Name: Tail Vein Puncture Without Anesthesia
Uploaded: 2023-06-30T14:30:00
Description: Watch this Scientific Journal Video about Tail Vein Puncture Without Anesthesia at JoVE.com

tail vein puncture without anesthesia

쥐의 혈액 채취 기술에 대한 비교 연구

Blood sampling is necessary for most animal studies, both in vivo and in vitro. In rats, as the frequency and amount of blood sampling can be significant, it is helpful to have different alternatives for collection. Various methods have been described in previous studies.
The most commonly used techniques are tail vein puncture and saphenous vein blood draw. Tail vein sampling is suitable for all rat strains. With proper training, the procedure is simple to perform and causes minimal distress to the animal1. Similarly, the saphenous vein blood draw, provided it is done properly, is also a quick and simple collection method. Neither method requires anesthesia, and both allow for repeated draws of small amounts of blood. However, the saphenous vein puncture usually yields a lower blood volume1 and requires the presence of two people to leave one hindlimb exposed for puncture2.
If large amounts of blood need to be collected from a single animal, cardiac puncture or puncture of the vena cava may be used (up to 10 mL of blood can be drawn from a 150 g rat with cardiac puncture2). These techniques require anesthesia and are terminal procedures. The animal must be euthanized after any of these two techniques2. The jugular stick is an alternative that can be used if large amounts of blood need to be collected in a study that has not yet reached its endpoint. However, this technique also requires significant technical skills to avoid harm to the animal; hence, its use should be limited3.
Other techniques, such as the subclavian vein blood draw, do not need the use of anesthetics before blood collection and allow for repeated sampling of small volumes of blood. However, restrained handling and appropriate needle incision are required for this technique. An improper operation may result in animal pain or even mortality, and the training for this method may be fastidious4. 
Other anecdotal procedures include the orbital puncture and the sublingual vein puncture, both of which require an anesthetic, and neither are recommended nor widely used. Though previous studies have shown faster blood collection by orbital puncture than by tail vein puncture, it was found that orbital puncture under diethyl-ether anesthesia was less well tolerated than the latter method (based on the animals' excitation scores and urine production)5. Moreover, this method is highly influenced by the skill of the person who performs the procedure and is mainly performed by experienced veterinarians. Comparably, the sublingual vein puncture is less distressing and is recommended for repeated blood sampling6. However, this technique presents severe adverse effects such as reduced food and water intake, which can lead to the animal's death7. 
This study describes two methods used in our laboratory for repeated blood sampling. Tail vein puncture can be performed on a conscious animal, and the tissue damage and adverse effects are minimal. The modification of this technique in this study includes stabilizing the tail with the index and middle finger, which allows a single operator to perform the blood collection. The dorsal penile vein puncture has already been described for simple intravenous injections. This technique is performed under anesthesia and allows for a reliable blood source in case of difficulties with other methods (e.g., during the immediate postoperative period, with a small animal, when performing perioperative blood draw under anesthesia). Similar to tail vein sampling, the injury at the puncture site will have a minor overall effect on the animal compared to the techniques mentioned above8. The aim of this methods paper is to offer inexperienced researchers simple and reliable blood sampling alternatives according to the context (e.g., for procedures done under anesthesia, for studies including male rats, for animals with thin tail veins).

저자 스포트라이트: 쥐의 상황에 따른 혈액 수집 방법 

쥐 모델에서 혈액 샘플링을 위한 수정된 꼬리 정맥 및 음경 정맥 천자

The issue of repeated blood collection methods needs to be addressed since the majority of studies on rats require them. It's important to take into account that a sampling method can be very efficient in a certain context, but become unsuitable for another study. This protocol aims to offer simple and reliable blood sampling alternatives to researchers.Both methods can be done by a single operator and require no extensive prior training, which allows for standardized procedure. When an animal is under anesthesia, the effect of isoflurane can lead to vessel spasm and make the tail vein puncture unsuitable for blood collection. Our protocol offers an alternative in this situation, the penile vein puncture, which presents a better success rate and yields significantly more amounts of blood in less time.

Watch this Scientific Journal Video about Tail Vein Puncture Without Anesthesia at JoVE.com

