This work was supported by the grant from the National Natural Science Foundation of China No. 81670269, No. 81500355 and No. 81500226.

This study was approved by the Central South University Ethics Committee for Animal Research from The Second Xiangya Hospital (Changsha, China). The manuscript was prepared according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines12.
1. Material and animal
<ol>
	<li>Prepare required materials: 75% ethanol, adhesive tape, epilating agent, 2 mL tube, 1.0 mL syringe connected with needle (26G), electronic scale, heparin and saline (see Table of Materials).</li>
	<li>Animals: Prepare 10 Kunming mice, 6-8 weeks old and weighing 21.6-28.3&#160;g (see Table of Materials). Maintain mice in accordance with the Guide for the Care and Use of Laboratory Animals13.</li>
</ol>
2. Anesthesia and animal positioning
<ol>
	<li>Weigh the mouse to calculate the required dosage of anesthetic agent.</li>
	<li>Inject sodium pentobarbital (60 mg/kg) through intraperitoneal injection to induce general anesthesia14 (Figure 1). Apply a sterile eye lubrication ointment at the start of the procedure to prevent damage to exposed eyes. 
	NOTE: Mice are considered to be sufficiently anesthetized when showing no motor response to testing of the pedal withdrawal reflex, tail pinch, or abdominal skin pinch.</li>
	<li>Place the mouse in the operation table in a supine position and 2-4 cm away from the edge of the table to facilitate vein puncture (Figure 2). Fix the limbs in a comfortable position as shown in Figure 2. No intubation or mechanical ventilation is needed in the whole procedure.</li>
	<li>Apply epilating agent around the infraclavicular space with a cotton swab.</li>
	<li>Three minutes later, wash the epilating agent with a wet cotton swab to remove the fur and any visible dirt.</li>
	<li>Sterilize the infraclavicular space with 75% ethanol and then dry with clean gauze.</li>
</ol>
3. Subclavian vein puncture and blood collection
<ol>
	<li>Identify the location of the clavicle bone and superior sternal fossa with a finger.</li>
	<li>Locate the puncture site close to the middle of the clavicle bone and caudal to it. Put the left index finger lateral to the puncture site to fix the skin and subcutaneous tissue (Figure 3).</li>
	<li>Move the needle upward and cranially toward the superior sternal fossa. Once the needle enters the subcutaneous tissue, move the ring finger of the right hand backward to form negative pressure (Figure 3). 
	NOTE: During this step, the position of the operator&#180;s right hand should be slightly lower than the operation table to make the needle move upward and superior to the horizontal plane. That is why the animal is placed near the edge of the table.</li>
	<li>Move the syringe forward 3-4 mm but stop if there is no blood drain into the syringe. Then slowly draw back the syringe and keep negative pressure in it. In most occasions, the blood would enter the syringe when drawing back.</li>
	<li>Once the blood enters the syringe, fix the syringe and maintain negative pressure until the required volume (200 &#181;L) of blood is collected in the syringe.</li>
	<li>After blood collection, withdraw the puncture needle, and press the puncture site with a cotton swab slightly for 1-2 minutes to stop bleeding. Then, return mice to the cage. 
	NOTE: Occasionally, one could not obtain the blood at his/her first attempt. Adjust the direction of the needle laterally and repeat steps 3.3-3.5. If 3 attempts fail to obtain blood sample, switch to the other side.</li>
	<li>Transfer the blood sample into a heparinized tube.</li>
</ol>
4. Mice recovery
<ol>
	<li>While the pentobarbital anesthesia is in effect, speed recovery by providing heat support until the mouse is moving again.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Submental+Blood+Collection+with+the+Retroorbital+and+Submandibular+Methods+in+Mice+(Mus+musculus).">Comparison of Submental Blood Collection with the Retroorbital and Submandibular Methods in Mice (Mus musculus).</a> Journal of the American Association for Laboratory Animal Science. 55 (5), 570-576 (2016).</li><li>Park, A. 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=Blood+collection+in+unstressed,+conscious,+and+freely+moving+mice+through+implantation+of+catheters+in+the+jugular+vein:+a+new+simplified+protocol.">Blood collection in unstressed, conscious, and freely moving mice through implantation of catheters in the jugular vein: a new simplified protocol.</a> Physiological Reports. 6 (21), e13904 (2018).</li><li>Heimann, M., Kasermann, H. P., Pfister, R., Roth, D. R., 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=Blood+collection+from+the+sublingual+vein+in+mice+and+hamsters:+a+suitable+alternative+to+retrobulbar+technique+that+provides+large+volumes+and+minimizes+tissue+damage.">Blood collection from the sublingual vein in mice and hamsters: a suitable alternative to retrobulbar technique that provides large volumes and minimizes tissue damage.</a> Laboratory Animals. 43 (3), 255-260 (2009).</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>Diehl, K. 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=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>van Herck, 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=Orbital+sinus+blood+sampling+in+rats+as+performed+by+different+animal+technicians:+the+influence+of+technique+and+expertise.">Orbital sinus blood sampling in rats as performed by different animal technicians: the influence of technique and expertise.</a> Laboratory Animals. 32 (4), 377-386 (1998).</li><li>Tsai, P. 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F., Tranholm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+blood+sampling+technique+on+blood+quality+and+animal+welfare+in+haemophilic+mice.">Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice.</a> Laboratory Animals. 45 (2), 114-120 (2011).</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=Safety+and+efficacy+of+a+modified+axillary+vein+technique+for+pacemaker+implantation.">Safety and efficacy of a modified axillary vein technique for pacemaker implantation.</a> Cardiology Plus. 3, 104-107 (2018).</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), (2018).</li><li>Kilkenny, C., 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:+ARRIVE-ing+at+a+solution.">Improving bioscience research reporting: ARRIVE-ing at a solution.</a> Laboratory Animals. 44 (4), 377-378 (2010).</li><li>Research, I. f. L. A. <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. , (1996).</li><li>Redel, 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=Impact+of+ischemia+and+reperfusion+times+on+myocardial+infarct+size+in+mice+in+vivo.">Impact of ischemia and reperfusion times on myocardial infarct size in mice in vivo.</a> Experimental Biology and Medicine (Maywood, N.J.). 233 (1), 84-93 (2008).</li><li>Tsukamoto, A., Serizawa, K., Sato, R., Yamazaki, J., Inomata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+signs+monitoring+during+injectable+and+inhalant+anesthesia+in+mice.">Vital signs monitoring during injectable and inhalant anesthesia in mice.</a> Experimental Animals. 64 (1), 57-64 (2015).</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), (2017).</li></ol>

