The authors have no acknowledgements to make.

All procedures were approved by the Tongji Medical College Council on the Animal Care Committee of Huazhong University of Science and Technology (Wuhan, China). The manuscript was prepared according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines6.
1. Husbandry
<ol>
	<li>Use Sprague-Dawley (SD) rats (n = 20, 10 males, 8 week-old, weighing 261 to 291 g, mean = 272.85 &#177;9.07 g) from the Experimental Animals Center of Huazhong University of Science and Technology and maintain rats in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No.85-23, revised 1996)</li>
	<li>House under standard conditions with free access to food and drinking water. Keep rats in 530 cm2 cages with wood-shaving bedding. House two rats in each cage. Keep all cages open to the room environment (no micro-isolation or ventilated caging)</li>
	<li>Ensure the staff involved in the experimental protocol regularly handle the rats.</li>
	<li>Maintain room temperature between 21 &#176;C and 23 &#176;C. Give rates a normal salt diet (0.3% NaCl) throughout the study.</li>
</ol>
2. Blood Collection Procedure
NOTE: The vacuum blood collection system consists of a double-pointed needle [22 G (0.7 mm x 25 mm)], a plastic holder, and a series of vacuum tubes (2 mL) with rubber stoppers (Figure 1).
<ol>
	<li>Place the rat in a plastic restraining holder and wash the tail with warm (20-30 &#176;C) water to remove any visible dirt or feces. While an assistant holds the tail in an extended position, wipe the tail with 70% ethanol, and clean with gauze to make the vein clear.</li>
	<li>Insert the 22 G butterfly needle tip into one of the lateral tail veins (around 5 mm) at a position approximately 2-3 cm away from the tip of the tail at angle of approximately 20&#176; (Figure 2). Collect blood into the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube (Figure 3). 
	NOTE: The maximal collection blood volume is 1.2 mL7.</li>
	<li>After blood collection, remove the needle and gently apply pressure to the puncture site with gauze for 15-30 s to stop the flow of blood. Then, release the rat from the plastic restraining holder and return the rat to its cage.</li>
	<li>Use tubes that contain EDTA as an anticoagulant to collect blood plasma. Gently invert the tube several times to mix anticoagulant in the blood and place the samples on ice vertically.</li>
	<li>Centrifuge the blood sample collection tubes in a refrigerated centrifuge at 2,000 x g for 10 min to separate plasma or serum.</li>
	<li>Extract the plasma/serum, taking care not to disturb the red and white blood cell layers. To collect blood serum, use tubes without anticoagulant. Both types of sample can be used immediately, or stored at -80 &#176;C for up to one year. 
	NOTE: Successful blood collection was defined as obtaining 1.2 mL blood each time. Maximal puncture time was set to three trials, if the total blood volume was lower than 1.2 mL after the third puncture, it was defined as a failed blood collection. The sampling duration starts from the tail vein puncture to the removal of the butterfly needle after blood collection.</li>
	<li>Collect blood twice within two weeks7.
	<ol>
		<li>To test and verify the feasibility of this method, train two medical students (beginners) for 2 h before performing this blood collection protocol. Use twenty rats and draw blood from 10 rats by 2 experienced fellows (experimental group) and from 10 rats by the 2 medical students (beginner group).</li>
	</ol>
	</li>
</ol>
3. Statistical Analysis
<ol>
	<li>Express data as a mean value &#177; standard deviation and analyze with SPSS Statistics 17. An &#945; value of 0.05 was chosen, therefore p &#60;0.05 was considered statistically significant.</li>
</ol>

