之间的潜在差异，这是本研究的一个局限性。然而，在不宜反复进行后肢注射或头部操作比骨盆肌肉操作更可行的临床或实验环境中（例如患有肌肉骨骼或后肢病变的动物），GV20穴位给药可能具有实际优势。比较小鼠皮下与腹腔注射氯胺酮-赛拉嗪方案的研究表明，背侧皮下给药可与腹腔注射同样有效，同时避免穿透腹腔脏器。这些发现支持以下总体概念：当背部操作更可行或应尽量减少肢体操作时，包括GV20穴位在内的背侧注射部位可能具有实际优势。然而，本研究未对这些实际获益进行专门量化。
本模型中没有证实GV20的优势，并不排除在不同剂量、其他药物组合或使用更敏感结局指标时可能存在获益，但表明在所测试的特定条件下未观察到优于肌内给药的效果。尽管本研究未显示GV20在任何评估变量上优于肌内给药，但对该穴位的穴位注射进一步研究，特别是在给药便利性及其对动物行为与福利的影响方面，可能支持其作为比肌内、腹腔或静脉注射创伤更小且可能应激更小的镇静给药替代途径。需要开展纳入应激相关结局指标的进一步研究，以扩展对GV20穴位注射在临床和实验环境中实施可行性的评估。
结论
在豚鼠GV20穴位给予右美托咪定，与肌肉注射途径相比，在最大镇静评分、起效时间或作用持续时间方面均未产生统计学显著差异。这些研究结果表明，对于豚鼠使用右美托咪定，在其他物种中于GV20穴位采用药物针灸所观察到的镇静效果和作用持续时间的增强，在本研究条件下未能得到证实。
利益冲突
作者无利益冲突需要披露。
资助
本工作由布宜诺斯艾利斯大学资助，项目编号UBACyT 20020220100199BA。
数据可用性
原始数据可在 https://doi.org/10.6084/m9.figshare.30609662 获取。
查看英文原文▾
Abstract
Recent research in both humans and animals has explored alternative routes for drug administration, such as pharmacopuncture, a technique involving the subcutaneous administration of low drug doses at acupuncture points. Among these points, the Governing Vessel 20 (GV20) has attracted particular interest in veterinary medicine due to its reported tranquilizing and sedative effects in humans and dogs. It has been proposed that administering sedative agents at this site may potentiate their effects, allowing for dose reduction while maintaining efficacy and minimizing adverse reactions. This study aimed to compare the sedative effects of medetomidine administered at the GV20 acupoint compared with the intramuscular route in adult guinea pigs ( Cavia porcellus ). Twenty animals received medetomidine (100 µg/kg) via both routes in a crossover design with a 2-week washout period. Sedation was assessed using a previously published scale (0-19). Sedation onset was defined as the time to achieve a score >3 of 19, and duration as the time to return to ≤3 of 19. Assessments were performed before and after administration at 1-minute intervals for the first 10 minutes, then every 2 minutes up to 30 minutes, and every 5 minutes thereafter until recovery. Comparisons of maximum sedation score, onset, and duration between routes were analyzed using the Wilcoxon signed-rank test. No significant differences were found in the maximum score, time to onset, or duration of sedation between the GV20 and intramuscular routes. These results indicate that, under the conditions of this study, medetomidine administration at GV20 did not enhance sedative efficacy or prolong the duration of action compared with the intramuscular route.
Abbreviation and Acronym: GV20, Governing Vessel 20
Introduction
Laboratory animals frequently require the administration of sedatives to minimize the anxiety and stress produced by handling or by maneuvers performed in an experimental model. Therefore, developing effective sedation strategies that reduce stress, facilitate handling, and promote animal welfare is of particular interest. Guinea pigs ( Cavia porcellus ) are increasingly used as laboratory and companion animals, and they frequently require reliable and safe sedation for procedures such as imaging, blood sampling, and minor interventions.
