Improving intravenous injection in black mice

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1 Improving intravenous injection in black mice Juerg Messer Roche Pharma Research & Early Development, Neuroscience, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland Correspondence to: Abstract: Biomedical research nowadays commonly involves the parenteral administration of large molecules to transgenic mouse models, preferably by intravenous (i.v.) injection. A convenient and routinely used route of i.v. administration is in the lateral tail veins. Particularly in transgenic mouse lines based on black strains, this technique is difficult to carry out simply because the veins contrast faintly to the skin. The vein visibility can be improved by utilizing a dedicated illumination device. In addition the i.v. injection procedure is facilitated if an optimal restrainer and effortless syringes are used. However, dedicated illumination device are rarely available on the market and the beneficial influence of a good restrainer and syringes might be underestimated. Here I introduce a special 180 LED illumination device to improve the visibility of the tail veins of black mice and report on the application of a Broome style restrainer as well as the selection of effortless insulin syringes for i.v. injection. The combined use of these devices and syringes improved the i.v. injection (Refine aspect). Introduction: Many of the potential drug candidates that are being developed in biomedical research are large molecules, e.g. proteins like antibodies. Unlike most small molecule drug candidates that are designated for enteral administration, i.e. oral (tablets, capsules), the large molecules would not get resorbed in the guts but get deactivated by digestion, therefore parenteral administration is required. Common parenteral routes of administration are intraperitoneal (i.p.), subcutaneous (s.c.) and intravenous (i.v.) injection. The latter is preferred in early research to ensure quick and complete bioavailability of the drug candidate in the blood circulation. Transgenic animals, mostly mice, are valuable animal models in biomedical research. Of these transgenic mouse lines, many are based on black strains, like the very common C57BL/6 strain. Mice feature two well accessible lateral tail veins, which makes these the prime target for the i.v. injection. In albino mouse strains, e.g. BALB/c, the lateral tail veins are easy to spot. This might not be the case in black mice. The skin of the tail is generally darker with a distinct black tinge of the top side, which subsides gradually in intensity to the sides. Even experimenters who are skilled in i.v. injection in albino mice may struggle when attempting to i.v. inject black mice due to poor contrast of the veins to the skin. The contrast also varies between the genders and age groups and you can be confronted by the situation that the veins can be seen hardly at all. The illumination in animal rooms with controlled environmental conditions is not adequate for i.v. injections and the use of an ordinary desktop lamp causes disturbing reflections on the tail. The illumination problem can be overcome with an illumination device that has an uniform light distribution over an angle of 180 around the mouse tail. The idea was to arrange small light emitting diodes (LED) in an arc, with the mouse tail right in the center of the arc, in order to achieve an uniform illumination. Another thought was inspired by the fact, that blue light in public toilets is applied to lower the visibility of the veins in an attempt to keep away heroin addicts. Why not trying the opposite, i.e. to optimize the light colour for best contrast of the

2 veins? RGB LEDs that generate any light colour, are readily available and cheap nowadays. According to these ideas, our in-house electronic workshop constructed a special LED lamp, using a piece of flexible RGB LED strip light and a RGB LED power supply. Another crucial requirement for successful i.v. injections is a suitable restrainer. Broome style restrainers are arguably the best choice, since they have a number of advantages over other types. Consider to try one of these instead of just using the old restrainer you might have at hand. Using a Broome restrainer alone might improve the procedure a lot. Also, the syringes used for the injection make a big difference. The plunger must slide with very low resistance, so that the experimenter has a chance to feel the resistance in case that the needle sticks in the tissue instead of the vein. Another factor to consider is the residual volume of the syringe, especially if precious large molecule solutions have to be injected. Syringes with integrated needle have a very small residual volume and are therefore highly recommended. Illumination: An uniform, reflection-free illumination is essential for successful i.v. administration in black mice. The standardized light conditions in the animal facilities are not sufficient for this purpose. Setting up an office lamp will not help, because it creates a bright reflection on the tail which obscures the tail vein. If the light sources are evenly distributed around the upper side of the tail, no reflections will occur. A simple way to implement this in practice is to mount a flexible LED strip light in an arc, with the tail in the center. Figure 1 shows an in-house built illumination device which consists of a RGB LED arc on a stand and a power supply. A Broome style restrainer is placed in the center. The arc has a diameter of 13 cm and is equipped with 10 RGB LED, one every ~2 cm (Osram Linearlight Colormix Flex LM10L-RGB2). It causes no reflections, and the light intensity is sufficient for the purpose. The enclosure contains a commercial power supply / RGB LED regulator rated for 230V mains. The light colour is determined by the intensity ratio of the primary colors. It is adjusted with the red, green and blue knob for best contrast of the veins. This illumination device has been in use for more than two years and has proved to be indispensable to i.v. inject black mice.