None declared.

This report represents an extension of previous research on blood sampling through the subclavian vein puncture in rats11. As the mouse is the most commonly used research animal, it would be valuable to see if this technique can also be applied to mice. The challenge comes from the relatively smaller diameter of the subclavian vein.
In the present research, we found that subclavian puncture in mice is a feasible and reliable way to collect blood. Compared to the conventional methods such as tail vein cutting or orbital blood collection, there are no ethical issues of the present method. All animals survived after this procedure without liquid supplements after around 200 &#181;L of blood extraction, and animals could be used for further experimental studies. Theoretically, the total sample volume extraction could not reach more than 10% of total circulating blood volume each time and the total volume of an adult animal is 55 to 70 mL/kg body weight5. For the mice used in this research (21.6-28.3 g), the maximum volume extraction should be around 200 &#181;L. We thus set the blood sampling amount around 200 &#181;L for each mouse. The inability to measure blood pressure, heart rate, and other stress parameters are the main limitations of this report.
Sufficient general anesthesia is another important issue to guarantee the success of the puncture. The mice should remain quiet during the puncture procedure to avoid the risk of injuring the vessel by the needle. We found that pentobarbital sodium at 60 mg/kg for general anesthesia could satisfy ideal depth of anesthesia. A downside of this agent is the relatively long recovery time (averaging about 30 minutes). In some institutes, isoflurane inhalation is used for the short recovery time and should be taken into consideration as an alternative anesthesia choice15.
The location of subclavian vein is shown in Figure 4. Because of the thin wall and low pressure in the vein, the anterior and posterior wall of the subclavian vein would attach to each other under the pressure of a needle while the needle is moving forward (Figure 5A). While withdrawing the plunger slowly backwards, the attached walls will separate spontaneously, and the tip of needle could enter the true cavity of vessel and blood could drain into the syringe under sustained negative pressure (Figure 5B). Use of a vacuum blood collection system16 might also favor the blood collection. Due to the relatively small diameter of the mice subclavian vein, there can be failures even for an experienced operator. For a novice, we recommend subclavian vein puncture in rats as reported11. After several successful puncture attempts in rats, the success rate of subclavian vein puncture in mice could be significantly enhanced. Make sure that the exact puncture location is just caudal to the middle of the clavicle bone. The needle moving upward and cranial to the superior sternal fossa are other key points to guarantee a successful puncture.
In conclusion, subclavian vein puncture is a safe and effective method for blood collection in mice. Besides tail vein cutting and retro-orbital plexus blood collection, this method is feasible, safe, and suitable for observational researches at several time points in mice.

