Name: Video: Auteur in de schijnwerpers: contextafhankelijke bloedafnamemethoden bij ratten
Uploaded: 2023-06-30T14:30:00.000+00:00
Duration: 6 min

This work was funded by Shriners Children&#39;s Boston (B. E. U., K.U., C.L.C.). L.C. is funded by &#34;La Bourse des Gueules Cass&#233;es&#34;, &#34;La Bourse Ann&#233;e Recherche&#34;, and &#34;La Bourse de l&#39;Amicale des Anciens Internes des H&#244;pitaux de Paris&#34;. Y.B. is funded by &#34;La Bourse des Gueules Cass&#233;es&#34;. Y.B. and I.F.v.R. are funded by the Shriners Hospitals for Children (Fellowships ID is #84308-BOS-22 #84302-BOS-21 respectively). This material is partially based upon work supported by the National Science Foundation under Grant No. EEC 1941543. Partial support from the US National Institutes of Health (R01EB028782, R56AI171958, and R01DK114506) is gratefully acknowledged.&#160;Figure 1 was created with BioRender.com.

The procedures were performed on 3 month old male Lewis rats, each weighing 300-400 g. A total of 24 animals were included, with three puncture conditions: 12 rats underwent tail vein puncture without anesthesia (group TV without anesthesia), and another 12 rats were anesthetized to undergo both tail vein puncture (group TV with anesthesia) and penile vein puncture (group PV with anesthesia). All the procedures were approved and respected the Institutional Animal Care and Use Committee (IACUC) guidelines. All the animals were euthanized at the end of the study (after a 1 month follow-up) by carbon dioxide overdose. See the Table of Materials for details related to all the materials and instruments used in this protocol.
1. General guidelines
<ol>
	<li>In line with the IACUC guidelines, ensure that the maximum blood volume drawn is no more than 10% of the total blood volume every 2 weeks9. For example, a rat of 300 g should have a total blood volume of approximately 19.2 mL. In the case of a protocol requiring four blood draws in the first week alone (day 0, day 1, day 3, day 7), limit the collection to a maximum of 250 μL of blood per sample.</li>
	<li>For procedures performed under anesthesia, administer isoflurane via a precision vaporizer to anesthetize the animal. Induce anesthesia in a chamber with a dose of 3%-5% isoflurane for 5 min, and maintain using a dose of 1%-3% isoflurane through a nose cone during the procedure. Adjust the isoflurane level based on continuous monitoring of the respiratory rate. Verify whether sedation is sufficient by toe pinch before starting the procedure.</li>
	<li>Do not leave the animal unattended during the procedure or until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>After the blood collection, monitor the animal until full recovery before returning it to its cage, and do not introduce it to the company of other animals until fully recovered. 
	​NOTE: In agreement with veterinary services, no post-procedure pain medication was necessary after tail vein or penile vein puncture.</li>
</ol>
2. Blood draw from the penile vein
<ol>
	<li>Preparation
	<ol>
		<li>Prepare the following equipment: sterile gauze, gloves, alcohol wipes, a microcapillary blood collection EDTA tube (purple cap), and a 30 G insulin syringe (30 U or 50 U).</li>
		<li>Take the rat out of its cage, and put it in a chamber for induction with isoflurane via a precision vaporizer (dose: 3%-5%). Once the animal is sedated, transfer it to the procedure table, and lay the animal on its back with its nose placed in the nose cone to maintain the anesthesia. Monitor the respiratory rate, and adjust the isoflurane level accordingly (maintenance dose: 1%-3%). Verify that the animal is sufficiently sedated by toe pinch before starting the procedure.</li>
	</ol>
	</li>
	<li>Blood sampling
	<ol>
		<li>Move the plunger back and forth in the syringe several times to smoothen the withdrawal. Create negative pressure in the syringe by pulling on the plunger to remove a couple of microliters.</li>
		<li>With the help of the non-dominant hand, retract the foreskin from the end of the penis, and hold the glans between the index and thumb, pulling gently. The dorsal penile vein will appear as a superficial blue cord. See Figure 1 and Figure 2.</li>
		<li>With the eye of the needle pointing upward, insert the insulin syringe into the vein at an angle of 35°. Once the needle has entered the vein, blood will flow into the syringe.</li>
		<li>Slowly withdraw the plunger of the syringe at a slow and steady rate to collect the desired volume. 
		NOTE: Do not withdraw the plunger too fast, as this will cause the vein to collapse and stop the blood flow.</li>
		<li>If the blood flow decreases, rotate the needle slightly clockwise or counterclockwise.</li>
		<li>Remove the syringe. A blood drop will form on the puncture site, the aspiration of which will allow the collection of a few more microliters of blood in the case of a non-sterile procedure.</li>
