A simple  device for injecting small volume 

(5-100 micro liter) of liquid in small laboratory animals

Jashdeep Bhattacharjee, Barun Das, P Nagarajan, Pramod Upadhyay

 National Institute of  Immunology

 Aruna Asaf Ali Marg

 New Delhi 110067, India

 Email : pkumar@nii.ac.in

Key words :        Injecting small volume, Small laboratory animal, tail vein injection, retro-orbital venous sinus injection

Abstract

Intense practice is required to achieve good success rate in injecting small volume (5-100μl) of liquid in small laboratory animals.  We have designed a device for making such injections incident free with minimal skills. In this device, the piston of the syringe is pushed by a pushing mechanism based on the cable release of SLR camera. To use this device, the experimenter holds the animal with one of the hands and the other hand holds the syringe with needle mounted on the device. After piercing the target site at blood vessel or the organ the experimenter becomes still and piston of the syringe is either pushed by another person from a distance or by a syringe pump energized through a foot switch. We have evaluated this device for delivering injections in the tail vein and retro-orbital venous sinus in mice. The accuracy of the delivered dose was high and the injection procedure was completed in lesser time when the injector device was used.

Introduction

Injecting small volumes of samples (drugs, plasmids, cell suspension etc.) at a precise location (tail vein, retro-orbital venous sinus, spleen, etc.) of small animal (1) such as mice is often challenging. Delivering such injections without any incidents requires intense practice and the success rate is not consistent. 

There are two critical steps which determine the success of these injections, piercing the needle at a very precise location followed by pushing the piston of the syringe to deliver the liquid (a drug or cell suspension) at the pierced location. Often, even after piercing the tissue at the accurate site incidents of wrong administration might occur due to dislocation of the needle during pushing the piston of the syringe. Normally we use one hand to adjust the animal in order to orient the site of injection towards the needle and the needle is pierced by the other hand. The difficulty comes when the experimenter needs to push the piston; to do this, hand holding the syringe has to be moved (Figure 1A). This movement causes most incident or failure of the injection.

Figure 1A. Difficulty experienced when an injection is delivered; one of the hands has to move to push the piston of the syringe.

There are a variety of syringe pumps which can hold 1ml syringe in the market and a few of those pumps are portable. But these syringe pumps are mostly infusion pumps and are designed to deliver the dose over a period of time. Most of the portable syringe pumps are sufficiently bulky and cannot be used directly to deliver injections to small animals.

Hammond and Bhattacherjee (2) described a very nice design of a micro-capillary injection apparatus which can deliver microliter volume to the anterior chamber or vitreous body of the eye of small animals. Recently, World Precision Instruments, Florida, USA has introduced a microsyringe pump, UMP-III which can deliver small volumes.  In these devices, fine size needles (33-36 gauge) are used and these are only suitable for delivering drugs etc. and are not appropriate for delivering cell suspensions.

To address this need, we have designed a device to deliver small volume (5-100ml) of liquid in small laboratory animal. In this device conventional 1 ml insulin syringe with 30 gauge needle is used and it can deliver small volumes of liquids (drug solutions, cell suspensions etc).  In order to use this device, the experimenter needs to hold and orient the site of injection of the anaesthetized animal towards the needle with one hand and uses other hand to hold the syringe mounted in the device to pierce the tissue and remains still, the piston of the syringe is pushed through a pushing mechanism (Figure 1B) made of a cable release used in SLR camera. The holding weight of this device with cable release is around 8 grams only which makes it as easy as holding a syringe.

Figure 1B. The piston pushing mechanism in which no movement is required to push the piston after piercing the retro-orbital venous sinus.

The cable release can also be pushed by mounting it on a typical syringe pump (Figure 1C) which has been adjusted to move the piston at a desired speed and is electrically energized by pressing a foot switch.

Figure 1C. The cable release mounted on a typical syringe pump.

Due to the simple design of this device, it can easily be fabricated for less than 10 USD and modified to meet unique experimental situations.

Materials and Methods

Construction of the device

Items required

1. Threaded cable release used in SLR camera of the type Nikon AR-3

2. Machined pieces of acrylic sheet of the dimensions given in Figure 2A.

Figure 2A. Dimensions of the different pieces of device.

