Name: Intravenous Injections in Neonatal Mice (Video) | JoVE Journal
Uploaded: 2014-11-11T13:00:00.000+00:00
Duration: 5 min 17 s
Description: Learn intravenous injection techniques for neonatal mice to deliver test articles in pediatric disease research. This non-invasive method enables precise therapy delivery in one to two-day-old mouse models.

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The authors wish to acknowledge the NINDS, FightSMA, and Families of SMA for financial support. SEGL is supported by NINDS training grant #5T32NS077984-02.

All procedures listed in the protocol have been approved Institute for Animal Use and Care committee (IACUC) of the Ohio State University.
1. Preparation of Workspace
<ol>
	<li>Gather wet ice to anesthetize the mouse pups, an empty cage to segregate the dam from the litter, a dissecting microscope, a light source that can be positioned at an angle to the injection (use of a light source at a 90&#176;&#160;angle to the injection site obscures the vein), a clean surface to place the animal for the injection, cotton swabs, 3/10 cc insulin syringe with 3/8&#8221; 30 G&#160;needle (one per animal) and 1% Evans Blue Dye (made with phosphate buffered saline (PBS)) solution for training.</li>
	<li>Remove the dam to a separate cage while manipulating the pups.</li>
</ol>
2. Injection Procedure
<ol>
	<li>Place a single pup directly on the wet ice for 30-60 sec&#160;to anesthetize the animal. Do not leave the animal on ice too long due to a risk of hypothermia related complications including ventricular fibrillation, tissue hypoxia and metabolic acidosis. 
	NOTE: Our experience is that 30-60 sec&#160;is sufficient to slow the pup movements to allow for injection. If deeper anesthesia is required, inhalants such as 1-2% isoflurane may be appropriate.</li>
	<li>While the animal is on ice, load the syringe with 30 &#956;l of Evans blue dye.</li>
	<li>When the animal is fully anesthetized, confirmed by lack of movement on the ice while still breathing, move it under the microscope. For a right-handed injection, face the animal&#8217;s muzzle to the right. Place the left index finger on the muzzle and the left middle finger caudal to the ear bud so that the ear bud is between the index and middle fingers (Figure 1).</li>
	<li>Examine just anterior to the ear bud for a superficial capillary that moves when the skin is manipulated. This capillary is NOT the target, however identification is important for temporal vein identification. Next, locate a dark, shadowy vein inferior to the capillary that remains fixed regardless of skin position. The temporal vein appears shadowy, runs dorsal to ventral, and feeds into the jugular vein (Figure 1).</li>
	<li>Enter the temporal vein with the needle bevel up. If correctly inserted, it is possible to view the needle bevel fill with blood through the skin. Then depress the plunger slowly and note blanching of the vein down the side of the face.</li>
	<li>Allow the needle to remain within the vein for an added 10-15 sec&#160;to prevent backflow of the injectant.</li>
</ol>
3. Post-injection
<ol>
	<li>After a proper injection, the pup should turn blue almost immediately. Remove the needle and use gentle force to apply a cotton swab to the injection site until the blood clots.</li>
	<li>Monitor the pup for signs of distress. Allow the pup 2-3 min&#160;to recover and rewarm, recognize when the pup is conscious, upright and moving, before returning to the cage. Cup the pups in the investigator&#8217;s gloved hands to provide appropriate warmth to aid in recovery if necessary. Alternatively, place a heating pad under the home cage to facilitate warming the injected pup. Generally after an Evans blue dye injection, euthanize pups. Do not include dye when injecting test material.</li>
	<li>Place the pup back into the home cage and ensure the pup is coated with bedding and/or nestlet to ensure reacceptance by the dam.</li>
	<li>Use a new syringe and cotton swab for each pup to maintain sterility.</li>
</ol>

