This research was funded by Guangdong Provincial Key Laboratory of Drug Non-clinical Evaluation and Research (No.2018B030323024) and Key Program &#34;New Drug Creation&#34; of Guangdong Key Research and Development Plan (No.2019B020202001), Guangzhou Fundamental and Application Foundation Research Project (No.202002030249 and No.202002030156).

All animal experiments described were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Lewwin Pharmaceutical Research Institute Co., Ltd. Male and female Sprague Dawley (SD) rats, ~6-11 weeks were used for the experiments. The rats were reared following the guidelines for the care and use of laboratory animals13.
1. Animal preparation
NOTE: All the SD rats used in this study were awake and were not anesthetized/euthanized. The skill of restraint handling by grasping the skin on the back of the animal is needed.
<ol>
	<li>Find the lowest point of the subclavian triangular fossa between the neck and the forelimb in a rat. Move ~2-3 mm toward the head and reach the needlepoint at the blood collection site (Figure 1). Remove the hair using an electric shaver. 
	NOTE: Depending on the experimental needs, serum or plasma may be required. Plasma is taken as an example in this protocol, and it will not affect the operation of blood collection.</li>
	<li>Prepare cotton swabs stained with 75% alcohol for wiping and disinfecting and dry cotton swabs for wiping.</li>
</ol>
2. Sampling of blood
NOTE: At least 2 people, both of whom must be experienced in blood collection and rat restraint techniques should carry out these steps.
<ol>
	<li>Grasp the skin on the back of the neck with one hand to keep the rat&#39;s head, neck, and breast upright and expose the injection site (Video 1). Straighten the forelimb on the side of the injection site to maintain its level.</li>
	<li>Keep the syringe parallel to the rat&#39;s head with the other hand and tilt the syringe outward 5&#176;-10&#176; so that the tip is tilted toward the ventral direction.</li>
	<li>Insert the needle fully into the anterior cavity. Draw back the syringe to maintain negative pressure within the tube.</li>
	<li>Slowly move the needle from deep to shallow and back up the same path. When blood has entered the syringe needle, fix the position of the needle (Video 2). 
	NOTE: The blood will then be filled at a constant rate in the syringe, as shown in Figure 2A (front view) and Figure 2B (side view).</li>
	<li>Monitor the maximum blood quantity in accordance with the standards set by the institution&#8217;s animal care and use committee. This depends on the weight and health of the animal. In absence of other requirements, do&#160;not remove more than 20% of the animal&#39;s total blood volume within 24 h, which requires ~3 weeks of recuperation14.</li>
	<li>When sufficient blood sample is collected, immediately withdraw the syringe and prepare for blood treatment (step 3).</li>
	<li>Pressurize the rat&#39;s injection site for ~1-2 min to stop the bleeding. Since the collection site is in the lower part of the neck, pinch the skin of the subclavian vein to stop the bleeding by pressing on it (Figure 3). 
	NOTE: Blood can be collected alternately from the bilateral subclavian vein when repeated blood collection is needed.</li>
	<li>Return the rat to the cage and observe its condition.</li>
</ol>
3. Processing the blood sample
<ol>
	<li>Remove the needle from the syringe and discard it in the sharp tool container. Slowly transfer the blood from the syringe to a 1.5 mL tube. Press the syringe against the wall to avoid bubble formations, if any. 
	NOTE: As pressure may cause red blood cells to rupture, remove the needle to prevent hemolysis14.</li>
	<li>Cover the microcentrifuge tube, gently flick it, and turn it upside down at least five times to thoroughly mix the blood with the anticoagulant.</li>
	<li>Centrifuge the whole blood sample at 2000 x g for ~10-15 min at room temperature within 120 min after collecting the plasma.</li>
	<li>Use a pipette gun (see Table of Materials) to transfer the upper plasma into a blank microcentrifuge tube. Do not touch the pipette head with the lower whole blood. Dispose of or re-centrifuge the plasma contaminated with the red blood cells.</li>
	<li>Use the samples immediately or store them at -30 &#176;C. 
	NOTE: The characteristics of the drug determine the storage time. The plasma specimens obtained from the subclavian vein are translucent and light yellow. Hemolysis may turn the plasma red.</li>
</ol>

