Summary
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.
Abstract
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.
Introduction
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 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' 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.
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Protocol
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.
- 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. - 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.
- 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.
- 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.
- Insert the needle fully into the anterior cavity. Draw back the syringe to maintain negative pressure within the tube.
- 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). - 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.
- When sufficient blood sample is collected, immediately withdraw the syringe and prepare for blood treatment (step 3).
- 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. - Return the rat to the cage and observe its condition.
3. Processing the blood sample
- 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. - Cover the microcentrifuge tube, gently flick it, and turn it upside down at least five times to thoroughly mix the blood with the anticoagulant.
- Centrifuge the whole blood sample at 2000 x g for ~10-15 min at room temperature within 120 min after collecting the plasma.
- 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.
- 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.
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Representative Results
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 1) and the last dosing day (Day 28). The dosage for the TKB, TKC, and TKD rats was 10 mg/kg, 30 mg/kg, and 100 mg/kg, respectively, and the dosage volume was 10 mL/kg. The blood collection volume of the first sampling was ~0.2 mL, the animal's age was ~6-7 weeks, the bodyweight of the male animal was ~268-297 g, and that of the female was ~214-239 g. The blood volume of the last sampling was ~0.3 mL, the animal's age was ~10-11 weeks, the bodyweight of the male animal was ~376-462 g, and that of the female animal was ~254-300 g. There was an interval of 28 days between the two times of TK blood collection. The animals' bodyweight increased steadily every week, and the clinical observation was normal. The total amount of blood of a 250 g rat is about 16 mL15, and the maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The body weight data of female animals are shown in Table 1, and that of male animals are shown in Table 2.
Figure 1: Depiction of the needlepoint of blood collection site. Please click here to view a larger version of this figure.
Figure 2: Blood extraction process. The blood was obtained smoothly. (A) Front view. (B) Side view. Please click here to view a larger version of this figure.
Figure 3: Representation of the hemostatic method. Please click here to view a larger version of this figure.
Figure 4: Specimen appearance after centrifugation. The left tube showed a qualified plasma image. The right tube showed a hemolysis sample. The plasma looked pink or red. The darker the color, the higher the hemolysis rate. Please click here to view a larger version of this figure.
Figure 5: The anatomy of the subclavian vein. This vein is located beneath the clavicle, and the needle can reach it just passed through the subclavian triangular fossa no more than 0.5 cm. It joins the internal jugular vein, vertebral vein, and external jugular vein to form the cranial vena vein. Please click here to view a larger version of this figure.
Table 1: Bodyweight data of female animals. Please click here to download this Table.
Table 2: Bodyweight data of male animals. Please click here to download this Table.
Video 1: Top-down view of the rat blood sampling process. Please click here to download this Video.
Video 2: Side view of the rat blood sampling process. Please click here to download this Video.
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Discussion
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 significantly reduces restless behaviors and prevents blood splashing. (4) It is a blood drawing method with high speed (20 s), high efficiency, low cost, and without anesthesia, which reduces the risk of animal anesthesia and the time of restraint in terms of animal welfare.
In this protocol, the rats are kept comfortable in the process of restraint, relaxing moderately when handling and avoiding a long-time touch. Loose handling allows rats to move quickly and may cause accidental injury to animals or people; on the contrary, it may cause hypoxia in rats. It is essential to stop bleeding while pressing on the sampling site. The operators hold the animal in their arms to smooth out them in the darkness. In addition, the negative pressure must not be too high during the blood collection; otherwise, it will lead to poor blood sampling. Since the PK and TK studies require repeated blood collection, the blood volume must be controlled according to different animal weight and health conditions; meanwhile, an alternate blood collection site will benefit animal welfare. If the animals are small or weak, they are provided with glucose and other supplements appropriately under the conditions permitted by the test. The needle inlet must be smooth and free from resistance, and when there is no blood to collect, or there is no resistance, the needle should be re-inserted in a slightly adjusted direction or changed sides. In the blood sampling process, it is necessary to avoid mixing blood taken twice from insertion. When the blood sample volume for the first injection is insufficient, the second injection is required, the syringe needs to be replaced, and the replacement tube is taken for the blood collected again in case of hemolysis. It is recommended to finish collecting blood at once. The blood in the syringe is attached to the wall and injected into the blood collection tube to avoid bubbles during the process. To collect blood timely, the operator needs to be fully prepared 2 min before the collection and inject 1 min before that. When the animal is less cooperative or challenging to operate, it must be soothed in advance. Hence, there needs to be a group of qualified technicians.
The brachial plexus is perpendicular to and behind the subclavian vein, so improper operation may lead to brachial plexus injury, forelimb pain, lameness, flinching, and other adversesymptoms16. This technique can also be applied to guinea pigs and meet blood collection needs in guinea pig experiments, but further research is needed to draw blood from guinea pigs by this method.
The anatomy of a subclavian vein is shown in Figure 5. The subclavian vein is located beneath the clavicle, and the needle can reach it just passing through the subclavian triangular fossa no larger than 0.5 cm. The method is therefore risky and calls for a qualified professional. An unqualified professional will result in animal hyperactivity, difficulty to control, poor blood circulation, animal weakness, or even mortality following a serial blood collection. Incorrect surgery will also damage the chest or artery. As a result, it is crucial to involve a qualified professional to achieve a successful experiment.
In conclusion, this protocol presents an alternative method for collecting blood from the subclavian vein of rats, which provides technical support for PK and TK studies.
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Disclosures
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.
Acknowledgments
This research was funded by Guangdong Provincial Key Laboratory of Drug Non-clinical Evaluation and Research (No.2018B030323024) and Key Program "New Drug Creation" of Guangdong Key Research and Development Plan (No.2019B020202001), Guangzhou Fundamental and Application Foundation Research Project (No.202002030249 and No.202002030156).
Materials
| 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. |
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