Currently, a wide range of drugs and combinations are available for the sedation of laboratory animals, among which α 2 agonists such as xylazine, medetomidine, and dexmedetomidine, known for their sedative, myorelaxant, and analgesic properties, stand out. Guidelines for laboratory animal anesthesia and comparative anesthesia reviews report medetomidine doses of 30-100 µg/kg by the subcutaneous or the intramuscular route, and dexmedetomidine doses of 15-50 µg/kg in small mammals and indicate that α 2 -adrenergic agonists are frequently administered in combination with ketamine or opioids to enhance sedation or to induce anesthesia. In certain rodent species, including rats and mice, injectable anesthetic protocols commonly rely on ketamine–xylazine or ketamine–medetomidine combinations, typically administered via intraperitoneal or subcutaneous routes, with α 2 -adrenergic agonist doses also within the mentioned range.
The known adverse effects of medetomidine and related α 2 agonists in small mammals and other species include dose-dependent bradycardia, reductions in cardiac output, peripheral vasoconstriction with increased systemic vascular resistance, and potential respiratory depression, particularly at higher doses or when used in combination with other agents. Gastrointestinal side effects such as vomiting, ptyalism, or altered motility have also been reported in dogs and other species receiving α 2 agonists.
The route of administration of α 2 agonists in dogs can modulate the onset, magnitude, and clinical expression of these cardiovascular, respiratory, and gastrointestinal effects. Intravenous administration is associated with rapid onset and more abrupt hemodynamic changes, whereas the intramuscular and subcutaneous routes generally produce slower onset and smoother cardiovascular profiles but may cause local pain or tissue irritation due to vasoconstriction. For instance, intramuscular dexmedetomidine shows variable absorption and a delayed peak effect occurring ∼10-20 minutes after administration, compared with an onset of ∼1 minute following intravenous injection, a difference attributed to local vasoconstriction and tissue perfusion effects.
The subcutaneous, intramuscular, and intraperitoneal routes are the most commonly used for administering sedatives in laboratory animals. However, recent research in humans and animals have explored alternatives for drug administration. In this sense, ‘pharmacopuncture,’ a technique of administering drugs in low doses subcutaneously at acupuncture points, has gained ground. It has been postulated that the use of pharmacopuncture may allow for a reduction in the doses typically required via other routes, while maintaining efficacy and minimizing the occurrence of side effects. Among the acupuncture points that have aroused the most interest in veterinary medicine is the Governing Vessel 20 (GV20) point, which has been studied in traditional oriental medicine for its therapeutic properties in humans and animals.
The GV20 point is located at the intersection between the dorsal midline of the skull and a line joining the rostral edge of the ears’ base at the top of the head. Studies in humans and dogs show that when this point is stimulated by acupuncture, electro acupuncture, or laser, it generates tranquilization and sedation. The mechanism of action remains unknown; however, some authors propose that acupuncture points may act as gateways for drugs, facilitating their absorption and enhancing their therapeutic effects.
Studies in dogs suggest that subcutaneous administration of dexmedetomidine at the GV20 acupoint offers advantages over intravenous administration by minimizing adverse effects and improving safety. In addition, a longer duration and higher efficacy of the sedative effect compared with the intramuscular route was observed, suggesting that administration at the GV20 acupoint can be an effective option for sedation in dogs. These studies used dexmedetomidine at clinical standard doses, rather than reduced doses, to initially establish the feasibility of the technique, reserving evaluation of potential dose-sparing effects for subsequent investigations. Recent studies in birds showed that the application of sedative drugs at the GV20 acupoint required a lower dosage, reduced the onset, and increased the degree of sedation compared with the intramuscular route. These findings show that the administration of sedatives at this point could be implemented in daily practice, facilitating management, reducing anxiety and stress, and improving animal welfare.
