None declared.

This study was approved by the Central South University Ethics Committee for Animal Research from the Second Xiangya Hospital (Changsha, China). The manuscript was prepared according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines9.
1. Material and Animal Preparation
<ol>
	<li>Prepare required materials: adhesive tape, epilating agent, 75% ethanol, blood collection tube, 2.5 mL syringe connected with needle (24 G, 0.6 mm x 25 mm), hair shaver, electronic scale, and gauge (see Table of Materials).</li>
	<li>Use 20 Sprague-Dawley (SD) rats, aged 8-10 weeks and weighing 153-200 g (see Table of Materials). Maintain rats in accordance with the Guide for the Care and Use of Laboratory Animals10.</li>
	<li>House rats under standard conditions with free access to food and drinking water. Keep them in 530 cm2 cages with wood-shaving bedding (2 rats per cage). Maintain animals in a temperature-controlled room temperature at around 25 &#176;C.</li>
	<li>Induce general anesthesia (sodium pentobarbital 40 mg/kg) in all animals through intraperitoneal injection before the subclavian vein puncture.</li>
	<li>Place the rat in a supine position. Fix the hind limbs in a comfortable position and fix the upper limb parallel to the body axis just beside the trunk. 
	NOTE: No mechanical ventilation is needed for this procedure (Figure 1).</li>
	<li>Clean both sides of the infraclavicular space to remove fur (with hair shaver and epilating agent, see Table of Materials) and any visible dirt. Wipe the neck and thoracic skin with 75% ethanol. Keep the area for puncture clean and dry with gauze.</li>
</ol>
2. Blood Collection Procedure
NOTE: Both sides of the subclavian vein are suitable for puncture, and here we choose the right side for puncture. The location and direction of the right clavicle are identified by the operator&#39;s left thumb (Figure 2). The suprasternal fossae are gently touched with the index finger as the suprasternal fossae is in the direction of the needle (Figure 2).
<ol>
	<li>Rinse a sterile 2.5 mL syringe with heparin solution (10 IU/mL).</li>
	<li>Locate the puncture site 0.5 mm caudal to the outer 1/3 region of the right clavicle (Figure 3). Move the syringe gently into the skin of the puncture site. Once the needle enters the infraclavicular skin, apply negative pressure with the operator&#39;s right hand (Figure 4). 
	NOTE: The average and maximal blood volume are 1.0 mL and 1.4 mL, respectively. The direction of the needle is positioned toward the suprasternal fossae and should be almost parallel to the clavicle just posterior to it. Usually, the subclavian vein can be reached by inserting the needle about 2 mm in this direction. Once the needle enters the vein, blood will enter the syringe under negative pressure.
	<ol>
		<li>Maintain negative pressure until enough volume of blood is drawn (Figure 5).</li>
	</ol>
	</li>
	<li>If the subclavian vein blood collection is not successful, withdraw the needle to the subcutaneous area and adjust the direction of the needle slightly interior to the sternum.
	<ol>
		<li>If three attempts fail, avoid the subclavian vein from the same side and use the contralateral side for blood sampling.</li>
	</ol>
	</li>
	<li>After blood collection, remove the puncture needle, and apply pressure to the puncture site for 1-2 minutes to stop bleeding. 
	NOTE: The pressure must be very gentle to prevent choking.</li>
</ol>
3. Blood Sample Processing
<ol>
	<li>Transfer the blood sample into an anticoagulant-coated vacuum tube (with EDTA) and centrifuge for 10 min at 1600 x g to collect plasma.</li>
	<li>Aspirate the supernatant to a clean tube and store at -80 &#176;C (Figure 6).</li>
</ol>

