Name: Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice (Video) | JoVE Journal
Uploaded: 2011-10-03T12:00:00.000+00:00
Duration: 5 min 55 s
Description: Learn intracerebroventricular and intravenous injection techniques for delivering therapeutic agents to the central nervous system using neonatal mice. These methods enable targeted brain delivery and systemic distribution for neuroscience research.

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5 minutes

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).

1. Intracerebroventricular injection
<ol>
<li>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. </li> 
<li>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.</li>
<li>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).</li> 
<li>Immobilize the PND 2 neonates via cryo-anesthesia for 1-2 minutes.</li>
<li>The needle used for this injection is a micro-liter calibrated sterilized glass micropipette that is attached to a 3ml 3 mL Hamilton syringe through a long tube.</li> 
<li>Break the tip of the needle (with spatula) to adjust for 2 mm penetration into the skull. Place the needle diagonally in the microcentrifuge tube containing the injection solution. Load the injection solution into the needle by carefully pulling the plunger of the syringe. </li>
<li>After the uptake of the solution, disassociate the syringe from the tube; pull the plunger up further and then attach to the needle again.</li>
<li>Hold the needle in right hand between the thumb and index fingers and place the syringe between the middle and ring fingers with the plunger touching the palm of the right hand.</li>
<li>Grasp the immobilized mouse firmly by the skin behind the head using the left hand and place on a fiber-optic light to illuminate relevant anatomical structures that can be used as a guide.</li>
<li>Insert the needle 2 mm deep, perpendicular to the skull surface, at a location approximately 0.25 mm lateral to the sagittal suture and 0.50–0.75 mm rostral to the neonatal coronary suture (Figure 1A).Then, push the plunger using the palm of the right hand very slowly and carefully. Monitor for ruptured vessels or facial swelling.</li>
<li>Remove the needle 15 seconds after discontinuation of plunger movement to prevent backflow.</li>
<li>To recover, keep the mice for 5–10 minutes in a warmed container until movement and general responsiveness is restored. </li>
</ol>
2. Intravenous injection via the temporal/facial vein
<ol>
<li>The solution to be injected should be prepared and supplemented with filtered green food dye at 1:100 dilution and injected under sterile conditions.</li>
<li>Attach a small plastic luer to the end of a 100 μL glass Hamilton syringe. To the luer, attach a 33 gauge 0.25 inch hypodermic needle. Insure that all parts are securely connected. </li> 
<li>Load the syringe with the volume to be injected, ensuring that there is no air in the syringe as injection of air into the vessel is lethal. It is often helpful to pipet the volume to be injected unto a piece of parafilm. This allows for the clear visualization of uptake of the solution into the syringe. </li>
<li>A Wee Sight Transilluminator is used to easily visualize the superficial temporal vein, or facial vein, in the neonate (Figure 2A). Before injection, secure the neonate to the transilluminator using surgical tape. Gauze should be placed between the neonate and the tape to prevent the tape from damaging the skin of the animal. Secure the animal to the transilluminator on its side, ensuring that the tape does not prevent respiration. The neonate’s neck should be gently turned so that the facial vein is easily visible and the nose should be taped to stabilize the head. </li>
<li>Using a 2.25X headband magnifier to allow for easier visualization, slowly insert the needle into the vein. The vein is very superficial so the needle should remain visible beneath the skin. </li>
<li>Slowly infuse the volume of solution into the vein. The green dye allows for easy visualization of the injection. </li>
<li>Wait 15 seconds before removing the needle as there is a lag between fully depressing the plunger of the syringe and the expulsion of the remaining solution due to the extremely narrow needle bore. </li>
<li>After removing the needle, use gauze to apply pressure to the injection site until the bleeding ceases.</li>
<li>Monitor the neonate for signs of distress. After a proper injection, no overt distress should be observed. The neonate should be given approximately 5 minutes to recover before it is returned to the cage.</li>
<li>Flush the needle/syringe unit with PBS in between injections as blood in the needle will quickly clog it due to small bore. </li>
</ol>
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. 
<img src="/files/ftp_upload/2968/2968fig1.jpg" alt="Mouse brain injection sites diagram; procedural images at 15 min, 60 min, and 12 hours post-injection." data-altupdatedat="1788235205000" data-alt="Figure 1"> 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.
<img src="/files/ftp_upload/2968/2968fig2.jpg" alt="Injection site diagram and lab result depicting experimental procedure on mouse, detailing facial veins." data-altupdatedat="1788235205000" data-alt="Figure 2"> 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.

