This research was funded by Guangdong Provincial Key Laboratory of Drug Non-clinical Evaluation and Research (No.2018B030323024) and Key Program &#34;New Drug Creation&#34; of Guangdong Key Research and Development Plan (No.2019B020202001), Guangzhou Fundamental and Application Foundation Research Project (No.202002030249 and No.202002030156).

All animal experiments described were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Lewwin Pharmaceutical Research Institute Co., Ltd. Male and female Sprague Dawley (SD) rats, ~6-11 weeks were used for the experiments. The rats were reared following the guidelines for the care and use of laboratory animals13.
1. Animal preparation
NOTE: All the SD rats used in this study were awake and were n...

<ol>
	<li>Shravya, 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=International+conference+on+harmonization+of+technical+requirements+for+registration+of+pharmaceuticals+for+human+use.+ICH+M2+EWG.">International conference on harmonization of technical requirements for registration of pharmaceuticals for human use. ICH M2 EWG.</a> Electronic common technical document. , (2014).</li><li>Tan, Y., 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> The China Food and Drug Administration (CFDA). , (2015).</li><li>McGuill, M. W., Andrew, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+effects+of+blood+loss:+Implications+for+sampling+volumes+and+techniques.">Biological effects of blood loss: Implications for sampling volumes and techniques.</a> ILAR Journal. 31 (4), 5-20 (1989).</li><li>Aguilar-Mariscal, H., 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=Oral+pharmacokinetics+of+meloxicam+in+the+rat+using+a+high-performance+liquid+chromatography+method+in+micro-whole-blood+samples.">Oral pharmacokinetics of meloxicam in the rat using a high-performance liquid chromatography method in micro-whole-blood samples.</a> Methods and Findings in Experimental and Clinical Pharmacology. 29 (9), 587-592 (2007).</li><li>Korfmacher, W., 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=Utility+of+capillary+microsampling+for+rat+pharmacokinetic+studies:+Comparison+of+tail-vein+bleed+to+jugular+vein+cannula+sampling.">Utility of capillary microsampling for rat pharmacokinetic studies: Comparison of tail-vein bleed to jugular vein cannula sampling.</a> Journal of Pharmacological and Toxicological Methods. 76, 7-14 (2015).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Blood+Withdrawal+I.">JoVE Science Education Database. Blood Withdrawal I.</a> JoVE. , (2021).</li><li>Yang, H., 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=Subclavian+vein+puncture+as+an+alternative+method+of+blood+sample+collection+in+rats.">Subclavian vein puncture as an alternative method of blood sample collection in rats.</a> Journal of Visualized Experiments. (141), e58499 (2018).</li><li>Zou, W., 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=Repeated+blood+collection+from+tail+vein+of+non-anesthetized+rats+with+a+vacuum+blood+collection+system.">Repeated blood collection from tail vein of non-anesthetized rats with a vacuum blood collection system.</a> Journal of Visualized Experiments. (130), e55852 (2017).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology &amp; Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Itziar, F., Arantza, P., Nahia, D., Virginia, P., Juan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+biochemistry+parameters+in+C57BL/6J+mice+after+blood+collection+from+the+submandibular+vein+and+retroorbital+plexus.">Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus.</a> Journal of the American Association for Laboratory Animal Science. 49 (2), 202-206 (2010).</li><li>Heimann, M., Roth, D. R., Ledieu, D., Pfister, R., Classen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublingual+and+submandibular+blood+collection+in+mice:+A+comparison+of+effects+on+body+weight,+food+consumption+and+tissue+damage.">Sublingual and submandibular blood collection in mice: A comparison of effects on body weight, food consumption and tissue damage.</a> Laboratory Animals. 44 (4), 352-358 (2010).</li><li>Wren-Dail, M. 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=Effect+of+isoflurane+anesthesia+on+circadian+metabolism+and+physiology+in+rats.">Effect of isoflurane anesthesia on circadian metabolism and physiology in rats.</a> Comparative Medicine. 67 (2), 138-146 (2017).</li><li>National Institute of Health. <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 for the Care and Use of Laboratory Animals. 8th edition. , (2011).</li><li>Lee, G., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+blood+from+the+lateral+tail+vein+of+the+rat.">Sampling blood from the lateral tail vein of the rat.</a> Journal of Visualized Experiments. (99), e52766 (2015).</li><li>Diehl, 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+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> Journal of Applied Toxicology. 21 (1), 15-23 (2001).</li><li>Casal, D., 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=Functional+and+physiological+methods+of+evaluating+median+nerve+regeneration+in+the+rat.">Functional and physiological methods of evaluating median nerve regeneration in the rat.</a> Journal of Visualized Experiments. (158), e59767 (2020).</li></ol>

There are certain benefits of collecting blood from the subclavian veins. (1) As the site of the blood collection is readily dissociated, and the venous plexus is not regular due to the different postures of the rats, the described method can easily localize the position of the venous plexus while maintaining the stable and comfortable postures of the rats. (2) The operation is easy and favorable to the rapid development of technicians' skills and less pain to animals. (3) An operating mode to make the animal comfortable...