		<li>If the first puncture fails, reinsert the needle more proximally on the vein. 
		NOTE: Unlike for tail vein sampling, the iterative puncture of the dorsal penile vein is usually unsuccessful.</li>
		<li>Apply light pressure to the puncture site to stop the bleeding, and wipe the area with a new alcohol wipe.</li>
		<li>Place the penis back in its neutral position.</li>
		<li>Turn the isoflurane off, and monitor the rat until complete recovery. Return the rat to its cage.</li>
	</ol>
	</li>
</ol>
3. Tail vein puncture
<ol>
	<li>Preparation
	<ol>
		<li>Prepare the following equipment: a plastic restraining holder, sterile gauze, gloves, alcohol wipes, a microcapillary blood collection EDTA tube (purple cap), and a 28 G 1/2 insulin syringe (30 U or 50 U).</li>
		<li>Take the rat out of its cage, and quickly secure it in a plastic restraining cone. Close the large end of the cone around the base of the tail. Ensure that the animal is comfortable and that breathing is unrestricted throughout the whole procedure.</li>
		<li>Dip the tail in warm water (37 °C) for about 1 min to dilate the vein. Dry the tail with a paper towel. Place the animal (in its restrainer) face down, with the tail lying on a heating pad.</li>
		<li>Select the right or left tail vein (blue line) for sampling by rotating the entire animal to either side (this avoids the twisting of the tail). Use the terminal third of the tail for blood vessel puncturing since the vessels become more superficial in this zone. The artery is ventral, and the two veins are lateral10.</li>
		<li>Wipe the tail with 70% ethanol wipes at the puncture site.</li>
		<li>Place the tail on the edge of the heating pad to create an angle in the terminal third of the tail. This brings the vein to the surface and creates more space for taking the sample.</li>
	</ol>
	</li>
	<li>Blood sampling
	<ol>
		<li>Move the plunger back and forth in the syringe several times to smoothen the withdrawal. Create negative pressure in the syringe by pulling on the plunger to remove a couple of microliters.</li>
		<li>With the help of the non-dominant index and middle finger, secure the tail flat on the heating pad. Place the middle finger proximally and the index finger distally, with the puncture site between these two fingers. Apply more pressure on the middle finger than on the index to secure the tail, occluding the vessel only proximally and allowing the blood to pool. See Figure 1 and Figure 3.</li>
		<li>With the eye of the needle pointing upward, slide the insulin syringe against the index finger until it is inserted into the vein (this creates an angle of 35° between the needle and the tail). Once the needle has entered the vein, blood will flow into the syringe. At this point, release the pressure on the index and middle finger to ensure that the blood flow is not occluded.</li>
		<li>Slowly withdraw the plunger of the syringe at a steady rate to collect the desired volume. 
		NOTE: Do not withdraw the plunger too fast; this will cause the vein to collapse and stop the blood flow.</li>
		<li>If the blood flow decreases, rotate the needle slightly in either direction.</li>
		<li>Remove the syringe from the tail. A blood drop will form on the tail's puncture site. The aspiration of this blood allows for the collection of a few more microliters of blood in the case of a non-sterile procedure.</li>
		<li>If the first puncture fails, reinsert the needle more proximally on the vein. 
		​NOTE: The vein becomes progressively more profound as it approaches the tail base. If there is no blood flow in the syringe, increase the angle between the syringe and the tail, or rotate the needle.</li>
		<li>Apply pressure to the puncture site to stop the bleeding, and wipe the area with a new alcohol wipe. Remove the rat from the plastic cone, and return it to its cage.</li>
	</ol>
	</li>
	<li>Tail vein puncture under anesthesia
	<ol>
		<li>Perform step 2.1.1 and step 2.1.2 for inducing and maintaining anesthesia.</li>
		<li>Perform steps 3.1.3-3.2.7 for blood collection; see Figure 1.</li>
		<li>Perform step 2.2.10 for animal recovery.</li>
	</ol>
	</li>
</ol>
<img alt="Injecting anesthesia into mice, illustration; laboratory method for animal study, syringe technique." class="xfigimg" src="/files/ftp_upload/65513/65513fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Schematics of the different puncture methods in this protocol. (A) Modified tail vein puncture on a conscious, restrained animal; (B) modified tail vein puncture and penile vein puncture under anesthesia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65513/65513fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Comparative Study of Blood Collection Techniques in Rats