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<li>Kienstra, K. A., Freysdottir, D., Gonzales, N. M., Hirschi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+neonatal+intravascular+injections%3A+modeling+newborn+disease%5BTitle%5D)+AND+%22J.+Am.+Assoc.+Lab.+Anim.+Sci%22%5BJournal%5D)">Murine neonatal intravascular injections: modeling newborn disease.</a> J. Am. Assoc. Lab. Anim. Sci. 46, 50-54 (2007).</li>
<li>Sands, M. S., Barker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Percutaneous+intravenous+injection+in+neonatal+mice%5BTitle%5D)+AND+%22Lab.+Anim.+Sci%22%5BJournal%5D)">Percutaneous intravenous injection in neonatal mice.</a> Lab. Anim. Sci. 49, 328-330 (1999).</li>
<li>Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rescue+of+the+spinal+muscular+atrophy+phenotype+in+a+mouse+model+by+early+postnatal+delivery+of+SMN%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.</a> Nat. Biotechnol. 28, 271-274 (2010).</li>
<li>Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li>
<li>Passini, M. A., Watson, D. J., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+delivery+to+the+mouse+brain+with+adeno-associated+virus%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Gene delivery to the mouse brain with adeno-associated virus.</a> Methods Mol. Biol. 246, 225-236 (2004).</li>
<li>Coady, T. H., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trans-splicing-mediated+improvement+in+a+severe+mouse+model+of+spinal+muscular+atrophy%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Trans-splicing-mediated improvement in a severe mouse model of spinal muscular atrophy.</a> J. Neurosci. 30, 126-130 (2010).</li>
<li>Baughan, T. D., Dickson, A., Osman, E. Y., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+bifunctional+RNAs+that+target+an+intronic+repressor+and+increase+SMN+levels+in+an+animal+model+of+spinal+muscular+atrophy%5BTitle%5D)+AND+%22Hum.+Mol.+Genet%22%5BJournal%5D)">Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy.</a> Hum. Mol. Genet. 18, 1600-1611 (2009).</li>
<li>Coady, T. H., Baughan, T. D., Shababi, M., Passini, M. A., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+single+vector+system+that+enhances+trans-splicing+of+SMN2+transcripts%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Development of a single vector system that enhances trans-splicing of SMN2 transcripts.</a> PLoS One. 3, e3468-e3468 (2008).</li>
<li>Dickson, A., Osman, E., Lorson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Negatively-Acting+Bifunctional+RNA+Increases+Survival+Motor+Neuron+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Hum.+Gene.+Ther%22%5BJournal%5D)">Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron in vitro and in vivo.</a> Hum. Gene. Ther. 19, 1307-1315 Forthcoming.</li>
<li>Mattis, V. B., Ebert, A. D., Fosso, M. Y., Chang, C. W., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+a+read-through+inducing+compound%2C+TC007%2C+lessens+the+severity+of+a+spinal+muscular+atrophy+animal+model%5BTitle%5D)+AND+%22Hum.+Mol.+Genet%22%5BJournal%5D)">Delivery of a read-through inducing compound, TC007, lessens the severity of a spinal muscular atrophy animal model.</a> Hum. Mol. Genet. 18, 3906-3913 (2009).</li>
<li>Williams, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligonucleotide-mediated+survival+of+motor+neuron+protein+expression+in+CNS+improves+phenotype+in+a+mouse+model+of+spinal+muscular+atrophy%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Oligonucleotide-mediated survival of motor neuron protein expression in CNS improves phenotype in a mouse model of spinal muscular atrophy.</a> J. Neurosci. 29, 7633-7638 (2009).</li>
<li>Passini, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> J. Clin. Invest. 120, 1253-1264 (2010).</li>
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No conflicts of interest declared.

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. ...

<table><tbody><tr><td>Green Food Dye</td><td>McCormick &amp;amp; Co.</td><td>n/a</td><td>Must be filtered</td></tr><tr><td>Hamilton Glass Syringe (100 &amp;mu;L)</td><td>Sigma-Aldrich</td><td>20702</td><td></td></tr><tr><td>LuerMxF Thread Style White Nylon</td><td>Small Parts, Inc.</td><td>VPLF-LC78-1-25</td><td></td></tr><tr><td>Fine gauge Hypodermic Needles</td><td>Popper &amp;amp; Sons, Inc.</td><td>7111</td><td>Size: 33(SWG) x &amp;#188;&quot; (6.35 MM)</td></tr><tr><td>Wee Sight Transilluminator</td><td>Respironics</td><td>1017920</td><td></td></tr><tr><td>2.25X Headband Magnifier</td><td>MagEyes</td><td>Model No. 5</td><td>Select magnification to fit individual preferences</td></tr></tbody></table>

delivery of therapeutic agents through intracerebroventricular (icv) and intravenous (iv) injection in mice

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

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.