According to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines1 and the National Medical Products Administration (NMPA) guidelines2, the number of blood collection time points of rats in the toxicokinetic (TK) study needs to meet the requirements of the dynamic drug exposure assessment. The approximate total blood volume of a rat is 55-70 mL/kg of body weight3. The collection time points are generally intensive within 30 min after dosing and decrease after that, and more than ten blood samples need to be collected within 48 h in routine testing4. For example, blood samples are collected at 12-time points (0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 8 h, and 12 h) in TK study of orally administered drugs. Researchers must repeatedly collect 200-250 &#181;L of blood in rats to obtain high-quality plasma for the TK testing5.
The blood collection sites in rats include tail blood vessels, retro-orbital plexus vein, submandibular vein, heart, abdominal aorta6,&#160;and so on. Among them, blood collection from the caudal vein of rats is a frequently used method, which requires experienced and skilled operators7,8. To collect blood from the retro-orbital plexus vein is less complicated; however, this method is not recommended as it may damage the rats&#39; eyesight9, and blood from the heart and abdominal aorta is only appropriate for final blood sampling10. Another method of collecting blood from the submandibular vein in a conscious rat has been shown to result in more complications and revealed insufficient blood sample quality11. Therefore, researchers may anesthetize the animal to reduce the difficulty of sampling. Still, the anesthesia also increases the cost of the experiment, and more seriously, it will affect the metabolic state of rats12. The present protocol uses a quick and straightforward method of collecting blood in the subclavian veins of rats without anesthesia, allowing accurate positioning and bilateral alternating blood collection to obtain high-quality samples in a timely and repeated manner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.0 mL syringe (with needle, 26 G, 0.45 mm x 15.5 mm)</td>
			<td>Jiangxi Hongda Medical Equipment Group LTD.(Nanchang, Jiangxi Province, China)</td>
			<td>20210629</td>
			<td></td>
		</tr>
		<tr>
			<td>75% alcohol</td>
			<td>Shandong Lierkang Medical Technology Co., Ltd. (Dezhou, Shandong Province, China)</td>
			<td>210717</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal source</td>
			<td>Hunan SJA Laboratory Animal Co., Ltd.</td>
			<td>grade: SPF</td>
			<td>laboratory animal production license number: SCXK (Hunan) 2019-0004, laboratory animal quality certificate number: 4307272101972847</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Caoxian Hualu Sanitary Materials Co., Ltd.(Heze, Shandong Province, China)</td>
			<td>20210301</td>
			<td>Need to be sterilized.</td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Shenzhen Codos Electric Appliance Co. , Ltd.(Shenzhen, Guangdong Province, China)</td>
			<td>CP-6800</td>
			<td></td>
		</tr>
		<tr>
			<td>EP tube, 1.5 mL</td>
			<td>Genetimes ExCell Technology,Inc.(Shanghai, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium (2 mL:12500 IU)</td>
			<td>Tianjin biochem pharmaceutical Co.,Ltd(Tianjing, China)</td>
			<td>51200702</td>
			<td>Prepare 1250 IU/mL heparin sodium solution. Add heparin sodium solution into the EP tube in advance and make it evenly distributed on the wall of the EP tube, and dry it at 60&#176;C for use.&#160;</td>
		</tr>
		<tr>
			<td>Labtip (volume range 5-200 &#956;L)</td>
			<td>Thermo Fisher Scientific Oy</td>
			<td>94300120</td>
			<td></td>
		</tr>
		<tr>
			<td>Low speed refrigerated centrifuge</td>
			<td>Hunan Xiangyi Laboratory Instrument Development Co., Ltd.(Changsha, Hunan Province, China)</td>
			<td>L535R</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette gun (20-200 &#956;L)</td>
			<td>BRAND</td>
			<td>12N92305</td>
			<td></td>
		</tr>
		<tr>
			<td>Rats (SD)</td>
			<td>Hunan SJA Laboratory Animal Co., Ltd. (Changsha, Hunan Province, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp tool container</td>
			<td>Taizhou Huangyan Yikang Plastic Factory (Taizhou, Zhejiang Province, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye drop</td>
			<td></td>
			<td></td>
			<td>This reagent is not a commodity and the manufacturer requires it to be tested. In the principle of confidentiality, the manufacturer and model cannot be provided.</td>
		</tr>
	</tbody>
</table>

repetitive blood sampling from the subclavian vein of conscious rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

