Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Summary

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.

Abstract

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.

Protocol

1. Intracerebroventricular injection

1. The first step is the preparation of the injection stock solutions; these solutions are viral vector, plasmid DNA, drugs, and should be injected under sterile conditions.
2. Mix a desired titer of viral vector (5-7 μL total) with 0.05% w/v trypan blue in PBS for visualization of the injection site.
3. Plasmid DNA solution (5 l μL total) contains D-(+)-glucose 20% (w/v) (1 μL), trypan blue (0.05%) PBS (1 μL), plasmid (˜5 ˜g/˜L) (2 ˜L), and 2.5 kDa linear PEI homopolymer (150 mM) (1 ˜L).
4. Immobilize the PND 2 neonates via cryo-anesthesia for 1-2 minutes.
5. The needle used for this injection is....

Discussion

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. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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