<ol>
	<li>Brown, C. <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+tail+of+a+rat.">Blood collection from the tail of a rat.</a> Lab Anim (NY). 35 (8), 24-25 (2006).</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> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</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> Lab Anim. 37 (2), 121-125 (2003).</li><li>Lee, G., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+blood+from+the+lateral+tail+vein+of+the+rat.">Sampling blood from the lateral tail vein of the rat.</a> J. Vis. Exp. (99), (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy WHO Guidelines Approved by the Guidelines Review Committee. , (2010).</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> Lab Anim. 44 (4), 377-378 (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> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Eder, J. M., Cutter, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+device+for+collecting+cord+blood.">A new device for collecting cord blood.</a> Obstet Gynecol. 86 (5), 850-852 (1995).</li><li>Reynolds, B. S., 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=Comparison+of+a+new+device+for+blood+sampling+in+cats+with+a+vacuum+tube+collection+system+-+plasma+biochemistry,+haematology+and+practical+usage+assessment.">Comparison of a new device for blood sampling in cats with a vacuum tube collection system - plasma biochemistry, haematology and practical usage assessment.</a> J Feline Med Surg. 9 (5), 382-386 (2007).</li><li>Wiwanitkit, V. <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+blood+specimens+from+plain+and+gel+vacuum+blood+collection+tubes.">Comparison of blood specimens from plain and gel vacuum blood collection tubes.</a> J Med Assoc Thai. 84 (5), 723-726 (2001).</li><li>Wollowitz, A., Bijur, P. E., Esses, D., John Gallagher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+butterfly+needles+to+draw+blood+is+independently+associated+with+marked+reduction+in+hemolysis+compared+to+intravenous+catheter.">Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter.</a> Acad Emerg Med. 20 (11), 1151-1155 (2013).</li></ol>

The authors have nothing to disclose

The present report describes an easy-to-learn blood collection method in rats. Our results showed that this method is feasible for repeatedly collecting a large volume of blood from rats. Moreover, this method could be easily mastered with a short-term learning curve. This procedure can be performed in non-anesthetized rats and only causes minimal stress to the animals.
Blood can be collected from rats in various locations. The tail vein is a superior location for blood sampling4. However, it is not always easy to obtain enough blood volume from the thin and low blood flow tail vein of the rats. The vacuum blood collection system is usually used for collecting blood samples from patients in daily clinical practice. This vacuum blood collection system has been used in patients and research animals for a long time8,9,10. This system consists of a vacuum blood collection tube with silicone coated inner tube wall and butterfly needle. It was based on the vacuum negative pressure principle and allows the continuous drawing of blood without the risk of blood collecting, which also reduces the chance of contamination and hemolysis11. The volume of blood sample in the catheter is about 0.3 mL. After withdrawing the needle from the tail vein, the blood blocked in the catheter will flow into the collecting vacuum tube due to the vacuum. This method is also applicable for collecting blood from rats weighing less than 100 g with the use of a smaller puncture needle.
This blood collection method has several advantages. This procedure only causes minimal injury to rats and blood collection can be performed without the use of anesthetics, thus the influence of the stress response and anesthetics on the blood sample can be avoided. Our observations showed that rats were quiet in the plastic restraining holder during the procedure and there were no deaths during these experiments. Secondly, repeated blood collection every one or two days is possible, which enables frequent sampling of blood with an adequate volume for various research purposes. Finally, this method is easy to learn and was easily mastered by beginners after a short (2 h) learning period, as indicated by the similar success rates and puncture times between the experimental and beginner groups in this report. In a previous report, Lee et al. demonstrated a similar method of sampling blood from the lateral tail vein of the rat4. Our method is simpler than that reported by Lee et al.4 in that we directly insert the puncture needle into the tail vein and the other end of the tube is directly connected to the vacuum tube for collecting the blood, without the use of an extra syringe and manual aspiration of the blood and without the need of &#39;milking&#39; the vein to facilitate blood flow. Collecting blood with this closed system reduces the chance of blood contamination. Use of the plastic restraining holder also reduces the stress to the rat and facilitates the fixation of the tail.
The following two steps are critical for the successful application of this method. 1) Extend the tail and avoid the movement of the tail; 2) Gently puncture the vein at an angle of 20&#176; to avoid puncture through the vein and keep the puncture needle in the vein.
In conclusion, the adopted vacuum blood collection method in rats is safe, feasible, and easy to practice. This method enables frequent sampling of blood with an adequate blood volume obtained from non-anesthetized rats.