The comparative sedative effect of subcutaneous (at the GV20 acupoint) compared with intramuscular administration of α 2 agonists remains uninvestigated in guinea pigs; consequently, it is unknown whether this route offers similar advantages to those reported in other species. The choice of guinea pigs as experimental animals was based on the relatively limited amount of available literature on sedation and anesthesia in this species compared with other experimental rodents. Furthermore, most of the published studies on pharmacopuncture at GV20 have been conducted in dogs, cats, horses, and avian species. The present work hypothesizes that medetomidine administered subcutaneously at the GV20 acupoint reduces the onset and increases the degree of sedation and the duration of the sedative effect compared with intramuscular administration.
The present study aimed to compare the maximum sedation score achieved, the onset, and the duration of action of medetomidine (100 µg/kg) administered at the GV20 acupoint compared with its intramuscular administration in adult guinea pigs.
Materials and Methods
Ethical review.
This study was approved by the IACUC of the Veterinary Sciences School, University of Buenos Aires (no. 2024/29).
Study design.
A prospective, blinded, randomized, crossover experimental study was conducted in 20 animals. Each animal received both treatments (a subcutaneous injection at the GV20 acupoint and an intramuscular injection) in a randomly assigned order, separated by a 2-week washout period.
Sample size.
A sample size of 20 guinea pigs was estimated using G*Power software based on data collected from a pilot study (unpublished data) conducted on 10 animals. The main outcome used to calculate the required sample size was the duration of action (mean 79.7 and 68.7 minutes, SD 13.99 and 14.41 minutes, for group intramuscular and GV20, respectively), with a power of 90% and a value of 0.05.
Inclusion and exclusion criteria.
Only animals in good health, as certified by the breeding facility, were included in the study. If an injection was suspected to have been misplaced (that is, administered outside the GV20 acupoint area), the data from that animal would be excluded from the analysis. There were no exclusions of animals in this study (n = 20).
Randomization.
The treatments were assigned according to a block randomization method, using the free Randomizer tool ( https://www.randomized.org/ ). To minimize potential confounding variables, the animals were individually housed in separate cages and clearly identified during the study using a nontoxic permanent marker.
Blinding.
Administration was consistently conducted by the same researcher, who was assisted by 2 investigators responsible for handling and restraint. An independent researcher, blinded to the treatment, conducted sedation score assessments, assisted by another researcher who recorded the data. Furthermore, all researchers involved in group allocation and drug administration were absent during the assessment phase.
Outcome measures.
Before and after the injection of medetomidine, the degree of sedation was assessed using a previously published scale that evaluates 5 specific aspects: movement, body tone, reaction to manipulation, posture, and righting reflex. By summing the values of these variables, a score ranging from 0 to 19 was assigned, where 0 indicates no sedation or normal behavior and 19 reflects the maximum level of sedation. In addition, according to the same assessment tool, the onset, a priori defined as the time required to achieve a score >3 of 19, was recorded, as well as the duration of action, defined as the time required to return to a score of ≤ 3 of 19. This threshold was established based on prior application of the scale, together with findings from preliminary pilot observations.
Statistical methods.
Data were analyzed using GraphPad Prism software (version 10.1.1) for macOS (GraphPad Software, San Diego, CA). The data were treated as nonnormally distributed, as the analyzed variables were discrete. Results are presented as median and IQR (Q3 − Q1). Comparisons between routes of administration for the maximum degree of sedation, onset, and duration of action were performed using the Wilcoxon signed-rank test for paired samples. Statistical significance was set at P < 0.05.
Experimental animals.
