良好的试验动物给药和采血包括途径和体积规范指引.docx

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1、A Good Practice Guide to the Administration of Substances and Removal of Bloodjncluding Routes andVolumes良好的实验动物给药和采血(包括途径和体积)规范指南Karl-Heinz Diehl1, Robin Hull2, David Morton3, Rudolf Pfister4, Yvon Rabemampianina5,David Smith6,*, Jean-Marc Vidal7 and Cor van de Vorstenbosch 8Aventis, PO Box 1140, D

2、35001 Marburg, Germany德国马尔堡市35001区1140信箱安万特公司2N I B S C, Blanch Lane, South Miimms, Potters Bar, Hertfordshire EN6 3QG英国赫特福德郡EN6 3QG波特斯巴镇South Miimms布兰奇道英国国家生物制品检定所3The University of Birmingham, Medical School, Edgbaston, Birmingham B15 2TT英国伯明翰市B15 2TT艾吉马斯顿伯明翰大学医学院4Novartis Pharma AG, CH-4002 Basel

3、, Switzerland瑞士巴塞尔CH-4002诺华制药公司,Centre de Recherche Pfizer, Etablissement dAmboise, Z1 Poce-sur-Cisse-BP 159 37401 Amboise Cedex, France 法国 Amboise Cedex Z1 Poce-sur-Cisse-BP 159 37401 Etablissement dAmboise 辉瑞研究中心 6AstraZeneca R&D Chamwood, Bakewell Road, Loughborough, Leics LEI 1 5RH英国莱斯特郡LEI 1 5R

4、H拉夫堡市贝克韦尔路Charnwood阿斯利康研 究中心7Aventis, 102 Route de Noisy, 95235 Romainville Cedex, France法国 Romainville Cedex 95235 Noisy 路 102 号安万特公司8N V Organon, PO Box 20, 5340 BH Oss, Netherlands荷兰BH Oss5340 20号信箱欧加农公司Key words: blood volumes; blood removal; administration substances; laboratory animals; refine

5、ment.关键词：血容量；采血；给药；实验动物；简化This article is the result of an initiative between the European Federation of Pharmaceutical Industries Associations (EFPIA) and the European Centre for the Validation of Alternative Methods (ECVAM).Its objectives are to provide the researcher in the safety evaluation labo

6、ratory with an up-to-date, easyto-use set of data sheets to aid in the study design process whilst at the same time affording maximum welfare considerations to the experimental animals.该文章为欧盟制药工业协会(EFPIA)和欧洲替代动物实验方法验证中4(ECVAM)之间的初步结果。其目的在 于为安全性评价实验室的研究者提供最新的易于使用的数据库以帮助研究设计过程，同时最大可能地考虑 到实 验动物的福利。Alth

7、ough this article is targeted at researchers in the European Pharmaceutical Industry, it is considered that the principles underpinning the data sets and refinement proposals are equally applicable to all those who use these techniques on animals in their taking such large volumes may well affect th

8、e calculated half-life Assessment of terminal half-life should be possible if final samples are taken within 24 h of the killing of an animal. These values do not include a terminal sample, which can be taken when the animal is terminally anaesthetized. Blood replacement has not been considered beca

9、use the volumes proposed do not warrant such intervention.计划采用较高的体积(占循环血容量的20%)以利于毒代或药代动力学的连续性采血，此处通常要求多次、 少量取样。但是，应该记住如此大体积采血对血液动力学的重大影响，可能影响半衰期的计算。如果最后一次 采样在处死动物的24小时内则有可能对终末半衰期进行评估。这些数据不包括最后一个样本，该样本为当动物 最终被麻醉时能够被采集的一个样本。未考虑使用血液代用品，因为在推荐的采血体积下不允许使用此 类人为 干预。Using the values from Table 4, an easy re

10、ference guide for the volumes that can be removed without significant disturbance to an animals normal physiology is presented in Table 5.采用表4中的数据得到不会明显干扰动物正常生理的采血体积的简易参考指南，见表5oSampling sites 采血部位Sites for venepuncture and venesection have been considered mainly in rodents and rabbit.23 This informa

11、tion has been reviewed in the light of technical advances in blood sampling procedures; the advantages and disadvantages of sites for each species are shown in Table 6, with the recommended ones for repeated sampling summarized in Table 7.静脉穿刺和静脉切开的部位主要考虑用于啮齿类动物和家兔。根据采血程序的技术进展对该信息进行了总 结；每种种属的采血部位的优势

12、和缺点见表6,重复采样的推荐部位总结于表7。It is important to note that samples taken from different sites may show differences in clinical pathology values and have implications for historical databases.For the more traditional routes, a description of the methodology can be obtained from the standard literature. Howev

