Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 µL 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, and so on. Among them, blood collection from the caudal vein of rats is a frequently used method, which requires experienced and skilled operators^7,8. To collect blood from the retro-orbital plexus vein is less complicated; however, this method is not recommended as it may damage the rats' 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.

Protokół

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.

1. 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.
2. Prepare cotton swabs stained with 75% alcohol for wiping and disinfecting and dry cotton swabs for wiping.

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.

1. Grasp the skin on the back of the neck with one hand to keep the rat'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.
2. Keep the syringe parallel to the rat's head with the other hand and tilt the syringe outward 5°-10° so that the tip is tilted toward the ventral direction.
3. Insert the needle fully into the anterior cavity. Draw back the syringe to maintain negative pressure within the tube.
4. 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).
5. Monitor the maximum blood quantity in accordance with the standards set by the institution’s animal care and use committee. This depends on the weight and health of the animal. In absence of other requirements, do not remove more than 20% of the animal's total blood volume within 24 h, which requires ~3 weeks of recuperation¹⁴.
6. When sufficient blood sample is collected, immediately withdraw the syringe and prepare for blood treatment (step 3).
7. Pressurize the rat'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.
8. Return the rat to the cage and observe its condition.

3. Processing the blood sample

1. 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.
2. Cover the microcentrifuge tube, gently flick it, and turn it upside down at least five times to thoroughly mix the blood with the anticoagulant.
3. Centrifuge the whole blood sample at 2000 x g for ~10-15 min at room temperature within 120 min after collecting the plasma.
4. 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.
5. Use the samples immediately or store them at -30 °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.

Wyniki

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 ...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
1.0 mL syringe (with needle, 26 G, 0.45 mm x 15.5 mm)	Jiangxi Hongda Medical Equipment Group LTD.(Nanchang, Jiangxi Province, China)	20210629
75% alcohol	Shandong Lierkang Medical Technology Co., Ltd. (Dezhou, Shandong Province, China)	210717
Animal source	Hunan SJA Laboratory Animal Co., Ltd.	grade: SPF	laboratory animal production license number: SCXK (Hunan) 2019-0004, laboratory animal quality certificate number: 4307272101972847
Cotton swab	Caoxian Hualu Sanitary Materials Co., Ltd.(Heze, Shandong Province, China)	20210301	Need to be sterilized.
Electric shaver	Shenzhen Codos Electric Appliance Co. , Ltd.(Shenzhen, Guangdong Province, China)	CP-6800
EP tube, 1.5 mL	Genetimes ExCell Technology,Inc.(Shanghai, China)
Heparin sodium (2 mL:12500 IU)	Tianjin biochem pharmaceutical Co.,Ltd(Tianjing, China)	51200702	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°C for use.
Labtip (volume range 5-200 μL)	Thermo Fisher Scientific Oy	94300120
Low speed refrigerated centrifuge	Hunan Xiangyi Laboratory Instrument Development Co., Ltd.(Changsha, Hunan Province, China)	L535R
Pipette gun (20-200 μL)	BRAND	12N92305
Rats (SD)	Hunan SJA Laboratory Animal Co., Ltd. (Changsha, Hunan Province, China)
Sharp tool container	Taizhou Huangyan Yikang Plastic Factory (Taizhou, Zhejiang Province, China)
Eye drop			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.