A total of 20 adult female Hartley guinea pigs, 952 ± 149 g body weight (mean ± SD) and aged between 1 and 2 years, enrolled in an unrelated study, were used. The breeding facility (National Administration of Laboratories and Health Institutes “Dr. Carlos G. Malbrán”, Buenos Aires, Argentina) certified the health status of the animals. The authors are not aware of which tests are routinely conducted by the breeding facility to assess the animals’ health status. The subjects were housed at the Anesthesia Department Laboratory of the Veterinary Sciences School, University of Buenos Aires within plastic cages measuring 80 × 50 × 43 cm (Savic, Kortrijk, Belgium), equipped with a grill top enclosure. During acclimatization and routine housing between procedures, guinea pigs were kept in groups of up to 3 animals per cage, in accordance with their social needs and institutional guidelines. For each sedation session, animals were temporarily housed singly in their original cages immediately before and after sedative administration to facilitate individual identification, handling, and data collection in the crossover design. This temporary single housing did not exceed the time required for sedation, scoring, and recovery during each session. As soon as recovery criteria were met and the animal had returned to baseline behavior, it was reintroduced to its group cage. The substrate consisted of autoclaved pine shavings. The room temperature was maintained between 19 and 23 °C, with a 12-hour light/12-hour dark cycle, with lights on during standard daytime working hours, and relative humidity was kept at ∼40%-60%. Their diet included pellets specially formulated for guinea pigs (Ganave, Grupo Pilar, Buenos Aires, Argentina), alfalfa hay and vegetables, with filtered water supplemented with ascorbic acid provided ad libitum.
Experimental procedures.
The study was conducted after a 2-week acclimatization period. The procedure room was maintained under quiet conditions with restricted personnel traffic, and no additional procedures were performed concurrently. Sudden auditory and visual disturbances were minimized by keeping doors closed and limiting movement and conversation during scoring periods.
Animals were fasted for 2 hours prior to the beginning of the study, and fresh vegetables were removed a few hours before sedation. This approach is aligned with laboratory animal anesthesia recommendations for guinea pigs and other small herbivores, which generally discourage prolonged fasting to reduce the risk of ileus.
Each guinea pig received a 0.05% medetomidine solution (Detor, Richmond Vet Pharma, Buenos Aires, Argentina) at a dose of 100 µg/kg, resulting in a total injection volume of 0.2 mL/kg, administered with 1 mL low–dead space syringes and 25-gauge, ⅝-in. needles, either intramuscularly into the semimembranosus muscle (intramuscular group) or subcutaneously at the GV20 acupoint (GV20 group) ( Figure 1 ). A medetomidine dose of 100 µg/kg was chosen based on available recommendations for α 2 agonist use in laboratory animals indicating that this range produces reliable moderate sedation. Although doses up to 500 µg/kg were suggested for guinea pigs, general laboratory animal anesthesia texts list medetomidine doses between 30 and 100 µg/kg SC in small mammals, including rodents and other small species, with higher doses primarily prolonging sedation rather than substantially increasing depth. Therefore, a 100 µg/kg dose was selected to ensure moderate sedation in all animals in both routes and provide an adequate range for comparing onset and duration between GV20 and intramuscular administration.
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The 2 administrations were separated by a 2-week washout period. All procedures were conducted during the light phase, within a relatively narrow, consistent daytime working window (8:00 am -1:00 pm ). Both sedative administrations in each guinea pig were scheduled within a comparable time range to minimize circadian variability. A team of 3 investigators was responsible for the restraint and injection procedures, while a blinded evaluator assessed the outcomes of the study. Animals were gently restrained manually by 2 researchers, familiar with guinea pig handling, using a combination of thoracic and pelvic support to minimize struggling and prevent excessive neck or spinal flexion. For intramuscular injections, the semimembranosus muscle was targeted, requiring moderate hindlimb extension and brief stabilization of the pelvic limb. For GV20 injections, the animal’s head was stabilized in a neutral position while the dorsal midline of the skull and a line joining the rostral edge of the ear bases were palpated to identify the acupoint, following descriptions of GV20 topography in other species. All injections were performed by the same experienced operator, assisted by 2 handlers, to minimize variability in restraint and drug administration.
Before each treatment was administered, the degree of sedation was evaluated using a previously established scale. This scale was then applied after drug administration at 1-minute intervals for the first 10 minutes after administration, followed by 2-minute intervals until 30 minutes, and finally, 5-minute intervals until the animal’s score returned to 3 of 19 or lower (endpoint). The following parameters were recorded for each administration route: depth of sedation, time to onset, and duration of action. Animals were observed continuously during the study for respiratory pattern and mucous membrane color.