<ol>
	<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology, Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Korfmacher, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+capillary+microsampling+for+rat+pharmacokinetic+studies:+Comparison+of+tail-vein+bleed+to+jugular+vein+cannula+sampling.">Utility of capillary microsampling for rat pharmacokinetic studies: Comparison of tail-vein bleed to jugular vein cannula sampling.</a> Journal of Pharmacological and Toxicological Methods. 76, 7-14 (2015).</li><li>Feng, J., 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=Catheterization+of+the+carotid+artery+and+jugular+vein+to+perform+hemodynamic+measures,+infusions+and+blood+sampling+in+a+conscious+rat+model.">Catheterization of the carotid artery and jugular vein to perform hemodynamic measures, infusions and blood sampling in a conscious rat model.</a> Journal of Visualized Experiments. (95), (2015).</li><li>Wickremsinhe, E. R., Renninger, M., Paulman, A., Pritt, M., Schultze, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Repeated+Tail+Clip+and+Saphenous+Vein+Phlebotomy+on+Rats+Used+in+Toxicology+Studies.">Impact of Repeated Tail Clip and Saphenous Vein Phlebotomy on Rats Used in Toxicology Studies.</a> Toxicologic Pathology. 44 (7), 1013-1020 (2016).</li><li>Takahashi, M., Makino, S., Kikkawa, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+suitable+for+mammalian+whole+embryo+culture.">Preparation of rat serum suitable for mammalian whole embryo culture.</a> Journal of Visualized Experiments. (90), e51969 (2014).</li><li>Cochran, B. J., 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=Determining+Glucose+Metabolism+Kinetics+Using+18F-FDG+Micro-PET/CT.">Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT.</a> Journal of Visualized Experiments. (123), (2017).</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>Zou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Blood+Collection+from+Tail+Vein+of+Non-Anesthetized+Rats+with+a+Vacuum+Blood+Collection+System.">Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System.</a> Journal of Visualized Experiments. (130), (2017).</li><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 Animal. 44 (4), 377-378 (2010).</li><li>National Institute of Health. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2011).</li><li>Antonelli, D., Feldman, A., Freedberg, N. A., Turgeman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axillary+vein+puncture+without+contrast+venography+for+pacemaker+and+defibrillator+leads+implantation.">Axillary vein puncture without contrast venography for pacemaker and defibrillator leads implantation.</a> Pacing and Clinical Electrophysiology. 36 (9), 1107-1110 (2013).</li><li>Luis, A. 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=Microscopic+magnetic+resonance+imaging+of+the+thoracic+venous+system+in+rats+with+congenital+diaphragmatic+hernia.">Microscopic magnetic resonance imaging of the thoracic venous system in rats with congenital diaphragmatic hernia.</a> Pediatric Surgery International. 27 (2), 175-180 (2011).</li><li>Tsukamoto, A., Serizawa, K., Sato, R., Yamazaki, J., Inomata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+signs+monitoring+during+injectable+and+inhalant+anesthesia+in+mice.">Vital signs monitoring during injectable and inhalant anesthesia in mice.</a> Experimental Animals. 64 (1), 57-64 (2015).</li></ol>

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

Here, we introduce an alternative method for blood extraction from the subclavian vein and we show that this method is feasible, safe, and applicable for blood sampling in rats. This method is derived from the subclavian vein puncture in pacemaker lead implantation for patients11. The subclavian vein is located just posterior and caudal to the clavicle and is a continuation of the axillary vein. It runs from the outer border of the first rib to the medial border of anterior scalene muscle12. From here, it joins with the internal jugular vein to form the brachiocephalic vein and drains into super vena cava, just beneath suprasternal fossae (Figure 8). The clavicle is easily identified and there is a clear anatomic relationship between the subclavian vein and the clavicle. Here, we applied this clinical skill to animal experiments. Due to the different orientation of the upper limb in humans and rats, the upper limb is placed just beside the trunk during the procedure to expose the space caudal to the clavicle, which will facilitate the puncture (Figure 1).
The subclavian vein is much larger than the tail vein. Therefore, it is easier to get enough blood from the subclavian vein than from the tail vein. The total sample volume could reach 10% of total circulating blood volume each time1. If conventional tail vein blood sampling or another route fails, researchers can consider the subclavian vein puncture. General anesthesia is required for this procedure, and this is a major limitation of this procedure. Here, we use pentobarbital sodium for general anesthesia. Compared to other agents, pentobarbital sodium is easy to inject, and no other special instruments are needed. Tsukamoto et al. reported that isoflurane inhalation had the most stable SpO2 than other agents and had short recovery times. In centers with inhalation equipment, isoflurane can be considered13. Moreover, as the subclavian vein is located near to the thoracic cavity, we must be careful to prevent severe procedure-related complications such as pneumothorax and hemopneumothorax. The direction of the needle must be carefully directed to avoid these complications. Here, the needle is always positioned parallel to the clavicle and just posterior to it and moved gently towards the suprasternal fossae. The results show that use of above technique can avoid potential complications. Avoid any downward direction of the needle toward the thoracic cavity in this procedure. Also, the depth of the subclavian vein must always be kept about 2 mm from the puncture site.
Occasionally, attempts from one side may fail. We do not suggest more than 3 attempts from the same side. In such conditions, use the other side for puncture, which may improve the success rate of blood sampling. To further illustrate the feasibility of our method, we tested the success rate and time expended between a beginner group and a skilled group. Interestingly, there were no statistical significance between these groups (p = 0.33), which proved the practicability of this newly developed method.
As discussed here, the main limitation is the success rate on one side. If the procedure fails, the other side of the subclavian vein must be considered. General anesthesia is used in this procedure and the recovery time averaged about 30 min with pentobarbital sodium. Isoflurane inhalation anesthesia has a much shorter recovery time and the adoption of new anesthesia methods could be helpful in reducing the duration of the procedure. We show that this method is applicable to rats, but we have not tested its applicability to smaller animals such as mice.
In conclusion, the study results indicate that the subclavian vein puncture method for blood sample collection is safe and feasible in rats. This method is especially valuable when large amounts of blood are needed. This method is an optimal alternative if conventional blood sampling procedures fail.