3. Cyanoacrylate adhesive to join the pieces of exactly cut acrylic sheet.

4. Plastic tube locker used in blood bags.

5. 1 ml Insulin Syringe, 26/30G (Dispo van, India)

6. Syringe Infusion Pump of type PHD 22/2000 (Harvard Apparatus, MA; US) operated by the foot switch of the type RS Stock No. 170-806 (Optional).

Get the exact sizes of acrylic sheet cut from a machining shop and carefully join them using the Cyanoacrylate adhesive. Fix the cable release in its hole and the device is ready to use.  Assembled device is shown in Figure 2B.

Figure 2B. The assembled device.

Using the device

1. Release the cable by pressing the hold rim.

2. Fill the desired dose (not more than 100 ml) in a 1 ml syringe and cap the needle.

3. Place the syringe in its slot on the device and apply the tube locker to firmly place the syringe in its slot.

4. Hold the device with filled syringe in one hand, uncap the needle and hold the animal by other hand. Pierce the desired tissue and become still. Instruct the other person to push the release cable to deliver the dose. 
Please see Video V1

Video V1. Video showing intrasplenic hepatocyte transplantation in mice carried out with injector device.

5. Alternatively, properly fix the free end of release cable in a typical syringe infusion pump which is wired through a foot switch. On the syringe pump, select the syringe size and flow rate so that the cable release moves at a desired speed. After piercing the tissue and becoming still, press the foot switch to energize the syringe pump so that it pushes the release cable to deliver the dose.

Results and Discussion

We have evaluated this device for performing injection in the tail vein and retro-orbital venous sinus in mice. These injection procedures have been approved by the Institutional Animal Ethics Committee of the National Institute of immunology, New Delhi, India. We have compared (i) the variability in the delivered dose and (ii) the time required to complete the injection procedure as measured by the time for which animal was held and the time for which the animal was pricked. These timings are very important for maintaining overall wellbeing of the animals during experiments. Figure 3 and 4 show the summary of results when tail vein and retro-orbital venous sinus injections were performed in NOD mice (N=10) with and without using this device.

Figure 3 shows the comparison of dose delivered.  For retro-orbital venous sinus injections, the variation in injected volume per injection, as indicated by standard deviation, was 10 times less when the injector device was employed. The scenario was comparable for tail vein injections.

Figure 3. A comparison of dose delivered when 100ml dose was delivered in retro-orbital venous sinus and tail vein in mice (N=10) with and without injector device.

A comparison of the time required for completing the injection procedure is shown in Figure 4. For tail vein injections, in both groups, on average the animals were held for around 25 seconds but the variations in the time durations was 4 times less when the injector device was employed.  Similarly, the animal was pricked for around 16 seconds and the standard deviation was half when the injector device was employed.  Also, noticeable reductions in the standard deviations of these timings were observed when the injector device was used for delivering retro-orbital venous sinus injections.

Figure 4. A comparison of the time required for completing the injection procedure with and without injector device.

Overall, when injector device was employed retro-orbital venous sinus injections the accuracy of the delivered dose was high and the injection procedure was completed in less time. The utility of this device is further enhanced when the retro-orbital venous sinus injections are reported to be less stressful than the tail vein injections (3).   

Further, due to high accuracy of delivered dose in less time, this injector device is very useful for delivering cells or bio-reagents during surgical procedures. The video V1 shows how this device was used for intrasplenic hepatocyte transplantation in mice.

References

1. Turner, P.V., Brabb, T., Pekow, C., et al.  Administration of substances to laboratory animals: routes of administration and factors to consider. Journal of the American Association for Laboratory Animal Science, 2011, 50, 600-613.

2. Hammond, B.R. and Bhattacherjee, P.,  A method for the intraocular injection of micro-volumes in small animal species. Current Eye Research, 1982, 1, 683-685.

3. Steel, C.D., Stephens, A.L., Hahto, S.M.,  et al.  Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model. Lab Anim (NY), 2008, 37(1), 26-32.