13、er, other methods require a special mention and have been reviewed below.值得注意的是从不同部位采集的样品在临床病理数据方面可能有所不同，且对历史数据库有影响。对于更加 传统的采血途径，可从标准文献中找到方法学描述。但是，对需要特别提及的其它方法综述如下。Lateral tarsal (saphenous) vein. This technique has been used in many laboratory animals, including rats,mice, hamster, gerbil, guinea p

14、ig, ferret, mink42 and larger animals, and volumes such as 5% of circulating blood volume may be taken. It does not require an anaesthetic and so is particularly suitable for repeated blood sampling as in pharmacokinetic studies. The saphenous vein is on the lateral aspect of the tarsal joint and is

15、 easier to see when the fur is shaved and the area wiped with alcohol. The animal is placed in a suitable restrainer, such as a plastic tube, and the operator extends the hind leg. The vein is raised by gentle pressure above the joint and the vessel is punctured using the smallest gauge needle that

16、enables sufficiently rapid blood withdrawal without haemolysis (e.g. 25-27 g for rats and mice). For small volumes,a simple stab leads to a drop of blood forming immediately at the puncture site and a microhaematocrit tube can be used to collect a standard volume. After blood has been collected, pre

17、ssure over the site is sufficient to stop further bleeding. Removal of the scab will enable serial sampling.跑外侧静脉（隐静脉）：该技术已经在许多实验动物中应用，包括大鼠、小鼠、仓鼠、沙鼠、豚鼠、雪貂、水 貂以及体型较大的动物，可以采集占循环血容量5%的血量。该方法不要求麻醉，因此特别适用于重复采血，如药代动力学研究。隐静脉在跑骨关节的旁边，当刮去被毛并用酒精擦拭后很容易看见。将动物置于合适的固定器 上，比如塑料试管，操作者分开动物的后肢。在关节上轻轻施压则该静脉凸起，然后采用能够满足快

18、速取血而不 会引起溶血的最小号的针头刺入血管(如25-27G针头适用于大鼠和小鼠)。对少量采血而言，可以采用简单的刺伤 从而在刺伤部位快速形成血滴，此时可使用微量血细胞比容试管收集标准体积的血液。采血后，在刺伤部位施加 足够的压力以止血。剥去结痂可连续取血。There appear to be no complications reported other than persistent (minor) bleeding and the method has the advantage that anaesthesia is not required. Even though no studi

19、es have been done on animal welfare in terms of body weight gain, diurnal rhythm, behaviour,etc, it seems unlikely that this route will seriously affect an animafs well-being.除顽固性(很少)出血外，目前看来没有并发症报道，该方法的优势在于不需麻醉。即使没有根据体重增长、昼 夜活动规律、行为等进行过有关动物福利的研究，但看来该途径不大可能严重影响动物的健康。Marginal ear vein/central ear art

20、ery. Blood sampling from the marginal ear vein is commonly used in rabbits and guinea pigs. This route may also be chosen in minipigs, often combined with the use of an intravenous cannula. Good restraint is necessary and the application of local anaesthetic cream some 20 -30 min before bleeding hel

21、ps to prevent an animal from shaking its head as the needle is pushed through the skin.Bleeds may also be taken by smearing the surface over the vein with petroleum jelly and then puncturing the vein and collecting the blood into a tube. For the removal of larger amounts of blood the central artery

22、in rabbits can be used, but afterwards it must be compressed for at least 2 min to prevent continuing bleeding and haematoma. The animal should be checked for persistent bleeding 5 and 10 min later.Repeated samples can be taken from this artery using an indwelling cannula, thus facilitating a kineti

23、c regimen over 8h.耳缘静脉/耳中央动脉：从耳缘静脉采血最常用于家兔和豚鼠。该途径也可在小型猪中选用，通常与静脉内 套管结合使用。必须进行良好的固定，在采血前大约20-30分钟应用局麻膏剂有助于防止针头穿透皮肤时动物 头部出现摇摆。也可用凡士林涂在静脉的表面，然后刺破静脉用试管收集血液。如果取血量较大，则可通 过家 兔耳中央动脉取血，但取后必须按压至少2分钟以止血和防止血肿。持续性放血5和10分钟后应对动物进 行检 查。使用留置套管能够重复从该动脉采血，因此有利于超过8小时的动力学研究。Sublingual vein. This technique is easy to per

24、form in rodents such as rats and is suitable for the removal of large volumes of blood (e.g. 0.2 -1 ml) at frequent intervals, limited only by the blood volume to be removed and by the necessary repeated anaesthesia. A refined method43 avoids some of the disadvantages previously seen and can be used

25、 for repeated sampling.Rats are anaesthetized and held by an assistant in a supine position. The loose skin at the nape of the neck is gathered up in order to produce partial stasis in the venous return from the head. A second person gently pulls out the tongue with a cotton-tipped applicator stick,