Blood collection is essential in animal research. The target veins for blood collection include the retro-orbital plexus, the jugular vein, the saphenous vein, tail blood vessels, and the carotid artery1,2,3,4. Sometimes, blood could be obtained from the abdominal aorta, vena cava or even the heart5,6,7. On such occasions, the animals must be sacrificed and cannot be used for subsequent observation; thus, these methods are used less in daily experimental work. Blood collection from the tail vein of rats is popular and less stressful compared to the above mentioned methods8.
However, blood collection from the tail vein is sometimes limited by an unsatisfactory success rate. Occasionally, it is also difficult to obtain enough blood volume by this method. As the subclavian vein is large enough and is located just under the clavicle bone, subclavian vein puncture can be an alternative method for blood sampling if routine blood collection methods are unsuccessful. Here, we introduce a method of blood collection by means of subclavian vein puncture in rats.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>rats</td>
			<td>Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan Province, China)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stastical software</td>
			<td>SPSS Statistics 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>epilating agent</td>
			<td>France Yi Sha Cosmetics Co.,Ltd(Guangzhou, Guangdong Province ,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mL syringe</td>
			<td>Shandong Weigao Group Medical Polymer Co.,Ltd(Weihai,Shandong Province ,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hair shaver</td>
			<td>Shanghai FLYCO Electric Co., Ltd(Shanghai,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>adhesive tape</td>
			<td>3M Deutschland GmbH(EdisonstraBe 6,59157 Kamen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Department of Pharmacy,The Second Xiangya Hospital of Central South Univesity</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>blood collection tube</td>
			<td>Hubei Jinxing Technology&#38;Development Co.,Ltd (Wuhan Hubei Province,China) (2ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electronic scale</td>
			<td>Dongguan Shengheng Electronics Co.,Ltd (Dongguan,Guangdng Province,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>canvas gloves</td>
			<td>for anethesia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hepain</td>
			<td>Nanjing Xinbai Pharmaceutical&#160; Co.,Limited (Nanjing, Jiangsu Province, China) (2mL, 12500 IU)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>physical saline</td>
			<td>Hunan Kelun Pharmaceutical Co., Ltd(Yueyang ,Hunan Province,China) (100ml)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A total of 20 SD rats (male n=10, female n=10) were used here.&#160;Blood collectionwas performed by skilled operators, who have performed more than 20 blood samplings from the subclavian vein in rats, and by the beginner operators, who have no subclavian vein puncture experience in rats or other animals. Before operations, beginners watched at least 3 procedures done by skilled operators.
Blood sampling succeeded in 17 rats through the right subclavian vein access. This procedure failed in 3 rats (1 in the skilled group and 2 in the beginner group), and blood sampling was successful through the left subclavian vein accesses in these 3 rats. The time course and blood volume collected for each case are illustrated in Table 1. The success rate was similar between the beginner group (80%) and the skilled group (90%, p = 0.531). The success rate was 100% for both groups when including the additional left subclavian vein accesses in the 3 rats. Two tailed Student&#39;s t-test showed that there was no significant difference on time expended between beginner group and skilled group (102.1 &#177; 14.9 s versus 84.3 &#177; 9.7 s, p = 0.33, Figure 7). No severe complications occurred in all procedures such as pneumothorax, hemopneumothorax, or major bleeding during or after operation. All animals survived and recovered within 30 minutes after the blood sampling.
To demonstrate the difficulties of learning the puncture procedure, we further compared the number of withdrawals between skilled and beginner groups. As shown in Table 2, the average number of withdrawals in the skilled group is slightly less than the beginner group (1.6 &#177;0.3 vs. 2.5 &#177;0.4), but there is no statistical significance (p = 0.0913).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58499/58499fig1.jpg" /> 
Figure 1: Position of rats. The rats are laid in a supine position, and the upper limbs are fixed parallel to the body axis, just beside the trunk. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig1large.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/58499/58499fig2.jpg" /> 