Blood sampling from rats is required for a wide variety of experimental studies. Techniques for blood collection from the rat include puncture of the heart, retro-orbital plexus, jugular vein, saphenous vein, tail blood vessels, carotid artery, abdominal aorta, and vena cava. Most techniques (except saphenous vein and tail blood vessel puncture) require anesthesia1,2. Veins of the rat tail are routinely punctured for blood sampling and vascular injection. After treating the tail with warm water, the blood vessels are well dilated and suitable for manipulation, even when rats are anesthetized3. 
Despite the development of new and improved techniques to obtain blood samples in small laboratory animals2, it is not always easy to obtain enough blood when sampling from small animals. The tail vein of rats is a superior location for blood sampling4. However, the tail vein is thin, thus it is sometimes hard to puncture and would take a long time to get enough blood due to its low blood flow. Blood collection via a vacuum extraction tube system is a typical method in daily clinical practice. This closed system for blood collecting reduces the risk of direct exposure to blood and has made it easier to take multiple samples from a single venipuncture5. The present report describes a simple tail vein blood sampling method in non-anesthetized rats using the vacuum extraction tube system. This method is easy to master and can be used to obtain a large volume of blood repeatedly in rats.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">a double-pointed needle</td>
			<td style="width:244px;">Shanghai Kang Nong medical instrument co., LTD, China</td>
			<td style="width:120px;">22G&#160;</td>
			<td style="width:79px;">(0.7&#215;25mm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:192px;">vacuum tubes</td>
			<td style="width:244px;">Wuhan Zhi Yuan, medical science and technology co., LTD, China</td>
			<td style="width:120px;">2 ml</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:192px;">rat restraining holder&#160;</td>
			<td style="width:244px;">Shanghai Kang Nong medical instrument co., LTD, China</td>
			<td style="width:120px;">250g rat hoder model</td>
			<td></td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:192px;">normal salt diet for rats</td>
			<td></td>
			<td></td>
			<td style="width:79px;">Rats received a normal salt diet (0.3% NaCl) throughout the study</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:192px;">SPSS software for statistical analysis</td>
			<td>SPSS Inc,&#160; Chicago; USA&#160;</td>
			<td style="width:120px;">Version 17.0.&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Sampling duration, body mass, and blood collection volume of the two groups 
Blood was collected from 10 rats (5 males) twice within two weeks in each group. The mean body mass in both groups was similar (272.00 &#177;9.66 g and 273.70 &#177;8.87 g). Sampling duration was significantly shorter in the experimental group compared to beginner group (2.77 &#177;0.53 min vs. 3.28 &#177;0.83 min, p &#60;0.05) (Table 1).
Success rate and puncture times of two groups 
The success rate was 95% (19/20) in the experimental group and 90% (18/20) in the beginner group (p &#62;0.05). Puncture times were not significantly different between the two groups (1.50 &#177;0.61 min vs. 1.85 &#177;0.75 min, p &#62;0.05) (Table 2). These results indicate this method is easy to learn, and the technique could be mastered after training for 2 h.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55852/55852fig1.jpg" /> 
Figure 1: 2 mL vacuum blood collection tube (left); 22 G butterfly needle (center); the plastic restraining holder (right). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55852/55852fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55852/55852fig2.jpg" /> 