Good plasma samples from the subclavian vein were translucent pale yellow (Figure 4, the left tube). Improper blood collection or manipulation resulted in hemolysis (Figure 4, the right tube).
The data of the testing facility showed that in a 4-week oral toxicity study of an eye drop in SD rats with TK, blood samples were collected twice at 9-time points (0 h, 0.167 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) between the first (Day ...

Watch this Scientific Journal Video about Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat at JoVE.com

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

In pharmacokinetics and toxicokinetics studies, we need to collect a certain amount of blood at specific time points in rats to detect drug exposure. This study describes a simple and efficient method for collecting blood from the subclavian vein in rats in this protocol. This technique enables repeat, timely, and easily identifiable sampling without anesthesia, and obtains high quality blood through repetitive sample collection.You should know the anatomy of the subclavian vein, capable rat restraint and the needle point. The method is risky, and it calls for a qualified professional. To begin, find the lowest point of the subclavian triangular fossa between the neck and the forelimb in a rat.Move approximately two to three millimeters towards the head and reach the needle point at the blood collection site. Remove the hair using an electric shaver. To keep the rat's head, neck, and breast upright, grasp the skin on the back of the neck with one hand and expose the injection site.Straighten the forelimb on the side of the injection site to maintain its level. Keep the syringe parallel to the rat's head with the other hand and tilt the syringe outward five to 10 degrees, so that the tip is tilted toward the ventral direction. Insert the needle fully into the anterior cavity.Draw back the syringe to maintain negative pressure with the tube. Slowly move the needle from deep to shallow and back up the same path. When blood has entered the syringe needle, fix the position of the needle.Depending on the weight and health of the rats, monitor the maximum blood quantity to be drawn in accordance with the standards set by the institution's animal care and use committee. In the absence of other requirements, do not remove more than 20%of the animal's total blood volume within 24 hours. When sufficient blood sample is collected, immediately withdraw the syringe, and prepare for blood treatment.Pressurize the rat's injection site for one to two minutes to stop the bleeding. Since the collection site is in the lower part of the neck, pinch the skin of the subclavian vein to stop the bleeding by pressing on it. Remove the needle from the syringe, and discard it in the sharp tool container.Slowly transfer the blood from the syringe to a 1.5-milliliter tube. Press the syringe against the wall to avoid bubble formations. Close the lid of the microcentrifuge tube and gently flick it.Mix the blood with the anticoagulant thoroughly by turning it upside down at least five times. Within 120 minutes after collecting the plasma, centrifuge the whole blood sample at 2, 000 RCF for 10 to 15 minutes at room temperature. Use a pipette gun to transfer the upper plasma into a fresh microcentrifuge tube.Do not touch the pipette head with the lower whole blood. Dispose of, or re-centrifuge the plasma contaminated with the red blood cells. Plasma samples accurately collected from the subclavian vein were translucent pale yellow.Improper blood collection, or manipulation resulted in hemolysis. During the first sampling, the body weight of the male animal was between 268 to 297 grams, and around 214 to 239 grams for the females. During the last sampling, the body weight of the male animal was between 376 to 462 grams, and around 254 to 300 grams for the females.There was a gap of 28 days between the toxicokinetic blood collection. The animal's body weight increased steadily every week, and the clinical observation was normal. The precise needle insertion method and angle can ensure smooth blood collection and avoid risks.This operation requires repeated practice.

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3.9K 次观看.  Guangdong Engineering Research Center for Innovative Drug Evaluation and Research. 在药代动力学和毒代动力学研究中，我们需要在大鼠的特定时间点收集一定量的血液来检测药物暴露。本研究描述了一种从该协议中大鼠锁骨下静脉收集血液的简单有效的方法。该技术无需麻醉即可重复、及时且易于识别的采样，并通过重复样本采集获得高质量的血液。您应该了解锁骨下静脉的解剖结构，有能力的大鼠约束和针尖。这种方法是有风险的，它需要一个合?...