We thank Virginia Doherty and Junmei Yao for technical assistance. This research was funded by NIMH (R01 MH084966), and the U.S. Army Research Office and the Defense Advanced Research Projects Agency (grant W911NF-10-1-0059) to KAG.

All experiments were done using adult male Long-Evans rats. All procedures were in accordance with the US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Massachusetts Institute of Technology and the Animal Care and Use Review Office of the USAMRMC.
1. Preparation
<ol>
	<li>Heparinise the catheter and syringe by placing the shielded needle in a 500 &#181;l tube containing heparin (1,000 USP units/ml) and then aspirating and expelling heparin solution through the needle.
	<ol>
		<li>Attach a butterfly catheter to the syringe. Keep the shield over the needle of the catheter to protect the sharp tip from damage.</li>
		<li>Withdraw a volume of heparin that is slightly greater than the volume of blood that will be collected. Detach the syringe and fill it with air.</li>
		<li>Re-attach the syringe to the catheter and use the air to expel excess heparin solution; ensure only trace amounts remain in the tubing, needle, and syringe.</li>
		<li>Place the sterile catheter, with the syringe still attached, on a sterile surface.</li>
	</ol>
	</li>
	<li>Quickly secure the rat in a clean cloth ensuring that fore- and hindpaws are in a comfortable position and breathing is unrestricted.
	<ol>
		<li>Secure the wrap with hook and loop tape; ensure that external genitalia are not constricted.</li>
		<li>Have an assistant gently and firmly restrain the rat (abdomen and base of tail) on a solid work surface with the tail hanging off the edge of the counter.</li>
	</ol>
	</li>
</ol>
2. Blood Sampling
<ol>
	<li>Immerse the tail in 42 &#176;C water for 40-50 sec to dilate blood vessels and dry the tail with a paper towel. Locate the tail vein to be bled (rotate the whole body with the tail to prevent twisting the tail). 
	Note: Sufficient warming of the tail is critical for the rapid collection of a blood sample. If the vasculature is constricted, proper placement of the catheter is difficult, and blood flow is vastly reduced. A heating pad may be used as an alternative to water immersion.</li>
	<li>Identify the sampling point. 
	&#8203;Note: The artery lies along the mid-dorsal aspect of the tail; do not use this for sampling.
	<ol>
		<li>Target either the left and right tail veins that lie lateral to the artery. Pigmentation of the tail, which varies by strain and increases with age, may obscure some of the vasculature. Target a portion of the vein in the lower portion of the tail.</li>
		<li>Wipe the target area with 2% chlorhexidine antiseptic solution.</li>
	</ol>
	</li>
	<li>Create negative pressure in the syringe and catheter by withdrawing the plunger from zero to approximately 50 &#181;l.</li>
	<li>Hold the tail gently and firmly near the tip to keep the tail straight throughout sample collection. Ensure that blood flow is not occluded by an overly tight grip.</li>
	<li>Slowly insert the catheter into the vein at a shallow angle approximately 5 cm from the tip of the tail. When the vein is penetrated, blood will flow into the catheter. Slowly withdraw the plunger of the syringe to collect the desired volume at a steady rate (~20 &#181;l per sec).
	<ol>
		<li>Consult the veterinary staff for information about the maximum blood volume that can be collected. The maximum amount of blood that should be collected depends on the weight and health status of the rat. Do not withdraw more than 15% of total blood volume in a 14 day period. 
		Note: Blood is much more difficult to collect from animals that were acutely stressed in the minutes prior to sample collection because stress hormones constrict the vasculature. For example, moving the rat&#39;s home cage to a novel room, taking several minutes to wrap the animal, or repeated insertion of the catheter into a vein are all likely to trigger an acute stress response.</li>
		<li>Facilitate blood flow by &#8216;milking&#39; the vein. Run a finger along the length of the vein, from the base towards the tip of the tail, but remain more than 2 cm from the tip of the inserted needle or the catheter may become dislodged from the vein.</li>
		<li>If blood cannot be successfully collected from the initial site of catheter penetration, re-insert the needle further up the vein. If blood was collected at the initial site, re-pressurize the needle by disconnecting and then reconnecting the catheter and syringe prior to re-insertion in the vein. In general, avoid additional penetrations.</li>
		<li>As multiple penetrations can cause tail vein collapse, in which the blood supply to the tail is cut off and the soft tail tissue becomes necrotized, euthanize the rat if there is tail vein collapse.</li>
	</ol>
	</li>
	<li>When adequate sample volume is collected, release pressure in the syringe by disconnecting and reconnecting the catheter. Aspirate slightly using the syringe plunger (~50 &#181;l), and withdraw the needle from the vein. 
	Note: If the needle is withdrawn without first releasing the pressure in the syringe, blood will drip from the needle.</li>
	<li>Briefly apply pressure to the insertion site to stop bleeding, and wipe the area with antiseptic solution. Return the rat to its home cage.</li>
</ol>
3. Processing the Blood Sample
<ol>
	<li>Aspirate air to ensure no blood remains inside the catheter needle, and use scissors to cut the catheter tubing just above the needle. Expel the blood into a sterile 1.5 ml microcentrifuge tube. 
	Note: If blood is pushed through the needle, the shearing force may cause red blood cells to rupture which can interfere with many downstream assays. Remove the needle to avoid hemolysis.
	<ol>
		<li>To collect blood plasma, use tubes that contain EDTA as an anticoagulant (here, use 10 &#181;l of 0.1 M EDTA for 200-400 &#181;l of blood; ensure the concentration of EDTA used does not interfere with the downstream assay) and place on ice.
		<ol>
			<li>Spin whole blood samples at 2,100 x g in a refrigerated centrifuge (4 &#176;C) for 10 min within 10 min of collection. Elute the plasma, avoiding disturbing the red and white blood cell layers.</li>
		</ol>
		</li>
		<li>To collect blood serum, place samples (without anticoagulant) at room temperature for up to 30 min to enable clotting. Spin the collection tubes in a refrigerated centrifuge (4 &#176;C) at 2,000 x g. The serum may then be eluted.</li>
	</ol>
	</li>
	<li>Use samples immediately, or store at -80 &#176;C for up to one year.</li>
</ol>