Figure 2: The needle tip was inserted into the lateral tail vein (around 5 mm) at the position approximately 2-3 cm away from the tip of the tail at angle of approximate 20&#176;. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55852/55852fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55852/55852fig3.jpg" /> 
Figure 3: Successful venipuncture. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55852/55852fig3large.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 colspan="5">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Experimental group</td>
			<td></td>
			<td colspan="5">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Beginner group</td>
		</tr>
		<tr>
			<td></td>
			<td>Gender</td>
			<td>Body weight (g)</td>
			<td>Collected blood&#160; volume (mL)</td>
			<td>sampling duration (min)</td>
			<td></td>
			<td></td>
			<td>Gender</td>
			<td>Body weight (g)</td>
			<td>Collected blood&#160; volume (mL)</td>
			<td>sampling duration (min)</td>
		</tr>
		<tr>
			<td>E1-1</td>
			<td>male</td>
			<td>291</td>
			<td>1.2</td>
			<td>3.12</td>
			<td></td>
			<td>B1-1</td>
			<td>male</td>
			<td>282</td>
			<td>1.2</td>
			<td>3.18</td>
		</tr>
		<tr>
			<td>E1-2</td>
			<td>male</td>
			<td>291</td>
			<td>1.2</td>
			<td>2.73</td>
			<td></td>
			<td>B1-2</td>
			<td>male</td>
			<td>282</td>
			<td>1.2</td>
			<td>2.18</td>
		</tr>
		<tr>
			<td>E2-1</td>
			<td>male</td>
			<td>276</td>
			<td>1.2</td>
			<td>2.3</td>
			<td></td>
			<td>B2-1</td>
			<td>male</td>
			<td>264</td>
			<td>1.2</td>
			<td>3.73</td>
		</tr>
		<tr>
			<td>E2-2</td>
			<td>male</td>
			<td>276</td>
			<td>1.2</td>
			<td>2.37</td>
			<td></td>
			<td>B2-2</td>
			<td>male</td>
			<td>264</td>
			<td>1.2</td>
			<td>2.47</td>
		</tr>
		<tr>
			<td>E3-1</td>
			<td>male</td>
			<td>267</td>
			<td>1.2</td>
			<td>2.73</td>
			<td></td>
			<td>B3-1</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>3.52</td>
		</tr>
		<tr>
			<td>E3-2</td>
			<td>male</td>
			<td>267</td>
			<td>1.0</td>
			<td>3.88</td>
			<td></td>
			<td>B3-2</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>2.53</td>
		</tr>
		<tr>
			<td>E4-1</td>
			<td>male</td>
			<td>263</td>
			<td>1.2</td>
			<td>2.41</td>
			<td></td>
			<td>B4-1</td>
			<td>male</td>
			<td>289</td>
			<td>1.2</td>
			<td>2.32</td>
		</tr>
		<tr>
			<td>E4-2</td>
			<td>male</td>
			<td>263</td>
			<td>1.2</td>
			<td>2.52</td>
			<td></td>
			<td>B4-2</td>
			<td>male</td>
			<td>289</td>
			<td>1.2</td>
			<td>3.62</td>
		</tr>
		<tr>
			<td>E5-1</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>3.61</td>
			<td></td>
			<td>B5-1</td>
			<td>male</td>
			<td>277</td>
			<td>1.2</td>
			<td>3.48</td>
		</tr>
		<tr>
			<td>E5-2</td>
			<td>male</td>
			<td>265</td>
			<td>1.2</td>
			<td>2.42</td>
			<td></td>
			<td>B5-2</td>
			<td>male</td>
			<td>277</td>
			<td>1.2</td>
			<td>3.12</td>
		</tr>
		<tr>
			<td>E6-1</td>
			<td>female</td>
			<td>285</td>
			<td>1.2</td>
			<td>3.33</td>
			<td></td>
			<td>B6-1</td>
			<td>female</td>
			<td>274</td>
			<td>0.9</td>
			<td>4.87</td>
		</tr>
		<tr>
			<td>E6-2</td>
			<td>female</td>
			<td>285</td>
			<td>1.2</td>
			<td>3.03</td>
			<td></td>
			<td>B6-2</td>
			<td>female</td>
			<td>274</td>
			<td>1.2</td>
			<td>3.37</td>
		</tr>
		<tr>
			<td>E7-1</td>
			<td>female</td>
			<td>271</td>
			<td>1.2</td>
			<td>2.82</td>
			<td></td>
			<td>B7-1</td>
			<td>female</td>
			<td>268</td>
			<td>1.0</td>
			<td>3.93</td>
		</tr>
		<tr>
			<td>E7-2</td>
			<td>female</td>
			<td>271</td>
			<td>1.2</td>
			<td>2.42</td>
			<td></td>
			<td>B7-2</td>
			<td>female</td>
			<td>268</td>
			<td>1.2</td>
			<td>3.82</td>
		</tr>
		<tr>
			<td>E8-1</td>
			<td>female</td>
			<td>268</td>
			<td>1.2</td>
			<td>1.93</td>