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	<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>Beeton, C., Garcia, A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drawing+blood+from+rats+through+the+saphenous+vein+and+by+cardiac+puncture.">Drawing blood from rats through the saphenous vein and by cardiac puncture.</a> Journal of Visualized Experiments. (7), 266 (2007).</li><li>Luzzi, M., 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=Collecting+blood+from+rodents:+A+discussion+by+the+Laboratory+Animal+Refinement+and+Enrichment+Forum.">Collecting blood from rodents: A discussion by the Laboratory Animal Refinement and Enrichment Forum.</a> Animal Technology and Welfare. 4 (2), 99-102 (2005).</li><li>Wang, L., 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=Repetitive+blood+sampling+from+the+subclavian+vein+of+conscious+rat.">Repetitive blood sampling from the subclavian vein of conscious rat.</a> Journal of Visualized Experiments. (180), e63439 (2022).</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=Blood+sampling+from+the+retro-orbital+plexus,+the+saphenous+vein+and+the+tail+vein+in+rats:+Comparative+effects+on+selected+behavioural+and+blood+variables.">Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: Comparative effects on selected behavioural and blood variables.</a> Laboratory Animals. 35 (2), 131-139 (2001).</li><li>Harikrishnan, V. S., Hansen, A. K., Abelson, K. S., S&#248;rensen, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+various+methods+of+blood+sampling+in+mice+and+rats:+Effects+on+animal+welfare.">A comparison of various methods of blood sampling in mice and rats: Effects on animal welfare.</a> Laboratory Animals. 52 (3), 253-264 (2018).</li><li>Zeller, W., Weber, H., Panoussis, B., B&#252;rge, T., Bergmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+blood+sampling+from+the+sublingual+vein+of+rats.">Refinement of blood sampling from the sublingual vein of rats.</a> Laboratory Animals. 32 (4), 369-376 (1998).</li><li>Nightingale, C. H., Mouravieff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+and+simple+method+of+intravenous+injection+into+the+laboratory+rat.">Reliable and simple method of intravenous injection into the laboratory rat.</a> Journal of Pharmaceutical Sciences. 62 (5), 860-861 (1973).</li><li>Blood collection: The rat. IACUC Guideline. UCSF Office of Research Institutional Animal Care and Use Program Available from: <a target="_blank" href="https://iacuc.ucsf.edu/sites/g/files/tkssra751/f/wysiwyg/guidelines%20-%20Blood%20Collection%20-%20Rat.pdf">https://iacuc.ucsf.edu/sites/g/files/tkssra751/f/wysiwyg/guidelines%20-%20Blood%20Collection%20-%20Rat.pdf</a> (2022)</li><li>Staszyk, C., Bohnet, W., Gasse, H., Hackbarth, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+of+the+rat+tail:+A+histological+re-examination+with+respect+to+blood+vessel+puncture+methods.">Blood vessels of the rat tail: A histological re-examination with respect to blood vessel puncture methods.</a> Laboratory Animals. 37 (2), 121-125 (2003).</li><li>Constantinides, C., Mean, R., Janssen, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane+anesthesia+on+the+cardiovascular+function+of+the+C57BL/6+mouse.">Effects of isoflurane anesthesia on the cardiovascular function of the C57BL/6 mouse.</a> ILAR Journal. 52 (3), e21-e31 (2011).</li><li>Hernaningsih, Y., Akualing, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+hemolysis+on+plasma+prothrombin+time+and+activated+partial+thromboplastin+time+tests+using+photo-optical+method.">The effects of hemolysis on plasma prothrombin time and activated partial thromboplastin time tests using photo-optical method.</a> Medicine. 96 (38), 7976 (2017).</li><li>Powles-Glover, N., Kirk, S., Jardine, L., Clubb, S., Stewart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+haematological+and+clinical+pathology+effects+of+blood+microsampling+in+suckling+and+weaned+juvenile+rats.">Assessment of haematological and clinical pathology effects of blood microsampling in suckling and weaned juvenile rats.</a> Regulatory Toxicology and Pharmacology. 69 (3), 425-433 (2014).</li></ol>

None of the authors have any conflicts of interest to declare.