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	<li>Vausort, M., Wagner, D. R., Devaux, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Noncoding+RNAs+in+Patients+with+Acute+Myocardial+Infarction.">Long Noncoding RNAs in Patients with Acute Myocardial Infarction.</a> Circ Res. 115 (7), 668-677 (2014).</li><li>Shah, R., 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=Biomarkers+for+Early+Detection+of+Colorectal+Cancer+and+Polyps:+Systematic+Review.">Biomarkers for Early Detection of Colorectal Cancer and Polyps: Systematic Review.</a> Cancer Epidemiol Biomarkers Prev. 23 (9), 1712-1728 (2014).</li><li>Chan, M. 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=Applications+of+blood-based+protein+biomarker+strategies+in+the+study+of+psychiatric+disorders.">Applications of blood-based protein biomarker strategies in the study of psychiatric disorders.</a> Prog Neurobiol. , (2014).</li><li>Cao, L., Li, L., Lin, D., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+learning+impairment+that+is+mediated+by+interleukin+1beta+in+rodents.">Isoflurane induces learning impairment that is mediated by interleukin 1beta in rodents.</a> PLoS One. 7 (12), e51431 (2012).</li><li>Culley, D. J., Baxter, M. G., Yukhananov, R., Crosby, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impairment+of+acquisition+of+a+spatial+memory+task+following+isoflurane-nitrous+oxide+anesthesia+in+rats.">Long-term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats.</a> Anesthesiology. 100 (2), 309-314 (2004).</li><li>Vahl, T. P., 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=Comparative+analysis+of+ACTH+and+corticosterone+sampling+methods+in+rats.">Comparative analysis of ACTH and corticosterone sampling methods in rats.</a> Am J Physiol Endocrinol Metab. 289 (5), E823-E828 (2005).</li><li>Kalinichenko, L. S., Koplik, E. V., Pertsov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+peripheral+blood+in+rats+with+various+behavioral+characteristics+during+acute+emotional+stress.">Cytokine profile of peripheral blood in rats with various behavioral characteristics during acute emotional stress.</a> Bull Exp Biol Med. 156 (4), 441-444 (2014).</li><li>McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+stress+hormones+in+health+and+disease:+Understanding+the+protective+and+damaging+effects+of+stress+and+stress+mediators.">Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators.</a> Eur J Pharmacol. 583 (2-3), 174-185 (2008).</li><li>Sanchez, A., Toledo-Pinto, E. A., Menezes, M. L., Pereira, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+norepinephrine+and+epinephrine+concentrations+in+adrenal+gland+of+the+rats+submitted+to+acute+immobilization+stress.">Changes in norepinephrine and epinephrine concentrations in adrenal gland of the rats submitted to acute immobilization stress.</a> Pharmacol Res. 48 (6), 607-613 (2003).</li><li>Meyer, R. M., Burgos-Robles, A., Liu, E., Correia, S. S., 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=A+ghrelin-growth+hormone+axis+drives+stress-induced+vulnerability+to+enhanced+fear.">A ghrelin-growth hormone axis drives stress-induced vulnerability to enhanced fear.</a> Mol Psychiatry. , (2013).</li><li>Knoll, A. T., Carlezon, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynorphin,+stress,+and+depression.">Dynorphin, stress, and depression.</a> Brain Res. 1314 (56-73), (2010).</li><li>Harvey, B. H., Brand, L., Jeeva, Z., Stein, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical/hippocampal+monoamines,+HPA-axis+changes+and+aversive+behavior+following+stress+and+restress+in+an+animal+model+of+post-traumatic+stress+disorder.">Cortical/hippocampal monoamines, HPA-axis changes and aversive behavior following stress and restress in an animal model of post-traumatic stress disorder.</a> Physiol Behav. 87 (5), 881-890 (2006).</li></ol>