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	<li>Shravya, 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=International+conference+on+harmonization+of+technical+requirements+for+registration+of+pharmaceuticals+for+human+use.+ICH+M2+EWG.">International conference on harmonization of technical requirements for registration of pharmaceuticals for human use. ICH M2 EWG.</a> Electronic common technical document. , (2014).</li><li>Tan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The China Food and Drug Administration (CFDA). , (2015).</li><li>McGuill, M. W., Andrew, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+effects+of+blood+loss:+Implications+for+sampling+volumes+and+techniques.">Biological effects of blood loss: Implications for sampling volumes and techniques.</a> ILAR Journal. 31 (4), 5-20 (1989).</li><li>Aguilar-Mariscal, 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=Oral+pharmacokinetics+of+meloxicam+in+the+rat+using+a+high-performance+liquid+chromatography+method+in+micro-whole-blood+samples.">Oral pharmacokinetics of meloxicam in the rat using a high-performance liquid chromatography method in micro-whole-blood samples.</a> Methods and Findings in Experimental and Clinical Pharmacology. 29 (9), 587-592 (2007).</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>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Blood+Withdrawal+I.">JoVE Science Education Database. Blood Withdrawal I.</a> JoVE. , (2021).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclavian+vein+puncture+as+an+alternative+method+of+blood+sample+collection+in+rats.">Subclavian vein puncture as an alternative method of blood sample collection in rats.</a> Journal of Visualized Experiments. (141), e58499 (2018).</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), e55852 (2017).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology &amp; Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Itziar, F., Arantza, P., Nahia, D., Virginia, P., Juan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+biochemistry+parameters+in+C57BL/6J+mice+after+blood+collection+from+the+submandibular+vein+and+retroorbital+plexus.">Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus.</a> Journal of the American Association for Laboratory Animal Science. 49 (2), 202-206 (2010).</li><li>Heimann, M., Roth, D. R., Ledieu, D., Pfister, R., Classen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublingual+and+submandibular+blood+collection+in+mice:+A+comparison+of+effects+on+body+weight,+food+consumption+and+tissue+damage.">Sublingual and submandibular blood collection in mice: A comparison of effects on body weight, food consumption and tissue damage.</a> Laboratory Animals. 44 (4), 352-358 (2010).</li><li>Wren-Dail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+isoflurane+anesthesia+on+circadian+metabolism+and+physiology+in+rats.">Effect of isoflurane anesthesia on circadian metabolism and physiology in rats.</a> Comparative Medicine. 67 (2), 138-146 (2017).</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. 8th edition. , (2011).</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> Journal of Visualized Experiments. (99), e52766 (2015).</li><li>Diehl, 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=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> Journal of Applied Toxicology. 21 (1), 15-23 (2001).</li><li>Casal, D., 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=Functional+and+physiological+methods+of+evaluating+median+nerve+regeneration+in+the+rat.">Functional and physiological methods of evaluating median nerve regeneration in the rat.</a> Journal of Visualized Experiments. (158), e59767 (2020).</li></ol>

Limei Wang, Jianmin Guo, Xiaoman Zhong, Yali Sheng, Qiwen Lai, Hui Song, and Wei Yang have a financial interest in Guangdong Lewwin Pharmaceutical Research Institute Co., Ltd, which, however, did not support this work. The other authors declare no competing interests.

There are certain benefits of collecting blood from the subclavian veins. (1) As the site of the blood collection is readily dissociated, and the venous plexus is not regular due to the different postures of the rats, the described method can easily localize the position of the venous plexus while maintaining the stable and comfortable postures of the rats. (2) The operation is easy and favorable to the rapid development of technicians' skills and less pain to animals. (3) An operating mode to make the animal comfortable...

According to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines1 and the National Medical Products Administration (NMPA) guidelines2, the number of blood collection time points of rats in the toxicokinetic (TK) study needs to meet the requirements of the dynamic drug exposure assessment. The approximate total blood volume of a rat is 55-70 mL/kg of body weight3. The collection time points are generally intensive within 30 min after dosing and decrease after that, and more than ten blood samples need to be collected within 48 h in routine testing4. For example, blood samples are collected at 12-time points (0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 8 h, and 12 h) in TK study of orally administered drugs. Researchers must repeatedly collect 200-250 &#181;L of blood in rats to obtain high-quality plasma for the TK testing5.
The blood collection sites in rats include tail blood vessels, retro-orbital plexus vein, submandibular vein, heart, abdominal aorta6,&#160;and so on. Among them, blood collection from the caudal vein of rats is a frequently used method, which requires experienced and skilled operators7,8. To collect blood from the retro-orbital plexus vein is less complicated; however, this method is not recommended as it may damage the rats&#39; eyesight9, and blood from the heart and abdominal aorta is only appropriate for final blood sampling10. Another method of collecting blood from the submandibular vein in a conscious rat has been shown to result in more complications and revealed insufficient blood sample quality11. Therefore, researchers may anesthetize the animal to reduce the difficulty of sampling. Still, the anesthesia also increases the cost of the experiment, and more seriously, it will affect the metabolic state of rats12. The present protocol uses a quick and straightforward method of collecting blood in the subclavian veins of rats without anesthesia, allowing accurate positioning and bilateral alternating blood collection to obtain high-quality samples in a timely and repeated manner.