The tail vein puncture is an efficient method to obtain blood from a conscious rat. However, when an animal is under anesthesia, the effect of isoflurane can lead to vessel spasms and make tail vein puncture unsuitable11. As shown in this study, an alternative in this situation is to collect blood from the penile vein, which is more successful and yields a significantly greater volume of blood in less time. It is important to remember that in the case of failure with this method on the first try, subsequent attempts may be unsuccessful. In contrast, tail vein puncture allows for several subsequent punctures in case of difficulty on the first try (there are two veins, and puncturing higher up on the tail can be attempted)1. However, iterative punctures can cause hemolysis in the sample, which distorts the results due to the release of hemoglobin and the internal components of erythrocyte membranes. This is especially true if the plasma is analyzed12. Avoiding multiple punctures and obtaining the sample in a single blood draw is preferable.
In this work, the blood sampling did not exceed 250 &#181;L per sample in accordance with this protocol and the IACUC guidelines to respect animal welfare in the case of multiple sampling. Advances in bioanalytical techniques have enabled the use of microsamples of less than 50 &#181;L to assess the blood biochemistry and metabolic parameters 13. Therefore, 250 &#181;L is sufficient to conclude that both methods are efficient for future studies. However, both the tail vein and the penile vein are small vessels and do not allow for the collection of large amounts of blood. These described methods are appropriate for repeated sampling and the monitoring of living animals. If large quantities of blood are necessary (for example, for end-of-study procedures), other methods yielding more blood-such as cardiac puncture-should be considered.
These described procedures are two among many; our choice of procedure was motivated by a few advantages. Both these techniques can be done by a single operator. Using the index and middle finger to stabilize the tail vein in the modified tail vein technique makes complementary human restraining unnecessary. However, it is necessary to assess the animal's welfare, and assistance by a professional (e.g., a veterinarian or veterinary technician) is always recommended to avoid subjecting the animal to unnecessary pain or distress2.
Moreover, previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal. In contrast, tail vein or dorsal penile vein puncture are rarely fatal to the rat, even if not well executed. This study had some failures with both methods, but no other adverse effects or deaths were observed. Some cases of urine retention after dorsal penile injections have been reported, but it is unclear whether the injection itself or the agent injected is responsible for this outcome. Neither this adverse effect nor abnormal healing or infection at the puncture site were noted during the study period. Furthermore, the daily assessments of the conditions of the animals did not reveal pain or distress in any groups (no porphyrin staining, no weight loss, animals judged to be comfortable by the research team and veterinary professionals). However, prolonged restraining and multiple punctures can cause animal distress. To avoid this, the needle should be rotated when blood flow slows down instead of repeating the puncture. Properly warming the tail with hot water and a heating pad to vasodilate the tail vein, as well as practicing these methods, are recommended to reduce the restraining time.
The tail vein puncture and dorsal penile vein puncture methods allow for fast sampling (under 6 min) and yield sufficient blood volumes for most assays. When done on a conscious animal, tail vein puncture is an efficient and reliable method to obtain blood. However, on a sedated animal, anesthetic agents tend to cause vessel spasms, and the tail vein is subject to important thinning11. In this scenario, penile dorsal vein puncture provides a better success rate than tail vein puncture, which tends to be unreliable for blood collection. However, one limitation of the penile vein blood draw is that it can only be performed on male rats and is, therefore, unsuitable for female rat studies. Therefore, according to the context (i.e., the sex of the animal, perioperative or post-operative blood draw, animals with thin tail veins), both penile vein puncture and tail vein puncture can be used alternately for repeated blood draws, even by researchers with little to no experience with animal studies.