<ol><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nature. 27, 59-65 (2009).</li>
<li>Dominguez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravenous+scAAV9+delivery+of+a+codon-optimized+SMN1+sequence+rescues+SMA+mice%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice.</a> Human molecular genetics. 20, 681-693 (2011).</li>
<li>Rahim, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravenous+administration+of+AAV2%2F9+to+the+fetal+and+neonatal+mouse+leads+to+differential+targeting+of+CNS+cell+types+and+extensive+transduction+of+the+nervous+system%5BTitle%5D)+AND+%22FASEB+journal+%3A+official+publication+of+the+Federation+of+American+Societies+for+Experimental+Biology%22%5BJournal%5D)">Intravenous administration of AAV2/9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 25, 3505-3518 (2011).</li>
<li>Foust, K. D., et al. <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>Duque, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol Ther. 17, 1187-1196 (2009).</li>
<li>Bostick, B., Ghosh, A., Yue, Y., Long, C., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+AAV-9+transduction+in+mice+is+influenced+by+animal+age+but+not+by+the+route+of+administration%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration.</a> Gene Ther. 14, 1605-1609 (2007).</li>
<li>Miyake, N., Miyake, K., Yamamoto, M., Hirai, Y., Shimada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+gene+transfer+into+the+CNS+across+the+BBB+after+neonatal+systemic+delivery+of+single-stranded+AAV+vectors%5BTitle%5D)+AND+%22Brain+research%22%5BJournal%5D)">Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors.</a> Brain research. 1389, 19-26 (2011).</li>
<li>Porensky, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+administration+of+morpholino+antisense+oligomer+rescues+spinal+muscular+atrophy+in+mouse%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse.</a> Human molecular genetics. 21, 1625-1638 (2012).</li>
<li>Hua, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+SMN+restoration+is+essential+for+long-term+rescue+of+a+severe+spinal+muscular+atrophy+mouse+model%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.</a> Nature. 478, 123-126 (2011).</li>
<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+%22Journal+of+the+American+Association+for+Laboratory+Animal+Science+%3A+JAALAS%22%5BJournal%5D)">Murine neonatal intravascular injections: modeling newborn disease.</a> Journal of the American Association for Laboratory Animal Science : JAALAS. 46, 50-54 (2007).</li>
<li>Glascock, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+therapeutic+agents+through+intracerebroventricular+%28ICV%29+and+intravenous+%28IV%29+injection+in+mice%5BTitle%5D)+AND+%22J.+vis.+Exp%22%5BJournal%5D)">Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.</a> J. vis. Exp. (56), (2011).</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+%22Laboratory+animal+science%22%5BJournal%5D)">Percutaneous intravenous injection in neonatal mice.</a> Laboratory animal science. 49, 328-330 (1999).</li>
<li>Bevan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+heart+failure+in+the+SMNDelta7+model+of+spinal+muscular+atrophy+and+correction+by+postnatal+scAAV9-SMN+delivery%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery.</a> Human molecular genetics. 19, 3895-3905 (2010).</li>
<li>Valori, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+delivery+of+scAAV9+expressing+SMN+prolongs+survival+in+a+model+of+spinal+muscular+atrophy%5BTitle%5D)+AND+%22Science+translational+medicine%22%5BJournal%5D)">Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy.</a> Science translational medicine. 2, (2010).</li>
<li>Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+intraperitoneal+or+intravenous+injections+of+recombinant+adeno-associated+virus+type+8+transduce+dorsal+root+ganglia+and+lower+motor+neurons%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.</a> Hum Gene Ther. 19, 61-70 (2008).</li>
<li>Guggenbuhl, P., Grosbois, B., Chales, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gaucher+disease%5BTitle%5D)+AND+%22Joint%2C+bone%2C+spine+%3A+revue+du+rhumatisme%22%5BJournal%5D)">Gaucher disease.</a> Joint, bone, spine : revue du rhumatisme. 75, 116-124 (2008).</li>
<li>Daly, T. M., Vogler, C., Levy, B., Haskins, M. E., Sands, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+gene+transfer+leads+to+widespread+correction+of+pathology+in+a+murine+model+of+lysosomal+storage+disease%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease.</a> Proc Natl Acad Sci U S A. 96, 2296-2300 (1999).</li>
<li>Daly, T. M., Ohlemiller, K. K., Roberts, M. S., Vogler, C. A., Sands, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevention+of+systemic+clinical+disease+in+MPS+VII+mice+following+AAV-mediated+neonatal+gene+transfer%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer.</a> Gene Ther. 8, 1291-1298 (2001).</li>
<li>Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Juvenile+neuronal+ceroid+lipofuscinoses%5BTitle%5D)+AND+%22Advances+in+experimental+medicine+and+biology%22%5BJournal%5D)">Juvenile neuronal ceroid lipofuscinoses.</a> Advances in experimental medicine and biology. 724, 138-142 (2012).</li>
<li>Forsythe, E., Beales, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bardet-Biedl+syndrome%5BTitle%5D)+AND+%22European+journal+of+human+genetics%22%5BJournal%5D)">Bardet-Biedl syndrome.</a> European journal of human genetics. 21, 8-13 (2013).</li>
<li>Bevan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+gene+delivery+in+large+species+for+targeting+spinal+cord%2C+brain%2C+and+peripheral+tissues+for+pediatric+disorders%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders.</a> Mol Ther. 19, 1971-1980 (2011).</li>
<li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retro-orbital+injections+in+mice%5BTitle%5D)+AND+%22Lab+animal%22%5BJournal%5D)">Retro-orbital injections in mice.</a> Lab animal. 40, 155-160 (2011).</li>
<li>Saunders, N. R., Joakim Ek, C., Dziegielewska, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neonatal+blood-brain+barrier+is+functionally+effective%2C+and+immaturity+does+not+explain+differential+targeting+of+AAV9%5BTitle%5D)+AND+%22Nature+biotechnology%22%5BJournal%5D)">The neonatal blood-brain barrier is functionally effective, and immaturity does not explain differential targeting of AAV9.</a> Nature biotechnology. 27, 804-805 (2009).</li>
<li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+AAV9-mediated+suppression+of+mutant+SOD1+slows+disease+progression+and+extends+survival+in+models+of+inherited+ALS%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.</a> Mol Ther. 21, 2148-2159 (2013).</li>
</ol>

The authors have nothing to disclose.