<table><tbody><tr><td>1.0 mL syringe (with needle, 26 G, 0.45 mm x 15.5 mm)</td><td>Jiangxi Hongda Medical Equipment Group LTD.(Nanchang, Jiangxi Province, China)</td><td>20210629</td><td></td></tr><tr><td>75% alcohol</td><td>Shandong Lierkang Medical Technology Co., Ltd. (Dezhou, Shandong Province, China)</td><td>210717</td><td></td></tr><tr><td>Animal source</td><td>Hunan SJA Laboratory Animal Co., Ltd.</td><td>grade: SPF</td><td>laboratory animal production license number: SCXK (Hunan) 2019-0004, laboratory animal quality certificate number: 4307272101972847</td></tr><tr><td>Cotton swab</td><td>Caoxian Hualu Sanitary Materials Co., Ltd.(Heze, Shandong Province, China)</td><td>20210301</td><td>Need to be sterilized.</td></tr><tr><td>Electric shaver</td><td>Shenzhen Codos Electric Appliance Co. , Ltd.(Shenzhen, Guangdong Province, China)</td><td>CP-6800</td><td></td></tr><tr><td>EP tube, 1.5 mL</td><td>Genetimes ExCell Technology,Inc.(Shanghai, China)</td><td></td><td></td></tr><tr><td>Heparin sodium (2 mL:12500 IU)</td><td>Tianjin biochem pharmaceutical Co.,Ltd(Tianjing, China)</td><td>51200702</td><td>Prepare 1250 IU/mL heparin sodium solution. Add heparin sodium solution into the EP tube in advance and make it evenly distributed on the wall of the EP tube, and dry it at 60&amp;deg;C for use.&amp;nbsp;</td></tr><tr><td>Labtip (volume range 5-200 &amp;mu;L)</td><td>Thermo Fisher Scientific Oy</td><td>94300120</td><td></td></tr><tr><td>Low speed refrigerated centrifuge</td><td>Hunan Xiangyi Laboratory Instrument Development Co., Ltd.(Changsha, Hunan Province, China)</td><td>L535R</td><td></td></tr><tr><td>Pipette gun (20-200 &amp;mu;L)</td><td>BRAND</td><td>12N92305</td><td></td></tr><tr><td>Rats (SD)</td><td>Hunan SJA Laboratory Animal Co., Ltd. (Changsha, Hunan Province, China)</td><td></td><td></td></tr><tr><td>Sharp tool container</td><td>Taizhou Huangyan Yikang Plastic Factory (Taizhou, Zhejiang Province, China)</td><td></td><td></td></tr><tr><td>Eye drop</td><td></td><td></td><td>This reagent is not a commodity and the manufacturer requires it to be tested. In the principle of confidentiality, the manufacturer and model cannot be provided.</td></tr></tbody></table>

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.

Good plasma samples from the subclavian vein were translucent pale yellow (Figure 4, the left tube). Improper blood collection or manipulation resulted in hemolysis (Figure 4, the right tube).
The data of the testing facility showed that in a 4-week oral toxicity study of an eye drop in SD rats with TK, blood samples were collected twice at 9-time points (0 h, 0.167 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) between the first (Day ...

Watch this Scientific Journal Video about Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat at JoVE.com

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

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 ...

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4.4K Views.  Guangdong Engineering Research Center for Innovative Drug Evaluation and Research. 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.

Video: Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

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

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

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

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

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

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 ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

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 ...

Sampling Blood from the Lateral Tail Vein of the Rat

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

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

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

Biology

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

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

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

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

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

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

Neuroscience

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

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

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

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

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