Blood sampling is necessary for most animal studies, both in vivo and in vitro. In rats, as the frequency and amount of blood sampling can be significant, it is helpful to have different alternatives for collection. Various methods have been described in previous studies.
The most commonly used techniques are tail vein puncture and saphenous vein blood draw. Tail vein sampling is suitable for all rat strains. With proper training, the procedure is simple to perform and causes minimal distress to the animal1. Similarly, the saphenous vein blood draw, provided it is done properly, is also a quick and simple collection method. Neither method requires anesthesia, and both allow for repeated draws of small amounts of blood. However, the saphenous vein puncture usually yields a lower blood volume1 and requires the presence of two people to leave one hindlimb exposed for puncture2.
If large amounts of blood need to be collected from a single animal, cardiac puncture or puncture of the vena cava may be used (up to 10 mL of blood can be drawn from a 150 g rat with cardiac puncture2). These techniques require anesthesia and are terminal procedures. The animal must be euthanized after any of these two techniques2. The jugular stick is an alternative that can be used if large amounts of blood need to be collected in a study that has not yet reached its endpoint. However, this technique also requires significant technical skills to avoid harm to the animal; hence, its use should be limited3.
Other techniques, such as the subclavian vein blood draw, do not need the use of anesthetics before blood collection and allow for repeated sampling of small volumes of blood. However, restrained handling and appropriate needle incision are required for this technique. An improper operation may result in animal pain or even mortality, and the training for this method may be fastidious4. 
Other anecdotal procedures include the orbital puncture and the sublingual vein puncture, both of which require an anesthetic, and neither are recommended nor widely used. Though previous studies have shown faster blood collection by orbital puncture than by tail vein puncture, it was found that orbital puncture under diethyl-ether anesthesia was less well tolerated than the latter method (based on the animals' excitation scores and urine production)5. Moreover, this method is highly influenced by the skill of the person who performs the procedure and is mainly performed by experienced veterinarians. Comparably, the sublingual vein puncture is less distressing and is recommended for repeated blood sampling6. However, this technique presents severe adverse effects such as reduced food and water intake, which can lead to the animal's death7. 
This study describes two methods used in our laboratory for repeated blood sampling. Tail vein puncture can be performed on a conscious animal, and the tissue damage and adverse effects are minimal. The modification of this technique in this study includes stabilizing the tail with the index and middle finger, which allows a single operator to perform the blood collection. The dorsal penile vein puncture has already been described for simple intravenous injections. This technique is performed under anesthesia and allows for a reliable blood source in case of difficulties with other methods (e.g., during the immediate postoperative period, with a small animal, when performing perioperative blood draw under anesthesia). Similar to tail vein sampling, the injury at the puncture site will have a minor overall effect on the animal compared to the techniques mentioned above8. The aim of this methods paper is to offer inexperienced researchers simple and reliable blood sampling alternatives according to the context (e.g., for procedures done under anesthesia, for studies including male rats, for animals with thin tail veins).

<table><tbody><tr><td>0.5 mL | 28 G &amp;frac12;&amp;nbsp; Insulin Syringes</td><td>BD</td><td>329424</td><td>for tail vein puncture</td></tr><tr><td>0.5 mL | 30 G x 5/16 Insulin Syringes</td><td>BD</td><td>320468</td><td>for penile vein puncture</td></tr><tr><td>250 L&amp;nbsp; Microtainer blood collection tubes with K2EDTA</td><td>BD</td><td>365974</td><td></td></tr><tr><td>Gauze Sponges</td><td>Curity</td><td>6939</td><td></td></tr><tr><td>Isoflurane Auto-Flow Anesthesia Machine</td><td>E-Z Systems</td><td>EZ-190F</td><td>for penile vein puncture</td></tr><tr><td>Isoflurane, USP</td><td>Patterson Veterinary</td><td>1403-704-06</td><td>for penile vein puncture</td></tr><tr><td>Nosecone for Anesthesia</td><td>World Precision Instruments</td><td>EZ-112</td><td>for penile vein puncture</td></tr><tr><td>Rodent Restraint Cone</td><td>Harvard Apparatus</td><td>ST2 52-95-86</td><td>for tail vein puncture</td></tr><tr><td>Small Animal Heated Operating Table (Adjustable)</td><td>Peco Services Ltd</td><td>69023</td><td></td></tr><tr><td>Webcol Alcohol prep pads</td><td>Simply Medical</td><td>5110</td><td></td></tr></tbody></table>

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.