Results
No clinically evident local or systemic adverse effects were observed, including tissue reactions, such as swelling, erythema, or ulceration. No episodes of visible cyanosis, apnea, or prolonged abnormal respiratory pattern were observed, and guinea pigs recovered uneventfully without apparent complications.
All animals and their complete individual datasets were included in the analysis, resulting in a total of 20 datasets per group. Maximum sedation score did not follow a normal distribution. Regarding the observed sedative effect, the maximum sedation score reached a median of 11.0 in the intramuscular group, with an IQR of 3.75 (Q3 − Q1 = 13.0-9.25) while the GV20 group achieved a median of 12.0, with an IQR of 3.75 (Q3 − Q1 = 13.0-9.25) ( Figure 2 ). The median onset of action was 7.5 minutes with an IQR of 7 minutes (Q3 − Q1 = 10-3) for the intramuscular group and 5.5 minutes (IQR = 5.5 minutes, Q3 − Q1 = 9.5-4.0) for the GV20 group ( Figure 3 ). Concerning the duration of action, the intramuscular group presented a median of 86 minutes (IQR = 23.5 minutes; Q3 − Q1 = 95.75-72.25), while the GV20 group showed a median of 82.5 minutes (IQR = 26.75 minutes; Q3 − Q1 = 89.0-62.25) ( Figure 4 ). The results obtained from the Wilcoxon test for the maximum score achieved ( P = 0.3237), as well as for the time to onset ( P = 0.5072) and the duration of action ( P = 0.0648), showed no significant differences between the 2 routes of administration.
[图表内容请查看原文]
[图表内容请查看原文]
[图表内容请查看原文]
Discussion
The present study aimed to compare the maximum sedation score, onset, and duration of action of medetomidine (100 μg/kg) administered subcutaneously at the GV20 acupoint and intramuscularly in guinea pigs. The results suggest that the sedative profile of medetomidine administered at the GV20 acupoint in guinea pigs is not significantly different from that obtained via the intramuscular route.
This outcome contrasts with previously published results in other species, suggesting improvement in sedative scores, onset, duration, or reduction in side effects when pharmacopuncture is employed. Pharmacopuncture has been proposed as an alternative to enhance the efficacy and/or reduce the dose of sedative drugs in veterinary medicine, potentially leading to a decrease in side effects. In the present study, the same medetomidine dose (100 µg/kg) was deliberately used for both routes to allow a controlled comparison of the sedative profile attributable to the route itself, independent of dose. This design choice follows the approach used in several pharmacopuncture studies in other species, where an initial comparison uses equivalent doses across routes to establish baseline equivalence or superiority before testing dose-reduction protocols. In guinea pigs, the administration at the GV20 acupoint might have the added benefit of being a more easily accessible route than intravenous administration.
Studies in dogs have shown that dexmedetomidine administered subcutaneously at the GV20 acupoint increased the sedative and analgesic scores and duration compared with an equal intramuscular dose, and it also showed a faster onset. In addition, fewer adverse events were observed after GV20 administration compared with the intravenous route. Similarly, in birds ( Amazona aestiva ), half-dose ketamine-midazolam pharmacopuncture at the GV20 acupoint achieved the same degree of sedation as a full intramuscular dose, with faster and more stable recovery. Authors suggest that the combination of acupuncture and sedatives might be a useful tool to design safer anesthetic protocols in avian species. These consistent findings across different species and drugs (α 2 agonists and others) highlight the potential of pharmacopuncture to enhance drug efficacy or reduce side effects.
On the other hand, certain studies have reported findings that align with the results of the present study. In cats, dexmedetomidine pharmacopuncture at the GV1 acupoint increased thermal antinociception but did not produce significant differences in sedation or muscle relaxation scores compared with the intramuscular route at the same dose. Similarly, a recent study in cats comparing subcutaneous dexmedetomidine administration at a site adjacent to the GV20 acupoint with intramuscular administration reported comparable levels of sedation.
