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

Vergelijkende studie van bloedafnametechnieken bij ratten

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).

Auteur in de schijnwerpers: contextafhankelijke bloedafnamemethoden bij ratten 

Gemodificeerde staartader en penisaderpunctie voor bloedafname in het rattenmodel

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  

Staartaderpunctie zonder anesthesie

Om te beginnen, zet de rat vast in een plastic fixatiekegel, omsluit het grote uiteinde van de kegel rond de basis van de staart. Zorg ervoor dat het dier comfortabel is en dat de ademhaling tijdens de hele procedure onbeperkt is. Dompel de staart ongeveer een minuut in warm water om de ader te verwijden en droog de staart af met een papieren handdoek.Plaats het dier in de fixator naar beneden met de staart op een verwarmingskussen. Om de juiste staartader voor bemonstering te selecteren, draait u het hele dier naar de rechter- of linkerkant. Gebruik het terminale derde deel van de staart voor het doorprikken van bloedvaten, omdat de bloedvaten in deze zone oppervlakkiger worden.Veeg de staart af met 70% ethanoldoekjes op de prikplaats en plaats deze op de rand van het verwarmingskussen, waardoor een hoek ontstaat in het terminale derde deel van de staart. Dit brengt de ader naar de oppervlakte en creëert meer ruimte voor het nemen van het monster. Voor bloedafname beweegt u de zuiger meerdere keren heen en weer in de spuit om de opzuiging glad te strijken en trekt u vervolgens aan de zuiger om negatieve druk in de spuit te creëren.Bevestig de staart plat op het verwarmingskussen met behulp van de niet-dominante wijs- en middelvinger. Plaats de middelvinger proximaal en de wijsvinger distaal met de prikplaats tussen deze twee vingers. Schuif de insulinespuit tegen de wijsvinger met het oog van de naald naar boven gericht totdat deze in de ader wordt ingebracht.Hierdoor ontstaat een hoek van 35 graden tussen de naald en de staart. Zodra de naald in de ader is gekomen, stroomt er bloed in de spuit. Trek de zuiger van de spuit langzaam op met een constante snelheid om het gewenste volume te verzamelen.Verwijder de spuit van de staart en laat een bloeddruppel vormen aan de punctiezijde van de staart. Zuig dit bloed aan om nog een paar microliter bloed te verzamelen, in het geval van een niet-steriele procedure. Oefen druk uit op de prikplaats om het bloeden te stoppen en veeg het gebied af met een nieuw alcoholdoekje.Nadat je de rat uit de kegel hebt verwijderd, breng je hem terug naar zijn kooi. De slagingspercentages met de staartaderpunctiemethoden werden vergeleken met het slagingspercentage van de penisaderpunctiemethode onder anesthesie. Interessant is dat onder anesthesie de staartader zeer onbetrouwbaar werd en een slagingspercentage van slechts 25% had. De slagingspercentages toonden bijna vergelijkbare resultaten voor de staartaderpunctie bij bewuste ratten en de penisaderpunctie onder anesthesie.De bloedafname werd in minder dan zes minuten uitgevoerd in de staartaderpunctie zonder anesthesie en de penisaderpunctiemethode onder anesthesie, terwijl de staartaderpunctiemethode onder anesthesie meer dan acht minuten duurde vanwege meerdere storingen.

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 ...

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.