3 Figure 1: Custom-made LED lamp for uniform, reflection-free illumination of rodent tails for the purpose of i.v. injections. Front view [a.] and top view [b.]. Note the power supply / control box that allows to adjust the intensity of the primary colors (RGB) separately for best contrast of the veins. A further improved version of the device would feature a broader arc with two stripes in parallel to increase the brightness. The LED will be shifted against each other by half the distance between two LED, so that the LED are interlocking. A connector that allows to separate the LED arc from the power supply for improved transportability would be added. Although our illumination device is a sturdy construction and made by professionals, it is quite simple to replicate. A do-it-yourself variant for costs as low as 40 US$, 40 or 50 CHF respectively, is shown in figure 2. A flexible RGB LED strip light, the length is one meter, self-adhesive, with a wall plug power supply and detachable control box is available from IKEA: Dioder, item # (Europe version) or # (US version). Alternatively, you can find an abundance of mail order suppliers of LED strip lights and accessories on the internet. To have several strips in parallel without the need to cut/solder the strip, just fold it twice by 90 between two LEDs. Since the gap between the LED is a bit wider than that of the Osram LED strip and to have a higher light intensity, the whole length of the LED strip light was used, folded into for parallel strips. The IKEA Dioder LED strip is coated with transparent soft plastic, which was carefully removed between those LEDs, where the strip will be folded. The folded LED strip light was then mounted on a piece of black cardboard. The arc can be made from a dry cleaner s wire coat hanger. The wire was bent using a bottle with 9 cm diameter which resulted in a radius of 13 cm. Fast hardening epoxy resin (Araldite Rapid ) was used to glue the cardboard/led assembly to the wire. In contrast to the popular hot glue, epoxy resin is heat resistant, sturdier and rigid. The resulting device is neither stylish nor as rugged as the one in figure 1, but does the job!

4 Figure 2: Low-cost and simple a do-it-yourself implementation of the illumination device for rodent tails. It is made of an IKEA LED strip light, a piece of cardboard, a wire coat hanger and some Araldite Rapid glue. On the right side the control box [a.]. The transparent coating has to be removed partially in order to fold the strip [b.]. Folded to four parallel strips [c.]. 36 LED in total, function control [d.]. There is adhesive tape on the back side to stick it to the cardboard [e.]. The wire frame, made from a coat hanger [f.]. The cardboard is hold in place by adhesive tape and then fixed with fast hardening epoxy resin. Note the reinforcing braces made from the remaining wire [g.].

5 Restrainers: A Broome style restrainer is an acryl glass cylinder which is closed on one end. An open groove extends from the opening to the center of the end plate, so that a mouse/rat can be pulled into the cylinder and the tail will hang out at the end. The animal is held in place by a flat or cone nose piece which is inserted into the cylinder. Broome restrainers usually have a separate heavy stand and are detachable easily. Figure 3 shows pictures of our custom-made Broome style restrainer with an inner diameter of 31mm and a flat nose piece, suitable for mice of approximately grams. It is important to use restrainers in sizes that fit the animal: if the diameter is too large the animal will turn around, if it is too small the animal will suffocate! Our restrainer is a simplified copy of the Broome style restrainers from Plas-Labs Inc. (plas-labs.com). The diameter lies in between the two smallest standard types from Plas-Labs Inc.. It is compliant with the stand from Plas-Labs Inc., which consists of a stainless steel base plate and adjustable aluminium pillars that hold the restrainer. There are other suppliers, just google for Broome restrainer or nose cone rodent restrainer. Some manufacturers are willing to offer you custom-sizes on request. Figure 3: Broome style restrainers with flat nose pieces (stand not shown), side view [a.] and top view [b.]. From the left to the right: Plas-Labs Inc. #551-BSRR, 1 diameter / custom-made (Roche), 31 mm diameter / Plas-Labs Inc. #552-BSRR, 1.5 diameter. The Broome style restrainer is arguably the most convenient type of restrainer to use from the view of the experimenter: the experimenter has both hands free once the mouse/rat is in the restrainer, the full length of the tail is accessible and the restrainer can be detached from the stand. Also, it is quite easy to insert the animal and to release it afterwards. It seems also to be beneficial for the animal: The