In a recent study conducted in mice, the subcutaneous interscapular route, the GV20 acupuncture point, and the intraperitoneal administration of a ketamine/xylazine sedative–anesthetic combination were compared. The results showed no significant advantage of administering this drug combination at the GV20 site compared with the other 2 routes evaluated. These findings are consistent with those of the present study. Based on the available evidence, no significant differences have been demonstrated between the GV20 site and other administration routes in rodents, as reported in other species.
Several factors may explain why medetomidine administration at the GV20 acupoint did not show significant advantages over intramuscular injection in guinea pigs. Anatomic and physiologic differences between species may influence drug absorption and distribution. In cats a greater irrigation in the subcutaneous tissue at the GV20 point was observed compared with the interscapular subcutaneous region, as assessed by infrared imaging. The authors suggested that this increased perfusion could account for enhanced drug absorption when sedatives are administered at the GV20 site in cats. To the best of our knowledge, no studies have evaluated regional differences in subcutaneous vascularization in guinea pigs, it is possible that, in this species, the perfusion pattern at GV20 does not differ significantly from that of other subcutaneous regions, limiting any acupoint-specific pharmacokinetic advantage. In addition, subtle differences in absorption rate or local blood flow may not translate into clinically meaningful changes in the sedation score. Findings in mice, where GV20 subcutaneous injection of ketamine–xylazine did not confer a clear advantage over interscapular subcutaneous or intraperitoneal routes, support the idea that potential benefits of GV20 pharmacopuncture may be species- or dose-dependent. Moreover, the sedation scale used, while previously applied in guinea pigs, has not been formally validated and may have limited sensitivity for detecting subtle differences in onset or peak effect between routes, especially at higher sedation levels. The absence of additional behavioral measures (for example, specific reflex tests) and the lack of physiologic monitoring might have reduced the ability to detect small route-dependent differences.
The exact mechanism underlying pharmacopuncture remains unclear. Some authors have suggested that acupuncture points may act as “gateways” for drugs, facilitating their absorption and enhancing their therapeutic effects. Acupuncture stimulation at GV20 has been shown to affect brain activity in humans, and it has also demonstrated a reduction in the excitability of the brain cortex in rats. The GV20 acupuncture stimulation can influence brain activity by increasing α band power and decreasing β band power, indicative of a “mood-modifying relaxation effect.” Alpha-2 adrenergic agonists, such as medetomidine, produce sedation by activating α 2a receptors in the locus coeruleus of the pons, blocking norepinephrine release, and modulating the autonomic nervous system. It has been suggested that acupuncture applied at the GV20 acupoint in dogs may induce sedative effects, at least partially, through the activation of the α 2 adrenergic system, as supported by evidence showing that the sedative response can be antagonized by atipamezole, whereas naloxone failed to reverse the effect.
Pharmacopuncture has been proposed as a viable alternative to enhance the absorption and efficacy of sedative drugs in veterinary medicine. However, in the specific case of medetomidine administration in guinea pigs, the results indicate that intramuscular injection produces a sedation profile comparable to that observed with subcutaneous injection at the GV20 acupoint. These findings highlight the importance of validating each route of drug administration according to the specific physiologic and behavioral characteristics of the species under study.