Figure 2: Identify the surface symbols of rats. (A) The left thumb of the operator is on the right clavicle to identify the location and array of this bone; (B) The suprasternal fossae are gently touched with the index finger. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig2large.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/58499/58499fig3.jpg" /> 
Figure 3: Location of puncture. The puncture site is located 0.5 mm caudal to the outer 1/3 site of the right clavicle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58499/58499fig4.jpg" /> 
Figure 4: Forming negative pressure in the syringe. Once the needle enters the infraclavicular skin, the fingers of right hand move to create negative pressure in the syringe. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58499/58499fig5.jpg" /> 
Figure 5: Withdrawing blood into the syringe. When the needle enters the subclavian vein, blood will enter the syringe under negative pressure. The negative pressure is kept until enough blood is obtained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58499/58499fig6.jpg" /> 
Figure 6: Plasma extraction after centrifuging. After centrifuging, the blood is separated into plasma and cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58499/58499fig7.jpg" /> 
Figure 7: The time course and success rate between different groups. This chart shows no significant differences in the time spent (left panel) or success rate (right panel) between the skilled group and the beginner group. The time course in the beginner group and the skilled group are 102.1 &#177; 14.9 s and 84.3 &#177; 9.7 s, respectively, p = 0.33 between groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58499/58499fig8.jpg" /> 
Figure 8: The anatomy of subclavian vein. The subclavian vein is located just posterior and caudal to the clavicle. It joins with the internal jugular vein to form the brachiocephalic vein and drains into super vena cava, just beneath the suprasternal fossae. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58499/58499fig8large.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">skilled group</td>
			<td></td>
			<td colspan="5">Beginner group</td>
		</tr>
		<tr>
			<td></td>
			<td>Gender</td>
			<td>Body weight (g)</td>
			<td>Blood volume (mL)</td>
			<td>Time course (S)</td>
			<td></td>
			<td></td>
			<td>Gender</td>
			<td>Body weight (g)</td>
			<td>blood volume (mL)</td>
			<td>Time course (S)</td>
		</tr>
		<tr>
			<td>S1</td>
			<td>male</td>
			<td>153</td>
			<td>1.6</td>
			<td>70</td>
			<td></td>
			<td>B1</td>
			<td>male</td>
			<td>149</td>
			<td>1.6</td>
			<td>72</td>
		</tr>
		<tr>
			<td>S2</td>
			<td>male</td>
			<td>168</td>
			<td>1.6</td>
			<td>60</td>
			<td></td>
			<td>B2</td>
			<td>male</td>
			<td>158</td>
			<td>1.6</td>
			<td>68</td>
		</tr>
		<tr>
			<td>S3</td>
			<td>female</td>
			<td>183</td>
			<td>1.6</td>
			<td>75</td>
			<td></td>
			<td>B3</td>
			<td>female</td>
			<td>190</td>
			<td>1.6</td>
			<td>83</td>
		</tr>
		<tr>
			<td>S4</td>
			<td>female</td>
			<td>170</td>
			<td>1.6</td>
			<td>74</td>
			<td></td>
			<td>B4</td>
			<td>female</td>
			<td>160</td>
			<td>1.6</td>
			<td>77</td>
		</tr>
		<tr>
			<td>S5</td>
			<td>female</td>
			<td>159</td>
			<td>1.6</td>
			<td>65</td>
			<td></td>
			<td>B5</td>
			<td>female</td>
			<td>153</td>
			<td>1.6</td>
			<td>200</td>
		</tr>
		<tr>
			<td>S6</td>
			<td>male</td>
			<td>200</td>
			<td>1.6</td>
			<td>83</td>
			<td></td>
			<td>B6</td>
			<td>male</td>
			<td>187</td>
			<td>1.6</td>
			<td>92</td>
		</tr>
		<tr>
			<td>S7</td>
			<td>male</td>
			<td>178</td>
			<td>1.6</td>
			<td>86</td>
			<td></td>
			<td>B7</td>
			<td>male</td>
			<td>164</td>
			<td>1.6</td>
			<td>99</td>
		</tr>
		<tr>
			<td>S8</td>
			<td>male</td>
			<td>187</td>
			<td>1.6</td>
			<td>168</td>
			<td></td>
			<td>B8</td>
			<td>male</td>
			<td>200</td>
			<td>1.6</td>
			<td>82</td>
		</tr>
		<tr>
			<td>S9</td>
			<td>female</td>
			<td>192</td>
			<td>1.6</td>
			<td>83</td>
			<td></td>
			<td>B9</td>
			<td>female</td>
			<td>198</td>
			<td>1.6</td>
			<td>70</td>
		</tr>
		<tr>
			<td>S10</td>
			<td>female</td>
			<td>157</td>
			<td>1.6</td>
			<td>79</td>
			<td></td>
			<td>B10</td>
			<td>female</td>
			<td>175</td>
			<td>1.6</td>
			<td>178</td>
		</tr>
	</tbody>
</table>
Table 1: The blood volume and time course from puncture to a finished blood collection. S: skilled group; B: beginners.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group&#160;</td>
			<td>n</td>
			<td>Mean</td>
			<td>SE</td>
			<td colspan="2">95% Conf. Interval</td>
		</tr>
		<tr>
			<td>B</td>
			<td>10</td>
			<td>2.5</td>
			<td>0.4</td>
			<td>1.59</td>
			<td>3.41</td>
		</tr>
		<tr>
			<td>S</td>
			<td>10</td>
			<td>1.6</td>
			<td>0.3</td>
			<td>0.91</td>
			<td>2.29</td>
		</tr>
	</tbody>
</table>
Table 2: The number of withdrawals between the two groups. B: beginners; S: skilled group. p = 0.0913