The authors have nothing to disclose.

Here, we describe a quick and simple procedure for obtaining a blood sample from a rat which offers significant advantages over other commonly used techniques. First, it does not require anesthesia, in contrast with sampling from the jugular vein or retroorbital sinus. When blood samples are collected surrounding behavioral procedures, administration of anesthetics is undesirable because it can interfere with learning and memory4,5. Second, it offers the ability to collect larger blood volumes than other venipuncture techniques, such as collection from the saphenous or dorsal pedal veins. Using the technique described here, up to 1.5 ml of blood may be collected from a rat at a single time point, a volume which readily allows multiple assays to be run in parallel. Finally, this procedure minimizes the potential for tissue damage compared to tail tip amputation or retroorbital bleeding. The use of this procedure facilitates compliance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals, which require minimizing the pain and distress that result from laboratory procedures performed on animals.
It is recommended that investigators new to this method practice the restraint and tail bleeding techniques in order to minimize the time that experimental animals are restrained. Blood collected in less than 3 min from the initiation of restraint provides optimal results.
The protocol described here may be used for sampling 1 to 4 times per week, but no more than twice per day. While repeated blood collections may be performed, different sampling sites moving upwards from the base of the tail should be used, and the left and right tail veins should be alternated as sampling sites. The total blood volume of rodents is 6-7% of their body weight, and no more than 15% of the total blood volume should be collected within a 2 week period. Serum or plasma comprises approximately 40-60% of the collected sample volume.
Blood sampling via the lateral tail veins may also be performed in the mouse as described here with a few minor modifications. First, only small gauge (27 G) catheters may be used. Second, it is recommended to use a tube restrainer, rather than a wrap, to immobilize the mice. The volume of blood that may be obtained from the mouse using venipuncture of the submandibular vascular bundle (200-500 &#181;l) is greater than can be safely collected from the tail vein (200 &#181;l maximum). Because sampling blood from the submandibular vascular bundle requires minimal restraint and may yield more blood, this is the preferred route for sampling in the mouse.
The rapidity with which this procedure may be performed, along with its minimally invasive nature, also minimizes the potential perturbation of blood-based measures by the acute stress response6. The acute stress response can alter circulating levels of many molecules, including interleukins and other immune-active factors7, hormones of the hypothalamic-pituitary-adrenal axis8, hormones in the sympathetic nervous system9, ghrelin10, endogenous opioids11, dopamine, and serotonin12. If resting circulating measures of these molecules or others regulated by these molecules are desired, it is important to minimize the stress response, which is triggered within as little as a minute of the start of stressor exposure.
Stress responses not only alter the composition of the blood, but also represent a technical obstacle for blood sampling because of the constriction of vasculature via increased drive from the sympathetic nervous system. It becomes increasing difficult to obtain steady blood flow from a rat that is mounting an acute stress response. Therefore, the animal&#39;s distress must be minimized in order to rapidly obtain samples that reflect the physiological state of interest.

