A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

The heating and restraining device offers a streamlined technique for successfully conducting intravenous tail vein injections, provides heating in a controlled environment, and effective handling to conduct the intravenous tail vein injection. Before attempting the technique, it’s important to familiarize yourself with the protocol and practice with saline injections. The visual demonstration offers the opportunity to show tips and caveats to help with the procedure.

Junko Yano, postdoc in my laboratory, will set up the equipment and I will demonstrate the procedure. To initialize the warming device, police the warming unit on a clean flat bench top and power it on, ensuring that the thermostat power indicator lamp is lit green. Then place bedding material inside the warming chamber to keep the area dry and retain heat.

For restraint device set up, place the restraint unit alongside the warming unit and determine the appropriate cone sizes for the animal. Manually adjust the base widths of the pliable aluminum cone to provide adequate restraint for the animal. Alternatively, replace the cone with custom fitted models to accommodate animals of varying sizes.

Set the thermostat to the desired temperature using the control dial, ensuring that the heater indicator is lit red and that the light bulb is on. Monitor the internal temperature display carefully while the bulb is on. It will activate automatically once the target temperature has been reached.

Position the restraint platform cone height to the optimum level for the user with the height adjustment knob. Once the target temperature has been reached, gently transfer the animals from the housing cage into the warming chamber. The warming chamber can safely contain four to six mice or one rat for a maximum of 20 to 30 minutes.

Heat treatment for 5 to 10 minutes is sufficient to induce vasodilation and enhance the visibility of tail veins. Monitor the animal for any signs of acute heat stress such as rapid respiration, lethargy, or jumping escape behavior. Remove the animal from the chamber by lifting it by the base of the tail and place it onto the cone opening of the restraining unit.

As the animal grasps on the far edge of the cone with its forelegs, gently pull the tail backwards and pass the tail through the open slit. Secure either of the hind legs protruding out from the cone so that the lateral vein is visible at the position of 12 o’clock. Grasp the tail at the mid to 2/3 length with the non-dominant hand between the thumb and the forefinger, putting slight tension on the lateral vein to maintain the tail positioning and vasodilation.

Wipe the skin of the injection site with a gauze sponge or pad moistened with 70%alcohol. Hold the syringe with the dominant hand and position the needle parallel to the tail. Insert the needle toward the direction of the blood flow at an angle of 10 to 25 degrees and advance further into the lumen of the vein by penetrating two to four millimeters.

Slowly inject the solution. In case of resistance or white blisters above the injection site, remove the needle and attempt a second injection at a site above the original needle placement. If injection of one lateral vein is unsuccessful, reposition the animal to the opposite side and make more attempts on the contralateral vein.

Remove the needle and firmly press the injection site with the thumb to prevent backflow of the injected solution or blood. Continue applying gentle compression with a clean gauze or tissue wipe until bleeding is stopped. Return the animal to its cage and monitor it for five minutes, ensuring that the animal resumes normal activity without further bleeding.

Once the heater of the warming chamber was activated, heat emission by the light bulb rapidly raises the internal temperature during the first 5 to 15 minutes, depending on the target temperature. The heater inactivates the light bulb if the detected internal temperature exceeded the set temperature. Subsequently, the device automatically repeats the heat cycle to maintain the internal temperature.

In a mouse model of bloodstream candidiasis resulting in sepsis, an intravenous challenge with Candida albicans in Swiss Webster mice caused a rapid onset of sepsis and dissemination of the organisms, leading to high mortality within three to four days. In contrast, animals could be protected from sepsis by pre-immunization or vaccination with an avirulent strain Candida dubliniensis, achieving more than 95%survival following the lethal IV challenge with virulent C.albicans. The unvaccinated animals with lethal infection had a significant increase in sepsis-induced morbidity whereas the vaccinated group exhibited minimal symptoms following the lethal challenge.

The results in progressive mortality versus vaccine-mediated protection were obtained reproducibly in four independent experiments. Be careful when positioning the animal in the cone that you correctly angle the needle to access the vein, adjust the needle into the vein and inject the vein. This streamline technique offers an efficient and effective means to conduct tail vein injections which will enhance the scientific outcomes of researchers.