			<td></td>
			<td>B8-1</td>
			<td>female</td>
			<td>262</td>
			<td>1.2</td>
			<td>2.07</td>
		</tr>
		<tr>
			<td>E8-2</td>
			<td>female</td>
			<td>268</td>
			<td>1.2</td>
			<td>3.05</td>
			<td></td>
			<td>B8-2</td>
			<td>female</td>
			<td>262</td>
			<td>1.2</td>
			<td>3.67</td>
		</tr>
		<tr>
			<td>E9-1</td>
			<td>female</td>
			<td>273</td>
			<td>1.2</td>
			<td>2.52</td>
			<td></td>
			<td>B9-1</td>
			<td>female</td>
			<td>281</td>
			<td>1.2</td>
			<td>4.38</td>
		</tr>
		<tr>
			<td>E9-2</td>
			<td>female</td>
			<td>273</td>
			<td>1.2</td>
			<td>3.17</td>
			<td></td>
			<td>B9-2</td>
			<td>female</td>
			<td>281</td>
			<td>1.2</td>
			<td>4.57</td>
		</tr>
		<tr>
			<td>E10-1</td>
			<td>female</td>
			<td>261</td>
			<td>1.2</td>
			<td>1.82</td>
			<td></td>
			<td>B10-1</td>
			<td>female</td>
			<td>275</td>
			<td>1.2</td>
			<td>2.53</td>
		</tr>
		<tr>
			<td>E10-2</td>
			<td>female</td>
			<td>261</td>
			<td>1.2</td>
			<td>3.25</td>
			<td></td>
			<td>B10-2</td>
			<td>female</td>
			<td>275</td>
			<td>1.2</td>
			<td>2.28</td>
		</tr>
		<tr>
			<td>Average</td>
			<td></td>
			<td></td>
			<td></td>
			<td>2.77</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>3.28</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="11">En-1: serial number of rats in the experimental group for the first blood collection.</td>
		</tr>
		<tr>
			<td colspan="11">En-2: serial number of rats in the experimental group for the second blood collection.</td>
		</tr>
		<tr>
			<td colspan="11">Bn-1: serial number of rats in the beginner group for the first blood collection.</td>
		</tr>
		<tr>
			<td colspan="11">Bn-2: serial number of rats in the beginner group for the second blood collection.</td>
		</tr>
	</tbody>
</table>
Table 1: Results of blood collected in the experimental group and the beginner group.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Blood collection times</td>
			<td>Average puncture times</td>
			<td>One time&#160; puncture</td>
			<td>Two times puncture&#160;</td>
			<td>Three times puncture&#160;</td>
			<td>Failed</td>
			<td>Success rate</td>
		</tr>
		<tr>
			<td>Experimental group</td>
			<td>20</td>
			<td>1.50 &#177;0.61</td>
			<td>11</td>
			<td>8</td>
			<td>0</td>
			<td>1</td>
			<td>95%</td>
		</tr>
		<tr>
			<td>Beginner group</td>
			<td>20</td>
			<td>1.85 &#177;0.75</td>
			<td>7</td>
			<td>9</td>
			<td>2</td>
			<td>2</td>
			<td>90%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">One time puncture: Number of rats with successful blood collection after the first&#160; puncture</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">Two times puncture:Number of rats with successful blood collection after the second puncture&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">Three times puncture:Number of rats with successful blood collection after the third puncture&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">Failed:Number of rats with failed blood collection after the third puncture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of the puncture times and success rates of the experimental group and the beginner group.

Watch this Scientific Journal Video about Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

35.4K Views.  Jianghan University. 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. 

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

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

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 ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

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 ...

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.

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 ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...