Biomarkers obtained from blood provide useful diagnostic, predictive, and stratifying measures in many clinical contexts, including cardiovascular disease1, cancer sciences2, and psychiatric disease3. They may also be used in basic science to assess the &#8220;state&#8221; of an organism, including the degree of hunger, inflammation, or stress present. Such measures can be influenced by variables that may or may not be critical to the question of interest, including the time of day that the sample is obtained and the gender of the subjects. It may also be influenced by the stress induced during the blood sampling procedures itself. Stress hormones and the perception of pain can rapidly alter the composition of the blood.
Rodents are the most commonly used laboratory animal, and multiple methods have been developed for blood collection. The ideal method of blood sampling should have minimal physiological impact on the animal, require no anesthesia, allow rapid and repeated sampling, and provide sufficient blood volume for numerous downstream applications. Popular techniques for collecting blood such as catheterization of the jugular vein or tail tip amputation do not meet these criteria.
The aim of this protocol is to describe a blood sampling technique for use in rats that is minimally stressful, does not require anesthesia, allows for multiple blood collections within a single subject, and provides a relatively large sample volume such that multiple assays may be performed on a single sample. The goal of this method is to obtain blood samples that are minimally influenced by the acute stress response.

<table><tbody><tr><td>Sodium heparin (1,000 USP units/ml)</td><td>Patternson Veterinary Supply</td><td>25021040010</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>JT Taylor</td><td>JT2020-01</td><td></td></tr><tr><td>Dermachlor rinse-chlorhexadine</td><td>Butler Schein</td><td>6356</td><td>Topical antiseptic solution, 2% chlorhexidine gluconate</td></tr><tr><td>SURFLO Winged Infusion Sets, Terumo, butterfly catheters</td><td>VWR Scientific</td><td>TESV25BLK</td><td></td></tr><tr><td>BD Tuberculin 1 cc syringes</td><td>VWR Scientific</td><td>BD309659</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>VWR Scientific</td><td>89202-682</td><td></td></tr><tr><td>500 &amp;mu;l microcentrifuge tubes</td><td>VWR Scientific</td><td>21150-330</td><td></td></tr><tr><td>Scissors, stainless steel, 5&quot;</td><td>VWR Scientific</td><td>82027-586</td><td></td></tr><tr><td>500 ml plastic beaker</td><td>VWR Scientific</td><td>414004-149</td><td></td></tr><tr><td>Clean cloth wrap</td><td>Butler Schein</td><td>2993</td><td></td></tr><tr><td>Velcro tape, 0.75 in width</td><td>Monoprice</td><td>B004AF9II6</td><td>Hook and loop tape</td></tr><tr><td>Timer</td><td>VWR Scientific</td><td>62344-641</td><td></td></tr></tbody></table>

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 plasma collected from the lateral tail vein as described in the protocol gives a plasma sample that was translucent and pale yellow in appearance. As shown in Figure 1, hemolysis in a sample imparts a red tint to the plasma. The acute stress response can rapidly alter the composition of blood. For example, circulating corticosterone concentration can markedly increase within 10 min of stressor exposure, as shown in Figure 2. The low basal levels of corticosterone obtained with this method prior to stressor exposure reveal that the sampling procedure itself is not a significant source of stress.
<img alt="Figure 1" src="/files/ftp_upload/52766/52766fig1.jpg" /> 
Figure 1: Sample appearance. (A) A hemolyzed sample is shown. After centrifugation, the plasma or serum layer (surface indicated by the black arrow) appears tinged with pink or red. Darker tints indicate greater levels of hemolysis. (B) After centrifugation, a properly collected sample will have a clear, yellowish appearance to the upper band (surface indicated by the black arrow), which corresponds to the non-hemolyzed plasma or serum. When removing this layer, it is important to not disturb the underlying whole blood, either by pushing the pipette tip into the whole blood layer or by aspirating some of the whole blood into the tip. Any plasma or serum contaminated with whole blood should be discarded.
<img alt="Figure 2" src="/files/ftp_upload/52766/52766fig2.jpg" /> 
Figure 2: Plasma corticosterone is rapidly elevated following a stressful experience. Blood was obtained from the lateral tail vein of adult female Long-Evans rats before and 10 min following exposure to 4 tones (10 sec, 2 kHz, 85 dB) co-terminating with footshocks (1 sec, 350 &#181;A). Blood plasma corticosterone at baseline (290.4 &#177; 138.8 pg / ml) was significantly less than the levels observed 10 min following presentation of the footshock stress (2204.8 &#177; 454.5 pg / ml, p = 0.02, n = 4), as determined by paired t-test. *, p &#60;0.05

Watch this Scientific Journal Video about Sampling Blood from the Lateral Tail Vein of the Rat at JoVE.com

Sampling Blood from the Lateral Tail Vein of the Rat

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, ...

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Research

JoVE Journal

Biology

102.4K Views.  Massachusetts Institute of Technology. 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.

Video: Sampling Blood from the Lateral Tail Vein of the Rat

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: ...

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

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 ...

Medicine

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 ...

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

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 ...

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 ...