Intravascular delivery of agents to the CNS or throughout the body is difficult in neonatal murine models of disease. The described protocol is a quick, relatively non-invasive way to intravenously administer solutions into neonatal mice with minimal equipment requirements. Though the temporal face vein can be viewed by the naked eye, injections may have greater accuracy with the use of the microscope and fiber-optic light source, especially for an unpracticed injector. Intravascular injections in neonatal mice have a hi...

Delivery of therapeutics to the central nervous system (CNS) in murine models of pediatric disease remains a challenge. Mice that model newborn disease states are undersized and developmentally immature, and therefore can be difficult to directly inject in appropriate structures within the CNS. Intravascular injection of therapeutic agents is a non-invasive, well tolerated method to deliver cells, drugs, or viral vectors to the entire body including the CNS1-5 and retina3,5-9. Previous publications describe temporal face vein injection using a transilluminator10,11, without a dissection microscope11,12, or requiring two individuals to inject10. The injection technique described in this protocol is advantageous because a single individual can inject pups, and the light source to view the temporal vein is not touching the pup, eliminating the need for surgical tape or the attachment of a pup to a fixed surface such as a transilluminator11. Delivery of adeno-associated viral vector serotype 9 (AAV9) in mice produces robust expression in neurons and astrocytes throughout the brain and spinal cord (Figure 1). Intravascular delivery of viral vectors into the superficial temporal facial vein has been reliably used in various studies in neonatal mice to treat the pediatric neuromuscular disorder Spinal Muscular Atrophy (SMA)2,4,13,14 and ultimately increased the lifespan of treated mice.
Intravascular injection of neonatal mice also effectively targets the peripheral nervous system and peripheral organs (Figure 2). Following injection of AAV, transduction of dorsal root ganglia, liver, heart, skeletal muscle, lung, and myenteric plexus of the gut has been observed1,3,6,7,15. Widespread transduction of the CNS and periphery makes this method of injection ideal for diseases requiring global expression of a transgene, such as Gaucher&#8217;s disease16 and other lysosomal storage diseases17,18, Batten&#8217;s disease and related neuronal ceroid lipofuscinoses,19 and Bardet-Biedl syndrome, a genetic multisystem disorder with onset of symptoms occurring in early childhood20. Intravascular injection into neonatal mice should also be considered as a novel method of modeling system-wide pediatric diseases. This technique has been translated to larger animal models5,21 and intravascular injection already exists as a clinically acceptable method of delivering therapeutics.
The current protocol describes a simple, efficient method of delivering agents to neonatal mice through the superficial temporal face vein no later than postnatal day 2. Injection can be completed by a single, practiced individual and is well tolerated by both the pups and the dams. Pups experience minimal distress and recover quickly. Importantly, successful injection will result in global delivery of the agent administered. This protocol is appropriate for delivery of viral vectors, pharmaceutical agents or cells to newborn mice.

<table><tbody><tr><td>Thinpro insulin syringe</td><td>Terumo</td><td>SS30M3009</td><td>3/10 cc, 3/8&quot; needle, 30 G, 1 per mouse</td></tr><tr><td>Evans blue dye</td><td>Sigma-Aldrich</td><td>E2129</td><td>Dilute to 1% with 1x Phosphate Buffered Saline</td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Fiber optic light source</td><td>Fisher Scientific</td><td>12-562-36</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td></td></tr></tbody></table>

intravenous injections in neonatal mice

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

During a proper injection, the vein should momentarily turn clear, or blanch. If injecting dye the entire pup should turn blue within seconds. If an improper injection has occurred, there is often a concentrated subcutaneous bolus in the head or neck and injectant may leak out of the injection site. Improper injections may also result in the appearance of bruising around the throat. Pups that receive subcutaneous injections (i.e. the injection was not fully delivered in the vein) generally experience no ill side...

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Research

JoVE Journal

Biology

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.

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

Source: Seman, B. G., et al. A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis. J. Vis. Exp. (2020)The video demonstrates a neonatal mouse model for studying systemic infection using encapsulated bioluminescent bacteria. Neonatal pups are injected with high- or low-dose bacterial suspensions and returned to their mother for postnatal care. The encapsulated bacteria evade early immune detection, spread systemically, and are visualized over time using luminescence imaging. This model...

Modeling Infection in Neonatal Mice Using Encapsulated Bioluminescent Bacteria

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An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain.  This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

Free-Hand Intracerebroventricular Injections in Mice

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

A Neonatal Rodent Model of Retroorbital Vein Injection

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

This protocol demonstrates temporal vein injection for intravenous delivery of pediatric cancer cells to neonatal mice, enabling precise, reproducible modeling of early-life immune responses against cancer. The method enables assessment of the impact of interventions, such as immunomodulation or therapy, when applied during the neonatal period.

A Neonatal Intravenous Transplant Model for Assessment of Early-Life Immune Activities Against Cancer Cells

Learn intravenous injection techniques for neonatal mice to deliver test articles in pediatric disease research. This non-invasive method enables precise therapy delivery in one to two-day-old mouse models.

Intravenous Injections in Neonatal Mice

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