Blood sample collection from mice is essential in most research laboratories. The conventional approaches for blood collection in mice is tail cutting when less than 100 &#181;L of sample is needed1. However, if more than 100 &#181;L of blood are required at a nonterminal time point, retroorbital, submandibular bleeding or submental blood collection are the most commonly considered techniques2. In some occasions, the jugular vein catheterization through a surgical incision was adopted as an alternative method3. 
Nevertheless, the above methods are harmful to the mice. To the best of our knowledge, the retroorbital method is not widely accepted because of the potential risk of complications4,5. Operation related trauma not only happens in the visible area6,7, but also deep within the orbit6. Besides, submandibular blood collection is stressful8 and might be associated with excessive bleeding2,9. Based on our prior research10,11, here we introduce a new strategy for blood collection from the subclavian vein in mice. The safety, feasibility, and obtained blood volume with this technique are presented and discussed.

<table><tbody><tr><td>1.0 mL syringe</td><td>Shandong Weigao Group Medical Polymer Co., Ltd (Weihai, Shandong Province, China)</td><td>20163151593</td><td></td></tr><tr><td>75% ethanol</td><td>Department of Pharmacy, The Second Xiangya Hospital of Central South University</td><td>made in Department of Pharmacy,The Second Xiangya Hospital of Central South Univesity</td><td></td></tr><tr><td>adhesive tape</td><td>3M Deutschland GmbH (Kamen, Germany)</td><td>1534-1</td><td></td></tr><tr><td>canvas gloves</td><td>for anesthesia</td><td></td><td></td></tr><tr><td>electronic scale</td><td>Dongguan Shengheng Electronics Co. Ltd (Dongguan, Guangdong Province, China)</td><td>KP-3000</td><td></td></tr><tr><td>epilating agent</td><td>France Yi Sha Cosmetics Co. Ltd (Guangzhou, Guangdong Province, China)</td><td>8281744</td><td></td></tr><tr><td>heparin (used concentration 10 U/mL, 2 mL, 12500 IU)</td><td>Nanjing Xinbai Pharmaceutical Co. Limited (Nanjing, Jiangsu Province, China)</td><td>H32025851</td><td></td></tr><tr><td>mice</td><td>Hunan SJA Laboratory Animal Co. Ltd (Changsha, Hunan Province, China)&amp;nbsp;</td><td>Kunming spcies</td><td></td></tr><tr><td>needle, 26G, 0.45 mm x 16 mm</td><td>Shandong Weigao Group Medical Polymer Co. Ltd (Weihai, Shandong Province, China)</td><td>20163151593</td><td></td></tr><tr><td>pentobarbital sodium (used solution 1%)</td><td>Merck</td><td>P-010-1ML</td><td></td></tr><tr><td>physiological saline, 100 mL</td><td>Hunan Kelun Pharmaceutical Co. Ltd (Yueyang, Hunan Province,China)</td><td>H43020454</td><td></td></tr><tr><td>stastical software</td><td>International Business Machines</td><td>SPSS Statistics 25</td><td></td></tr><tr><td>tube, 2 mL</td><td>Hubei Jinxing Technology &amp;amp; Development Co. Ltd (Wuhan Hubei Province, China)</td><td>MCT-150-C</td><td></td></tr></tbody></table>

blood collection through subclavian vein puncture in mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