6 animal will not attempt to run away all the time because the way out is blocked. Also, less handling is required: once the animal is in the restrainer, you can simply detach the restrainer from the stand and immerse the tail in warm water to dilate the veins, making it unnecessary to grasp the animal firmly by the scruff. Other types of restrainers like injection cones require the experimenter to firmly hold the animal by its tail all the time, while the animal attempts to run away. It seems obvious but is speculative if this causes more stress for the animal, but certainly it bears a high risk that the needle will slip out of the vein when the animal is jumping in a desperate attempt to escape. Flat bottom restrainers have no metal stand, not the full length of the tail might be accessible and you have to pull out the animal in order to release it. Syringes: Decades ago, glass syringes with metal plungers were the preferred tool for i.v. injections, because they have a very low resistance of the plunger. However, glass syringes also have disadvantages: heavy, need to be cleaned & autoclaved after use, expensive. For these reasons, glass syringes were mostly replaced by sterile single-use plastic syringes, which have the disadvantage of having a higher plunger resistance due to the rubber/silicone plunger seal. Keep in mind that standard syringes with attached needles (Luer, Luer-Lock) have quite a high residual volume. For research on large molecules, it is mandatory to use syringes with integrated needles to minimize losses, see figure 4. Integrated needles also simplify the process of removing air bubbles: just fill/eject twice while the needle is immersed in the solution and the air is mostly removed. Figure 4: U-100 insulin syringes with integrated needle. The small residual volume minimizes losses of (often precious) injection solution. Removing air bubbles is very simple and convenient. A number of different brands of insulin-100 type single-use syringes with integrated needle were evaluated in our lab for i.v. injections: BD Micro-Fine ml, Insulin Syringe, U100, 0.33 mm (29 G) x 12,7 mm BD Micro-Fine + 1 ml, Insulin Syringe, U100, 0.33 mm (29 G) x 12,7 mm Terumo Myjector, Insulin Syringe, 1 ml, U100, 0.33 mm (29 G) x ½ B Braun Omnican 100, 1 ml, Insulin Syringe, U100, 0.3 mm (30 G) x 12 mm, # B Braun Omnican 50, 0.5 ml, Insulin Syringe, U100, 0.3 mm (30 G) x 12 mm, # S The main focus was on plunger resistance and handling aspects. This assessment was of course highly subjective, and was later supplemented by more objective force measurements of the plunger resistance (see figure 5). The 0.5 ml syringe from B.Braun turned out to have the lowest plunger

7 resistance. It has to be noted that the plunger resistance can vary from syringe to syringe, even if the batch is the same. This is true for all brands and types. a. b. Figure 5: Simple setup to measure the plunger resistance. Water is slowly poured into a beaker which depresses the plunger [a.]. Table [b.] with measurements of n=2 syringes per type, which confirmed the subjective findings. The BD 0.5 ml syringes are not listed in the table, because we had none left for this test. * Force in Newton was recalculated from the weight (volume) of the water. The diameter of the tip of the barrel of the 1 ml BD Micro-Fine + syringe is smaller than any of the other tested types, which allows inserting the needle 1 or 2 mm further into the vein before the barrel touches the tail. However, since the animals hardly move in the Broome restrainer, slipping out of the vein is a low risk. Therefore it is no significant disadvantage that the needles of the other syringes can only be inserted ~6mm. If it is an issue for you, try to bend the needle a few degrees upward.

8 Procedure: Our standard procedure for i.v. injections in mice is as follows: 1. Weigh the animals and calculate the application volumes. Tip: Check the balance before use with an adequate calibration weight, even if you are not working under GLP conditions. An unnoticed malfunction of the balance may cause serious implications. 2. The light colour of the LED lamp is set for best contrast of the tail veins. 3. Fill the syringe and remove air bubbles. We apply an injection volume of 5 ml/kg [1]. With smaller volumes, your relative error in dosing will increase, because the graduation of a 0.5 ml syringe is only 0.01ml/unit and 0.02ml/unit for the 1 ml syringes. The injection solution must not be cold! Injecting a large volume of cold solution might decrease the body temperature and this could have an influence on the efficacy of the drug candidate. 4. Carefully insert the mouse in the Broome restrainer by pulling the tail through the slot. Make sure the mouse retracts the hind legs. Insert and fasten the nose piece (do not compress the animal), then release the tail. 5. Remove the restrainer from its stand and immerse the tail in warm water for approx. 45 seconds to dilate the veins. Use a water bath set to 42 C. Tip: Use a beaker in the water bath, to contain the contamination by urine and faeces. Using a heat lamp to warm up the whole animal might be more efficient but takes longer and bears the risk of heatstroke. 6. Dry the tail with a paper tissue and quickly check which lateral vein, the left or the right, is better visible. Rotate the restrainer ±90 accordingly and put it back on the stand, so that the chosen lateral tail vein faces you. 7. With one hand, bend down the tail over the index finger and hold it in place with the thumb. Hold the syringe in your preferred hand, parallel to the vein, the bevel facing upward. Insert the needle at almost a 0 angle for 6-7mm. Keep in mind that the vein is just below the skin! Do not attempt to aspirate blood as this may cause the vein to collapse! 8. Carefully push the plunger. The resistance you feel should be low, i.e. the plunger should move effortless. If the resistance is high and/or if a bulge forms at the tip of the needle, you have missed or pierced the vein try again 1 cm further up. Otherwise you might be able to see how the injection solution fills the vein towards the body. Inject slowly - be aware that 5 ml/kg is quite a large volume! 9. Cover the injection site with a swab or paper tissue before retracting the needle to avoid loss of blood and drug. Hold the swab in place while you carry the restrainer back to the cage. Remove the nose piece and let the mouse slide out of the restrainer into the cage. 10. The restrainer might be dirty with urine and faeces. Rinse it with water before you treat the next mouse. Comparable protocols and instructional videos can be found on the internet, e.g. [2], [3], [4].