Success was defined as a blood draw yielding at least 100 µL of blood in under 10 min (from the puncture time to the end of the blood collection), and failure was defined as a blood draw yielding less than 100 µL of blood or taking more than 10 min to retrieve the required blood volume. A maximum of 250 µL of blood per sample was allowed. The statistical analyses were conducted using a one-way ANOVA test for multiple comparisons and the chi-squared test. The data were presented as mean value ± standard deviation, and p &lt; 0.05 was used as the cut-off for determining statistical significance.
The comparison of the success rates showed similar results for tail vein puncture in conscious rats (92%) and penile vein puncture under anesthesia (83%)(p = 0.0543), as shown in Figure 4. Interestingly, under anesthesia, the tail vein became very unreliable, and tail vein puncture under anesthesia had only a 25% success rate in this study, probably due to thinning of the vein. In the case of anesthesia, the penile vein puncture was more successful than the tail vein puncture for sampling (p &lt; 0.0001).
We compared the blood volumes collected and the procedure durations among tail vein and dorsal penile vein puncture performed on rats under anesthesia and tail vein puncture performed on conscious rats. Figure 5 shows that tail vein puncture without anesthesia (217.5 μL ± 69.04 μL) and penile vein under anesthesia (185.8 μL ± 66.4 μL) yielded comparable quantities of blood (p = 0.4966), and these volumes of blood were significantly higher than the volume collected with tail vein puncture under anesthesia (54.4 μL ± 68.8 μL) (p &lt; 0.0001).
The duration of the procedure was similar in the penile vein puncture under anesthesia group (315.2 s ± 160 s) and the tail vein puncture without anesthesia group (262.5 s ± 171 s) (p = 0.6632). Figure 6 shows that sampling was performed in less than 6 min in both groups, whereas the tail vein puncture under anesthesia took more than 8 min (500.8 s ± 196 s) due to multiple failures (p &lt; 0.0382).
<img alt="Laboratory injection procedure in mice, experiment setup with syringe, gloved hands, research context." class="xfigimg" src="/files/ftp_upload/65513/65513fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Dorsal penile vein puncture method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65513/65513fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Injecting snake, gloved hands, medical experiment, using syringe, controlled environment." class="xfigimg" src="/files/ftp_upload/65513/65513fig03v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Modified tail vein puncture method. Note that the tail is held down and the puncture site is located between the index and middle fingers. The syringe should rest and slide against the index finger to maintain a stable puncture angle. The use of the non-dominant hand allows for the stabilization of the tail on a conscious animal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65513/65513fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Success rate comparison bar chart; anesthesia sampling, statistical significance indicated." class="xfigimg" src="/files/ftp_upload/65513/65513fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Success rates with tail vein puncture under anesthesia, penile vein puncture under anesthesia, and tail vein puncture without anesthesia. **** p &lt; 0.0001 with the chi-squared test. Abbreviations: TV = tail vein; PV = penile vein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65513/65513fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph comparing blood volume under different sampling methods; anesthesia impact analysis." class="xfigimg" src="/files/ftp_upload/65513/65513fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Comparison of the blood volumes (in μL) obtained in the three groups. ***p &lt; 0.001; ****p &lt; 0.0001 with ANOVA analysis for multiple comparisons. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65513/65513fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar chart comparing duration in seconds for TV and PV under anesthesia and TV without anesthesia." class="xfigimg" src="/files/ftp_upload/65513/65513fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Comparison of the sampling duration (in seconds), defined as the time from the moment of puncture to the end of the blood draw, in the three groups. Failure was defined as a blood draw duration lasting longer than 600 s (10 min). *p &lt; 0.05; **p &lt; 0.01 with ANOVA analysis for multiple comparisons. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65513/65513fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Context Dependent Blood Collection Methods in Rats at JoVE.com

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.

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 ...

modified-tail-vein-penile-vein-puncture-for-blood-sampling-rat

Nanyang Technological University

Research

JoVE Journal

Medicine

6.2K Views.  Massachusetts General Hospital. Het probleem van herhaalde bloedafnamemethoden moet worden aangepakt, aangezien de meeste studies bij ratten dit vereisen. Het is belangrijk om er rekening mee te houden dat een bemonsteringsmethode in een bepaalde context zeer efficiënt kan zijn, maar ongeschikt kan worden voor een ander onderzoek. Dit protocol is bedoeld om eenvoudige en betrouwbare alternatieven voor bloedafname te bieden aan onderzoekers.Beide methoden kunnen door één operator worden uitgevoerd en vereisen geen ...

Video: Auteur in de schijnwerpers: contextafhankelijke bloedafnamemethoden bij ratten 

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Biology

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

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.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat&#39;s recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Detailed microsurgical techniques are demonstrated to establish a longer-term jugular vein cannulation rat model for sequential blood collection in the same animal. Physiological and hematological parameters have been monitored during the rat's recovery phase. This model has been applied to study pharmacokinetics of orally administered polyphenol without inducing animal stress.

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, ...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Neuroscience

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 ...

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.

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

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