We repeated this procedure in 10 Kunming mice (male n = 5, female n = 5, weight 25.4 ± 2.0 g). Nine procedures succeeded on the right side. One case failed within 3 attempts in the right side and blood collection succeeded on the left side. The time course (from puncture to obtaining required blood volume) ranged between 35-126 seconds (average 68.4 ± 26.4 s). Blood collection volume was set to around 200 µL (average 203 ± 11.6 µL). Blood collection succeeded within 1 to 4 attempts. Blood sampling succeeded on the first attempt in two mice and after 4 attempts in one mouse; the sampling took 2-3 attempts in the other mice. All data were illustrated in Table 1. All animals survived and recovered within 30 minutes after the blood sampling. There was no significant difference between sexes for all observed parameters (Table 1).
<img alt="Laboratory mouse injection procedure for research, experimental setup, genetic study, animal testing." class="xfigimg" src="/files/ftp_upload/59556/59556fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: General anesthesia through intraperitoneal injection <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59556/59556fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mouse surgery preparation; taped limbs on sterile mat for experimental procedure analysis." class="xfigimg" src="/files/ftp_upload/59556/59556fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: The supine position of mouse. Note the distance between margin and mouse is 2-4 cm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59556/59556fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rodent injection procedure; lab technique; anesthetized mouse in sterile environment; research study." class="xfigimg" src="/files/ftp_upload/59556/59556fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: The puncture site and posture of operator's hands. Note that the ring finger of right hand moves backward to form negative pressure. The left hand is placed lateral to the puncture site to fix the skin and subcutaneous tissues. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59556/59556fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mouse anatomy diagram showing clavicle bone, subclavian vein, and suprasternal fossa." class="xfigimg" src="/files/ftp_upload/59556/59556fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: The location of the subclavian vein, clavicle bone and superior sternal fossa. The subclavian vein courses beneath the clavicle bone and drains into the superior vena cava under the superior sternal fossa. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59556/59556fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Balloon angioplasty process, catheter insertion diagram, blood flow restoration, cardiovascular method." class="xfigimg" src="/files/ftp_upload/59556/59556fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: The relation between the vessel wall and needle during the procedure. (A) The walls of the subclavian vein attach to each other as the needle moves forward, so no blood could be drawn out. (B) While withdrawing the needle backward, the walls separate from each other and the blood could be drawn out. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59556/59556fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>variable</td>
			<td>Total (n=10)</td>
			<td>Male (n=5)</td>
			<td>Female (n=5)</td>
			<td>P Value</td>
		</tr>
		<tr>
			<td>Body Weight (g)</td>
			<td>25.3 ± 2.2</td>
			<td>25.3 ± 3.0</td>
			<td>25.4 ± 1.3</td>
			<td>0.926</td>
		</tr>
		<tr>
			<td>Blood Volume (μL)</td>
			<td>203.0 ± 11.6</td>
			<td>208 ± 13.0</td>
			<td>198 ± 8.4</td>
			<td>0.187</td>
		</tr>
		<tr>
			<td>Time Course (s)</td>
			<td>68.4 ± 26.4</td>
			<td>70.6 ± 35.1</td>
			<td>66.2 ± 18.0</td>
			<td>0.809</td>
		</tr>
		<tr>
			<td>Withdrawals</td>
			<td>2.3 ± 0.9</td>
			<td>2.4 ± 1.1</td>
			<td>2.2 ± 0.8</td>
			<td>0.760</td>
		</tr>
		<tr>
			<td colspan="4">There were no significant sex differences for all observed parameters, all P value &gt;0.05.</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Observed parameters between the sexes

Watch this Scientific Journal Video about Blood Collection Through Subclavian Vein Puncture in Mice at JoVE.com

Blood Collection Through Subclavian Vein Puncture in Mice

Here, we present a protocol to take blood samples from the subclavian vein of mice.