9 Results: The presented LED illumination devices provide an uniform illumination of the mouse tail, without disturbing reflections. The effect is difficult to recognize on pictures of a mouse tail, though. For a comparison of different lighting conditions, a black electric cable was used instead. Figure 6 shows, that the two LED illumination devices do not cause a prominent longitudinal reflection on the surface of the cable, where in case of a mouse tail one of the lateral veins would be located. Figure 6: Comparing different illuminations. The disturbing reflections are difficult to image on a mouse tail, instead a black electric cable was used for this comparison. [a.] Office lamp, causing a prominent reflection exactly where the vein would be. [b.] In-house built LED illumination [c.] Do-it-yourself LED illumination, based on IKEA LED strip light. Particularly for black mice like the C57BL/6 strain, the LED illumination device enabled us to successfully carry out i.v. injections independently from the available lighting conditions. We are not aware of a commercial source for such a LED lamp for the illumination of mouse and rat tails, but it was demonstrated that the construction is simple and can be reproduced easily at low costs. The switch from old injection cones and other restrainer types at hand to a Broome style restrainer simplified the procedure and might reduce the stress for the animals. Furthermore the incidences when the needle slipped out of the vein due to a sudden movement of the mouse were virtually eliminated. It is also convenient that the tail can be immersed into warm water to dilate the veins while the animal is in the restrainer. This might convince some people who currently warm up the animal under a heat lamp, to use a water bath instead, avoiding to expose the animal to the risk of burnings or a heatstroke. As a side note: We have to admit, that for blood sampling from the tail vein, using the water bath is not as efficient as the heat lamp. When we introduced insulin syringes with integrated needle to reduce the volume of required injection solutions, the aspect that the plunger must move effortless was a bit neglected. Sometime later several brands and types were evaluated in order to select the one with the lowest plunger resistance.

10 The subjective comparison was verified with a simple force measurement. The 0.5 ml B.Braun Omnican syringes turned out to be not far away in this regard from glass syringes. To be able once more to feel if the needle is in the vein or not, helps to avoid that some of the solution is injected into the tissue. The invention of the LED lamp and introduction of the Broome restrainer at Roche was awarded 2 nd place in the Rs Roche Award in the category Surgery, Methodology, Training and Techniques. Conclusion: Apart from the skills of the experimenter, using the right technical aids and materials is crucial for a successful intravenous injection in mice and rats. In particular for black mice, the presented LED illumination device for uniform illumination of the tail and optimal contrast of the veins proved to be indispensable. Replacement of our old restrainers, e.g. injection cones, by a Broome style restrainer turned out to facilitate the i.v. injection procedure a lot. Syringes with integrated needle minimise the required amount of injection solution. It is worth to compare several brands and types of syringes to find the one with the lowest plunger resistance, so that the experimenter can feel if the injection is ok or not. In our evaluation, the B.Braun Omnican syringes turned out to be better than others in this regard, especially the 0.5 ml type. References: [1] Society for Laboratory Animal Science (SOLAS), Empfohlene Volumina für die Applikation von Substanzen bei Versuchstieren, March 2010:4 [2] University of Delaware Office of Laboratory Medicine, Intravenous (IV) Tail Vein Injection SOP #PRO-005, Rev 5/2010, [3] Intravenous Injection in the Mouse, [4] Acknowledgment: Rainer Bosshart (construction of the LED lamp) Dominik Mangold (CAD construction of the restrainer) Dr. med. vet. Aziz Cetinsu (review) Bernd Bohrmann, Ph.D. (review) Completed on 09-Jan-2015.

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