26、 grasps it with thumb and forefinger and one of the sublingual veins (there is one on each side of the midline) is punctured with a 23 -25 g hypodermic needle as near to the tip of the tongue as possible.The rat is turned over to allow blood to drip into a tube and after the requisite volume of bloo

27、d has been obtained the compression at the scruff of the neck is released and the animal is placed in a supine position.The tongue is again extended in order to stem the flow of blood with a dry cotton-tipped applicator stick; usually there is no need to use any haemostatic agent.舌下静脉：该操作在啮齿类动物如大鼠中易

28、于进行且适合于较大体积(如I 0.2-1 ml)的频繁性采血，仅受限 于 采血体积和必要的重复性麻醉。一种简化的方法避免了以前的方法中所见到的缺点且能够用于重复性采血。大鼠 麻醉后，由助手握持住大鼠，使其处于仰卧位。聚拢颈背部松弛皮肤以便部分阻塞静脉血从头部回流。第二名操 作者用顶端被棉花包裹的涂抹棒轻轻拉出舌头，用拇指和食指抓住舌头，用23-25G皮下注射针头刺破舌下静脉 之一（中线的左右侧各有一根舌下静脉），尽可能靠近舌尖。将大鼠翻转以便于血液流入试管中，采集到需要量的 血后，松开施加在后颈部的压力，将动物置于仰卧位。再次将舌头拉出用顶端被干棉花包裹的涂抹棒止血；通常 没有必要使用任何抗凝

29、剂。With this technique, rats do not show any significant differences in food or water consumption or body weight gain compared with untreated anaesthetized control animals. Moreover, there appear to be fewer pathological changes than with retrobulbar sampling44.However, anaesthesia may still be a lim

30、iting factor.采用该技术，大鼠在摄食和饮水或体重增长方面与未采血的麻醉对照动物相比没有明显差异。止匕外，该法较 眼球后采血所引起的病理变化更少。但是，麻醉可能仍然是一个限制性因素。Lateral tail vein. In principle this route is similar to the lateral tarsal vein but tends to yield smaller blood volumes （0.1-0.15 ml in mice; up to 2 ml in warmed rats）. Blood is removed either by syrin

31、ge/needle or stab puncture of a lateral tail vein. Anaesthesia is unnecessary, which makes this route particularly suited for repeated blood sampling. Vasodilatation may be necessary to promote bleeding and can be caused by exposing an animal to 37 for 5-8 min or by local warming of the tail. There

32、appear to be few disadvantages that affect animal well-being, but animals must be closely monitored for signs of distress if heat exposure is used.尾侧静脉：原则上该途径与附外侧静脉相似，但倾向于获得较小的采血量小鼠0.1-0.15 ml；处于暖和条件下 的大鼠最高可达2ml）。可以通过注射器/针头或刺穿尾侧静脉采血。由于勿需麻醉，这使得该途径特别适用于重 复采血。有必要使血管扩张以促进血流，可以将动物暴露于37C的温度中共5-8min或加热尾部。该

33、方 法损伤很 小，基本不会影响动物健康，但在加热时必须严密监测动物的不适症状。Cranial vena cava. Minipigs may be restrained in a sling or on their backs with the forelegs retracted caudally. Other methods, sometimes used in agricultural settings （snout tying, hog tying, suspending animals by their rear legs） , are stressful and are inapp

34、ropriate for laboratory animals because of the potential adverse effects on the science. In order to avoid injury to the vagus nerve, the needle is inserted into the right side of the neck, lateral to the manubrium sterni, and directed at a 30-45 angle towards the left shoulder.A popping sensation w

35、ill be felt by the sampler when the needle enters the vein, and then blood can be readily withdrawn. This method can also be utilized for sequential venipuncture but haematomas form in the area after the needle is withdrawn, therefore it is best reserved for procedures that do not require withdrawal

36、 more often than weekly.4颅大静脉：小型猪可以用绳套固定或将其前肢后缩固定于其背部。农业上有时采用的其它方法（捆绑嘴部、 捆绑猪躯干、通过后肢悬吊）会引起动物紧张，因此不适用于实验动物，因为会对研究的科学性产生潜在的不 利影响。为避免损伤迷走神经，应将针头插入颈部右侧，胸骨柄旁，以30-45。角向左肩方向进针。当针头进 入 血管的时候通过取样器可以觉察到破裂的感觉，然后可以轻易地采集到血样。该方法亦被用来作连续性静脉穿 刺，但抽出针头后穿刺部位形成血肿，因此在不要求每周频繁采血的情况下，该方法为一种最佳候选程序。Amputation of the tail tip. T

37、his technique is commonly used in rats and mice, with sample volumes of 0.1-0.2 ml beingobtained. Amputation should be restricted to the tail tip (0.5-1 mm should be adequate,and over time a maximum of 5 mm can be removed) and repeat bleeding is feasible in the short term by removing the clot. Seria