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

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Universidad San Sebastian

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Medicine

34.7K Views.  Central South University. Here, we present a protocol to take blood samples from the subclavian vein of mice.

Video: Blood Collection Through Subclavian Vein Puncture in Mice

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

Behavior

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

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Improved Blood Sampling Technique to Assess Gut Microbial Metabolites in Mice

Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous distant organ systems. Short-chain fatty acids (SCFA) are one class of metabolites produced by gut microbes during the fermentation of indigestible dietary fiber. They are now recognized&#160;as important contributors to how the gut microbiome influences extra-intestinal organ systems via the gut-lung, gut-brain, and other gut-organ axes throughout the host. SCFAs are absorbed from the colon, through intestinal tissue,&#160;into the portal vein (PV). They then pass through the liver, and are consumed in various organs such as the brain, muscle, adipose tissue, and lungs. SCFAs are most easily measured in the expelled fecal material however, more accurate measurements have been obtained from intra-colonic fecal contents. Here we propose that sampling PV and systemic circulating plasma of a single subject may be preferable for studying the&#160;absorption, transport, and systemic levels of SCFAs in mice. We present a new technique for efficient blood sampling from the PV and inferior vena cava (IVC) that allows for the collection of relatively large volumes of blood from the portal and systemic circulations. This is accomplished by ligating the PV, thereby allowing&#160;for the dilation or enlargement of the PV as it backfills from the mesenteric veins that drain into it. Using this method, we were able to improve the rate of successful collection as well as the total amount of blood collected (up to 0.3 mL from IVC and 0.5 mL from PV).

Author Spotlight: Gut Microbiome-Lung Tissue Communication Through Short-Chain Fatty Acid Analysis

The protocol demonstrates a method to collect blood from portal veins and inferior vena cava from mice sequentially to evaluate the production and absorption of gut microbial metabolites.

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous ...

Immunology and Infection

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.

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 ...

Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Author Spotlight: Developing an Efficient Blood Collection Method in Non-Anesthetized Rats 

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...

Puncture Wound Hemostasis and Montaged Electron Microscopy 

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

Author Spotlight: Revealing Platelet Dynamics Through Advances in Structural Hematology

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds ...

Blood Withdrawal I

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Blood collection is a common requirement for research studies that involve mice and rats. The method of blood withdrawal in mice and rats is dependent upon the volume of blood needed, the frequency of the sampling, the health status of the animal to be bled, and the skill level of the technician.1 All methods discussed-retro-orbital sinus bleeds, initial tail snip bleeds, and intracardiac bleeds-require the use of a general anesthesia.

Retro Orbital, Tail and Intra-cardiac Blood Collection in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Blood collection is a common requirement for ...

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Blood Withdrawal II

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for analysis can be done through a variety of methods. Each method of collection has variations in the type of restraint required, the invasiveness of the procedure, and the necessity of a general anesthetic.1Historically, the use of the retro-orbital sinus cavity has been used, but not without debate. The controversy related to the potential tissue damage,or even blindness,caused by retro-orbital bleeds has led to the development of facial and submandibular vein bleeding methods in mice.Blood collection from the saphenous vein in both mice and rats is another technique that has been developed. These procedures do not require anesthesia and therefore are suitable when the use of anesthetics may confound blood results or other data.

Collecting Blood from Facial, Submandibular, Saphenous &amp; Femoral Veins in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for ...

Catheter Based Intravenous Injection of Experimental Agents: A Technique for Delivering Experimental Agents Intravenously Through Jugular Vein in Mouse Model

Source: Bornert, A. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62515">In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow.</a> J. Vis. Exp. (2021)
In this video, we describe a method for implanting a catheter into the jugular vein of a mouse model through which experimental agents such as drugs and tracer dyes can be delivered intravenously in vivo. Such an arrangement when connected to other systems can also be used for monitoring hemodynamics and blood sampling.

Source: Bornert, A. et al. In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow. J. Vis. Exp. (2021)
In this ...