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.

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 ...

In this video, intra cerebro ventricular and intravenous injection methods will be demonstrated in neonatal mice. For intra cerebro ventricular injection, the anesthetized mouse is first placed on top of a fiber optic light source to illuminate the sagittal and transfer sinuses. The inoculum is then carefully injected at a point lateral to the sagittal suture and rostral to the coronary suture.If performed correctly, the inoculum will be distributed first in the injected ventricle and will eventually reach the other ventricle for intravenous injection. The mouse is secured to a transluminator in such a way that the facial vein is exposed. The inoculum is then slowly injected directly in the vein and becomes distributed systemically.The implications of these techniques extend toward therapy of many diseases and disorders because they allow for the optimal placement of therapeutics. The ICV technique provides a means to cross the blood-brain barrier, while the IV technique allows for systemic therapeutic delivery. First, prepare the injection solution in a micro centrifuge tube.In this example, the inoculums contain triam blue for better visualization of the injection site. To inject mice use a sterilized and calibrated glass micropipet needle attached to a series of plastic tubing, gradually increasing in diameter and ending in a three milliliter plastic syringe. Using flat end tweezers, break off the tip of the glass needle so that it's three millimeters long.This will allow at least two millimeter penetration into the brain. Carefully load the solution to be injected into the glass micropipet needle by placing it diagonally in the micro centrifuge tube and pulling on the syringe plunger. Stop pulling the plunger as soon as the needle loading is completed.To avoid contaminating the tubing, remove the syringe from the tubing system attached to the glass micro pipet. Extend the plunger completely and reattach the needle firmly. Hold the micro pipee glass needle between the thumb and index finger.Place the syringe between the ring and little fingers with a plunger touching the palm of the injecting hand cryo anesthetize the two day old mouse. Then firmly grasp the cry anesthetized mouse by the skin at the base of the head, and place it on a fiber optic light source to illuminate the superior sagittal and transfer sinuses. The injection site is located about a quarter of a millimeter laterally to either side of the confluence of these sinuses holding the syringe perpendicular to the skull surface.Insert the needle penetrating two millimeters deep. Using the palm of the injecting hand, slowly push the plunger down to inject the solution. Wait 15 seconds before removing the needle to prevent backflow and monitor the mouse for cranial swelling or signs of ruptured vessels.Once the mouse has been injected, place it in a prewarm container for five to 10 minutes to allow it to recover. Compare the injected mouse to a non injected mouse. 10 to 15 minutes after intra cerebro ventricular injection of inoculum containing triam blue.The fluid should be distributed in the injected side of the brain after 60 minutes due to the connectivity of the cerebral ventricles, uniform distribution of triam blue in both cerebral hemispheres and olfactory bulbs should be visible. Finally, after 12 hours, the inoculum will be visible in the rostral central spinal canal. Prepare the injection solution and add green food dye at a one to 100 dilution.To visualize the injection, attach a small plastic lure to the end of a 100 microliter glass Hamilton syringe. Fit the syringe with a 33 gauge 0.25 inch hypodermic needle and ensure that all parts are securely connected. Load the syringe with the volume to be injected.Be sure to remove any air bubbles from the inoculum as injecting air intravenously is lethal. Use surgical tape and gauze to secure the neonate to the transluminator. By first securing the forearm and body to the transluminator and then securing the head so that the facial vein is visible.Ensure that the tape is loose enough that the animal can still breathe. Using a 2.25 x headman magnifier to allow better visualization, slowly insert the needle into the vein. Because the vein is superficial, the needle will remain visible under the skin.Slowly infuse the inoculum into the vein. Wait 15 seconds before removing the needle to allow all fluid to be expelled. Once the needle has been removed, use a piece of gauze to apply pressure to the injection site until the bleeding stops.Return the neonate to a warm place and allow five minutes for it to recover. Monitor it for signs of distress. If the injection has been performed correctly, the animal should not show signs of discomfort.Once the mouse is recovered, return it to its cage. If the intravenous injection has been performed correctly, the neonate will become green. After watching this video, you should have a good understanding of how to administer ICV and IV injections in neonatal mice.These techniques can be used as a means of injecting the therapeutic agent of your choice.

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Learn intracerebroventricular and intravenous injection techniques for delivering therapeutic agents to the central nervous system using neonatal mice. These methods enable targeted brain delivery and systemic distribution for neuroscience research.

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

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