Watch this Scientific Journal Video about Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats at JoVE.com

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection 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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

13.9K Views.  Central South University. Here, we present a protocol to collect blood samples from the rat subclavian vein.

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

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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 ...

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 ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

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. ...

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 ...

The Rigid Tube as an Alternative in Controlling the Problematic Airway

The unexpected problematic airway represents a large proportion of anesthesia-related morbidity and mortality. The retromolar or paraglossal approach is an alternative to the majority of the rigid instruments used for tracheal intubation, which follow the midline to access the glottis. This single-center, prospective case-series study offers an option to conventional laryngoscopy in case of a poor glottic view, introducing an instrument (the rigid tube for laryngoscopy) that uses the retromolar approach to accomplish tracheal intubation. If after anesthesia induction, the modified Cormack-Lehane glottis view grade &#62;2b, the intubation is carried further with the rigid tube. The tube follows the direction of the thyroid cartilage while advancing from the labial commissure, displacing the tongue to the contralateral side. Adjusting the position of the larynx with the nondominant hand by gently pushing the thyroid cartilage and following an imaginary line towards it while advancing it improves the time needed for proper glottis visualization. Once the epiglottis is in sight, the practitioner progresses slowly, lifting the epiglottis and aiming the tip of the tube more anteriorly. When the glottis appears in the visual field, the intubating tube introducer is placed in the trachea, and a lubricated cuffed intubating tube is advanced over the introducer after the rigid tube is extracted. This tool was tested on 30 patients with an unsatisfactory glottic view when using the Macintosh laryngoscope and obtained excellent results with respect to intubation time and complications. The reduced visual field is the main limitation of this method, which requires a training period for reasonable expertise. This simple, robust, and cheap instrument could be a rescue option in case of a difficult airway.

Presented here is a protocol of tracheal intubation over an intubating tube introducer using a rigid tube for laryngoscopy with an attached light source. The main characteristics of this maneuver are the retromolar approach and the use of the thyroid cartilage as a landmark while advancing the rigid tube.

The unexpected problematic airway represents a large proportion of anesthesia-related morbidity and mortality. The retromolar or paraglossal approach is ...

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, ...