This study presents certain limitations. First, a non-GV20 subcutaneous site was not evaluated, which limits conclusions about whether any differences are due to acupoint-specific properties or more generally to subcutaneous administration. Future work may add another subcutaneous administration (that is, interscapular subcutaneous) to determine whether any benefits are related to the acupoint or to differences in subcutaneous blood flow or tissue structure. In addition, although the sedation scale employed was previously described, it has not been formally validated. Moreover, the sedation scale does not include specific facial or oral cavity parameters. Rodent grimace scales and facial-based pain scoring evidence facial and oral features that might be sensitive indicators of altered sensory and affective states. Therefore, future studies in guinea pigs might incorporate additional items, including standardized evaluation of oral cavity withdrawal or facial responses, to enhance the sensitivity and construct validity of sedation assessment tools in this species. The study included only female subjects, and physiologic parameters such as hemodynamic and respiratory variables were not monitored, preventing the assessment of potential treatment-related differences in the incidence of adverse effects. In addition, while pharmacopuncture is generally associated with a reduction in undesirable side effects and overall drug consumption, this study did not specifically evaluate these aspects, focusing primarily on sedative efficacy, onset, and duration. In dogs, certain studies evaluating pharmacopuncture at the GV20 acupoint also used dexmedetomidine at clinical standard doses rather than reduced doses when first establishing the feasibility of the technique, reserving dose reduction evaluation for subsequent work. The absence of significant differences in the present study does not rule out the possibility of dose-reduction benefits, but such benefits were not demonstrated under the specific conditions (dose and scale) of this study. Further investigations, exploring lower (subclinical) doses of medetomidine, or assessing physiologic parameters and side effects, may be conducted to elucidate the potential benefits of GV20 pharmacopuncture in guinea pigs.
Cardiovascular and respiratory parameters were not quantitatively monitored, and therefore potential differences in cardiorespiratory depression between routes could not be assessed. No obvious clinical signs of cardiorespiratory compromise or delayed recovery were observed, but this does not exclude mild or subclinical effects, and the absence of quantitative cardiorespiratory data prevents a definitive assessment of subtle cardiovascular or respiratory depression. Future studies might incorporate basic physiologic monitoring and consider the possibility of oxygen supplementation to enhance safety.
In general, animals exhibited slight movement at the time of needle insertion with both administration routes. Although the researchers’ subjective impression suggested greater ease of administration at the GV20 point and a comparatively lower observable reaction following injection, no objective indicators of stress (for example, corticosterone concentrations or heart rate variability) were measured to assess potential differences between GV20 and intramuscular administration, which represents a limitation of the study. Nevertheless, administration at the GV20 point may potentially offer practical advantages in clinical or experimental settings in which repeated hindlimb injections are undesirable or when access to the head is more feasible than access to the pelvic musculature, for example, in animals with musculoskeletal or hindlimb pathology. Studies comparing subcutaneous and intraperitoneal ketamine–xylazine protocols in mice have shown that dorsal subcutaneous administration can be as effective as intraperitoneal injection while avoiding penetration of the abdominal viscera. These findings support the general concept that dorsal injection sites, including the GV20 site, may offer practical advantages when access to the back is more feasible or when limb manipulation should be minimized. However, such practical benefits have not been specifically quantified in this study.
The absence of demonstrated advantage of GV20 in this model does not exclude potential benefits at different doses, with other drug combinations, or when using more sensitive outcome measures, but indicates that no improvement over intramuscular administration was observed under the specific conditions tested. Although the present study did not show significant advantages of GV20 over intramuscular administration for any of the evaluated variables, further investigation of pharmacopuncture at this acupoint, particularly in terms of ease of administration and its impact on animal behavior and welfare, may support its use as a less invasive and potentially less stressful alternative route for sedative administration compared with intramuscular, intraperitoneal, or intravenous injection. Further work including stress-related outcome measures is needed to expand the assessment of the feasibility of implementing GV20 pharmacopuncture in clinical and experimental settings.
Conclusion
Medetomidine administration at the GV20 acupoint in guinea pigs did not result in statistically significant differences in maximum sedation score, onset of action, or duration of action compared with the intramuscular route. These findings suggest that for medetomidine in guinea pigs, the enhancements in sedative efficacy and duration observed with pharmacopuncture at the GV20 site in other species were not confirmed under the conditions of this study.
Conflict of Interest
The authors have no conflicts of interest to disclose.
Funding
This work was funded by the University of Buenos Aires, Project UBACyT 20020220100199BA.
Data availability
Raw data are available at https://doi.org/10.6084/m9.figshare.30609662 .