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1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University, 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri
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This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.
Glascock, J. J., Osman, E. Y., Coady, T. H., Rose, F. F., Shababi, M., Lorson, C. L. Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice. J. Vis. Exp. (56), e2968, doi:10.3791/2968 (2011).
Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.
1. Intracerebroventricular injection
2. Intravenous injection via the temporal/facial vein
3. Representative results:
ICV injection: The brain can be harvested at different time points post-injection and visualized to demonstrate successful injection technique. If the dye is not added to the injection solution, it will be difficult to check for the accuracy of the injection. Proper injection of one of the ventricles will allow distribution of the trypan blue on the injected side of the brain approximately 10-15 minutes post-injection (Figure 1B). A uniform distribution of trypan blue in both right and left cerebral hemispheres and olfactory bulbs will be visible approximately 60 minutes post injection (Figure 1C). This is due to connectivity of the cerebral ventricles that will permit the dissemination of the blue dye to the adjacent ventricle. Accurate injections can also be visualized by dispersal of the blue dye in the rostral central spinal canal approximately within 12 hours post-injection (Figure 1D). Inaccurate injections can be distinguished by lack of blue color in cerebral hemispheres. In this case, the depth of the needle was not sufficient and the injection solution has probably distributed under the skin. An alternative possibility is that the needle penetrated too deep in the brain, thereby passing beyond the ventricle and the solution is emptied underneath the cerebral ventricles.
IV injection: Accurate and efficient temporal/facial vein injection can be visualized immediately following the injection. As the solution is injected, the green dye allows for the visualization of the solution entering and flowing through the facial vein (Figure 2A). Additionally, altered skin color of the mouse will be observed following injection. The neonate’s normal pink color is converted to green, due to presence of green food dye in the injection solution (Figure 2B). The extent of the green color change observed in the neonate is dependent upon the concentration of dye used. For simple visualization of injection and during a practice period, a 1:50 dilution is recommended. However, the dramatically altered skin tone often results in the pup being rejected by the mother and therefore, removal of the dye or a significantly reduced concentration should be considered during experimental procedures. An inaccurate injection will result in the accumulation of green dye under the skin at the site of the injection. Incomplete injection, which occurs when the opening of the needle is not fully inserted within the vein, is seen when some solution flows in the vein and some accumulates under the skin. If this is observed, the needle should be inserted further into the vessel.
Figure 1. Demonstration of successful I.C.V. injections in PDN2 mice. A) Schematic of injection sites utilized in I.C.V. injections into the ventricles. B) Brain from I.C.V. injected Pup with trypan blue in PBS on left ventricle, photo taken 15 minutes post injection. C) Brain from I.C.V. injected Pup with trypan blue in PBS on left ventricle, photo taken 60 minutes post injection. D) Rostral central spinal canal is shown in I.C.V. injected pup with trypan blue in PBS on left ventricle, photo taken 12 hours post injection.
Figure 2. Demonstration of successful I.V. injections in PDN2 mice. A) Schematic of facial vein utilized for I.V. injections. B) Pup injected I.V. with green dye in PBS compared to un-injected littermate, Photo taken immediately post injection.
Research using mouse models of disease often requires the administration of drugs or other substances to neonates. In this video, we demonstrate the step-by step procedures involving two types of injection strategies that can be used to target the CNS: 1) direct injection into the CNS utilizing intracerebroventricular (ICV) injection; or 2) IV injection targeting the temporal/facial vein. The timing of these injections is of great importance. Since the ICV injections are freehand, the skull must be relatively malleable. This type of injection is only possible through the first week of life, and becomes increasingly more challenging and detrimental to the animals as the animals age. However, the primary benefit is that direct delivery to the CNS is achieved, effectively bypassing the blood brain barrier. Therefore, if an investigator is developing CNS therapeutics, it is possible to analyze compounds that are not fully optimized for drug-like properties such as blood brain barrier permeability, however, compound activity can be measured in disease-relevant tissues. Use of the temporal vein during the first few days of life has proven valuable as this is a relatively non-invasive technique for general IV administration. At this developmental stage, no additional veins are easily accessible, such as the tail vein which becomes more accessible during the second week of life 5. Both injection techniques are safe and can be performed freehand, with mice recovering shortly after injection without any detrimental side effects. Both techniques are suitable for rapid injection of a group of animals in a short period of time. They require proper visualization of the targeted area to ensure a successful injection. This is provided by the transilluminator and magnifying headband in IV injection (Table 1) and a fiber-optic light in ICV injection.
One of the advantages of IV injection is that a larger volume (100 ∞Ll) can be injected with ˜90% success rate 6. Common problems encountered while learning the IV technique include needle misplacement resulting in subcutaneous injections and/or incomplete injections. These problems can be countered by ensuring the bevel of the needle is thoroughly threaded through the vessel. The needle should be inserted into the vessel far enough that the bevel of the needle is completely surrounded by the vessel. Additionally, the vessel is extremely superficial, thus the injector must ensure that the needle is in the vessel and not beneath it. To check if the needle is in the vessel, after placement the needle should be gently moved side to side. If the needle has penetrated the vessel, the vessel will move along with the needle. If the vessel does not move, the needle is not inserted properly. Performing this check before injection will increase the number of successful injections. Neonatal delivery of AAV9 vectors has been recently utilized to successfully transduce a high percentage of motor neurons using recombinant AAV vectors in SMA mice 7,8.
The most important consideration during the ICV injection involves the length of the needle tip. The tip must be long enough such that it can penetrate at least 2 mm in the skull. If the tip is not long enough, the needle will not be able to efficiently enter into the ventricles. Alternatively, if the tip is too long, this hinders the uptake of the solution to be injected and also creates a fragile tip that is difficult to guide correctly through the skull. The angle of the needle is important to ensure exact penetration into the ventricle by holding it perpendicular to the skull with the tip toward the sagittal suture. ICV injections have been utilized in numerous studies to introduce compounds, drugs, therapeutic RNAs, plasmid DNAs, and viral vectors into the CNS of the diseased mice models 4,9-17. However, the limiting factor in ICV injection is the small capacity of the cerebral ventricle in neonatal mice that may not allow penetration of a desired volume of the therapeutic agents. Therefore, the ability to reach therapeutic expression levels using a lower vector dose in a short period of time is particularly important where the delivery volume is restricted.
No conflicts of interest declared.
The authors would like to thank John Marston for expert animal husbandry and Dr. Marco A. Passini for technical assistance at the early stages of this project. This work was funded by grant from the National Institutes of Health to C.L.L. (R01NS41584; R01HD054413).
|Green Food Dye||McCormick & Co.||n/a||Must be filtered|
|Hamilton Glass Syringe (100 μL)||Sigma-Aldrich||20702|
|LuerMxF Thread Style White Nylon||Small Parts, Inc.||VPLF-LC78-1-25|
|Fine gauge Hypodermic Needles||Popper & Sons, Inc.||7111||Size: 33(SWG) x ¼" (6.35 MM)|
|Wee Sight Transilluminator||Respironics||1017920|
|2.25X Headband Magnifier||MagEyes||Model No. 5||Select magnification to fit individual preferences|
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