38、l amputations resulting in a significant shortening of the tail (i.e.5mm) are not acceptable. The technique may not be suitable for older animals. Anaesthesia is recommended.尾尖切断术(断尾法)：该技术通常用于大鼠和小鼠，采血量为切断术应仅限于尾尖(每次0.5. 1mm应当足够，随试验需要最多只能切除5mm),通过去除血凝块可在短期内重复采血。可明显缩短尾部(如5mm)的连续性切断术不能被接受。该技术不适合老龄动物。建议对

39、动物进行麻醉。Cardiac puncture. This should always be carried out under general anaesthesia and in the past it has been used with recovery in small rodents due to the lack of alternative routes. However, other methods are now available and because of potentially painful and fatal sequelae, such as pericard

40、ial bleeding and cardiac tamponade,this technique should only be used for terminal bleeds.心脏穿刺：该方法通常在麻醉条件下进行，在过去由于缺乏替代途径，因此被用于对小型啮齿类动物采血。 但是,现在有其它方法可供利用，并且由于潜在性疼痛和致命性后遗症,如心包出血以及心脏压塞，因此该技术应 仅用于末期采血。Retrobulbar plexus. The retrobulbar route has been commonly used by researchers in the past but has bee

41、n observed to cause adverse effects. Concern has therefore arisen because of these effects and their potential severity. Recently, however, other methods have been developed that meet the scientific requirements and also improve the welfare of the animals. Nevertheless,the Technical Subgroup felt th

42、at it was worth reviewing in detail some of the advantages and disadvantages of retrobulbar bleeding in the context of the new methods.眼球后静脉丛：研究者过去普遍采用眼球后采血途径，但会引起的不良反应。因为这些不良反应及其潜 在 的严重性，因此该方法所涉及到的利害关系正日渐突出。但是，最近已开发出其它符合科学性要求的方法，由 此改善了动物福利。然而，技术小组感到在新方法的上下文中应详细回顾眼球后采血的优势和缺点。Bleeding from this plexu

43、s always should be carried out under general anaesthesia in all species and anaesthesia is a requirement in some national regulations.The method has been described in detail by a number of workers46-48对于所有种属的静脉丛采血均应该在常规麻醉条件下进行，在某些国际注册中要求对动物进行麻醉。大量 工作者对该方法均进行了详尽的描述。There is little published work on r

44、efining the method.The approach (lateral or access via the dorsal or upper aspect of the eye in rats) as the optimal way to penetrate the conjunctiva in order to minimize tissue damage has been discussed.23 An interval of 2 weeks between bleeds at the same site should allow damaged tissue to repair

45、in most cases,49 but this does not mean that the animals do not experience some discomfort during the early stages before healing is complete;there are, however, concerns over repeated retrobulbar punctures. Whereas some studies have shown that repeated orbital bleeds do not affect the animalsiurnal

46、 rhythm0,51 or the histology of the orbital tissue long term52,49 (i.e. both showed that any tissue damage healed), other studies have found histological changes, abnormal clinical signs and evidence of discomfort3 55 which has led to animals having to be killed on humane grounds and so lost from th

47、e study. There are also other serious potential adverse effects:仅有少量有关简化该方法的研究工作被公开。作为穿透结膜的最佳方法以尽量减轻组织损伤的途径(从腹 侧、背侧和上方进入大鼠眼球)已有所讨论。在同一部位的两次采血之间必须有2周间隔期以使损伤组织可以在 大多数情况下恢复，但这并不意味着动物在完全恢复前的早期不会经历某些不适。然而某些研究已表明重复眼 窝采血不会长期影响动物的昼夜节律或眼窝组织结构(如这两篇文献中的研究均表明任何组织损伤均痊愈)，其 它研究发现了组织变化、异常临床表现和明显不适，从而出现人道立场不得不处死动物从而

48、给研究工作带来损 失。该方法还存在其它严重的潜在性不良反应：(i) retrobulbar haemorrhage resulting in haematoma and excessive pressure on the eye, which is almost certainly painful for the animal;(ii) any pressure required to stem persistent bleeding (e.g. by pressing on the eye) or pressure from a haematoma can lead to corneal u

49、lceration, keratitis,pannus formation, rupture of the globe and micro-ophthalmia;(iii) damage to the optic nerve and other intra-orbital structures, which can lead to deficits in vision and even blindness;(iv) fracture of the fragile bones of the orbit and neural damage by the micropipette; and penetration of the eye globe itself with a loss of vitreous humour.引起血肿和眼压过高的眼球后出血，必须会使动物感到疼痛；(ii)任何为止血而施加的压力(如压迫眼部)或来源于血肿的压力均能导致角膜溃疡、角膜炎、角膜翳形成、眼球破裂和小眼畸形；(iii)视神经和其它眼窝内结构损伤可导致视力降