Tail Vein Puncture Without Anesthesia  

마취 없이 꼬리 정맥 천자

시작하려면 쥐를 플라스틱 구속 원뿔에 고정하고 원뿔의 큰 끝을 꼬리 밑 부분에 둘러 쌉니다. 동물이 편안하고 절차 전반에 걸쳐 호흡이 제한되지 않는지 확인하십시오. 꼬리를 따뜻한 물에 약 1 분 동안 담그면 정맥이 확장되고 종이 타월로 꼬리를 말립니다.구속기에서 꼬리가 가열 패드에 놓이도록 동물을 아래로 향하게 놓습니다. 샘플링에 적합한 꼬리 정맥을 선택하려면 전체 동물을 오른쪽이나 왼쪽으로 회전시킵니다. 꼬리의 말단 1/3은 혈관 천공을 위해 사용하는데, 이 구역에서 혈관이 더 표면적이 되기 때문입니다.펑크 부위에서 70 % 에탄올 물티슈로 꼬리를 닦고 가열 패드의 가장자리에 놓고 꼬리의 끝 1/3에 각도를 만듭니다. 이렇게 하면 정맥이 표면으로 나오고 샘플을 채취할 수 있는 더 많은 공간이 생깁니다. 혈액 샘플링의 경우 주사기에서 플런저를 앞뒤로 여러 번 움직여 금단을 부드럽게 한 다음 플런저를 당겨 주사기에 음압을 생성합니다.주로 사용하지 않는 검지와 중지를 사용하여 꼬리를 가열 패드에 평평하게 고정합니다. 가운데 손가락을 근위부로, 검지를 이 두 손가락 사이에 천자 부위가 오도록 말단에 놓습니다. 인슐린 주사기가 정맥에 삽입 될 때까지 바늘의 눈이 위쪽을 향하도록 집게 손가락에 대고 밉니다.이것은 바늘과 꼬리 사이에 35 도의 각도를 만듭니다. 바늘이 정맥에 들어가면 혈액이 주사기로 흘러 들어갑니다. 주사기의 플런저를 일정한 속도로 천천히 빼내어 원하는 부피를 모으십시오.꼬리에서 주사기를 제거하고 꼬리의 구멍이 뚫린 쪽에 혈액 방울이 형성되도록합니다. 비멸균 절차의 경우 이 혈액을 흡인하여 몇 마이크로리터의 혈액을 더 수집합니다. 천자 부위에 압력을 가하여 출혈을 멈추고 새 알코올 물티슈로 해당 부위를 닦습니다.원뿔에서 쥐를 제거한 후 새장으로 되돌립니다. 꼬리 정맥 천자 방법의 성공률은 마취 상태에서 음경 정맥 천자 방법의 성공률과 비교되었습니다. 흥미롭게도 마취 상태에서 꼬리 정맥은 매우 신뢰할 수 없었고 성공률은 25%에 불과했습니다.성공률은 의식이 있는 쥐의 꼬리 정맥 천자와 마취 상태에서 음경 정맥 천자에 대해 거의 유사한 결과를 보였습니다.혈액 채취는 마취 없이 꼬리정맥 천자법, 마취 하에 음경정맥 천자 방법은 6분 이내에 시행한 반면, 마취 하에 꼬리정맥 천자 방법은 다발성 실패로 인해 8분 이상 소요되었다.

Research

JoVE Journal

Medicine

Tail Vein Puncture Without Anesthesia

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...

연구

의학

To begin, secure the rat in a plastic restraining cone, enclose the large end of the cone around the base of the tail. Ensure that the animal is comfortable and breathing is unrestricted throughout the procedure. Dip the tail in warm water for about one minute to dilate the vein, and dry the tail with a paper towel.In the restrainer, position the animal facing down with the tail lying on a heating pad. To select the appropriate tail vein for sampling, rotate the entire animal to the right or left side. Use the terminal third of the tail for blood vessel puncturing since the vessels become more superficial in this zone.Wipe the tail with 70%ethanol wipes at the puncture site and place it on the edge of the heating pad, creating an angle in the terminal third of the tail. This brings the vein to the surface and creates more space for taking the sample. For blood sampling, move the plunger back and forth in the syringe several times to smoothen the withdrawal and then pull on the plunger to create negative pressure in the syringe.Secure the tail flat on the heating pad using the non-dominant index and middle finger. Place the middle finger proximally and the index finger distally with the puncture site between these two fingers. Slide the insulin syringe against the index finger with the eye of the needle pointing upward until it is inserted into the vein.This creates an angle of 35 degrees between the needle and the tail. Once the needle has entered the vein, blood will flow into the syringe. Slowly withdraw the plunger of the syringe at a steady rate to collect the desired volume.Remove the syringe from the tail and allow a blood drop to form on the puncture side of the tail. Aspirate this blood to collect a few more microliters of blood, in case of a non-sterile procedure. Apply pressure to the puncture site to stop the bleeding and wipe the area with a new alcohol wipe.After removing the rat from the cone, return it to its cage. The success rates with the tail vein puncture methods were compared with the success rate of the penile vein puncture method under anesthesia. Interestingly, under anesthesia, the tail vein became very unreliable and had a success rate of only 25%The success rates showed almost similar results for the tail vein puncture in conscious rats and the penile vein puncture under anesthesia.The blood sampling was performed in less than six minutes in the tail vein puncture without anesthesia, and the penile vein puncture method under anesthesia, whereas the tail vein puncture method under anesthesia took more than eight minutes due to multiple failures.

Drawing Blood from the Penile Vein

Move the plunger back and forth in the syringe several times to smoothen the withdrawal and then pull on the plunger to create negative pressure in the syringe. Retract the foreskin of the anesthetized rat from the end of the penis and hold the glands between the index and thumb pulling gently. The dorsal penile vein will appear as a superficial blue cord.With the eye of the needle pointing upward, insert the insulin syringe into the vein at an angle of 35 degrees. Once the needle has entered the vein, blood will flow into the syringe. Slowly withdraw the plunger of the syringe at a slow and steady rate to collect the desired volume.Remove the syringe. A blood drop will form on the puncture site. Aspirate the drop to collect a few more microliters of blood in case of a non-sterile procedure.Apply light pressure to the puncture site to stop the bleeding and wipe the area with a new alcohol wipe. Place the penis back in its neutral position. After turning off the isoflurane, monitor the rat until complete recovery and return it to its cage.The blood volume collected in the dorsal penile vein puncture method under anesthesia was compared with the blood collected in the tail vein puncture method with and without anesthesia. The result shows that the penile vein puncture method under anesthesia and the tail vein puncture method without anesthesia yielded comparable quantities of blood.