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>

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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 border="0" cellpadding="0" cellspacing="0" width="625">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thinpro Insulin Syringe</td>
			<td style="width:103px;">Terumo</td>
			<td>SS30M3009</td>
			<td style="width:188px;">3/10cc, 3/8&#34; needle, 30G, 1 per mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Evans Blue Dye</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2129</td>
			<td style="width:188px;">Dilute to 1% with 1X Phosphate Buffered Saline&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cotton Tipped Applicators</td>
			<td style="width:103px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">23-400-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fiber Optic Light Source&#160;</td>
			<td style="width:103px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">12-562-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting Microscope</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Intravenous Injections in Neonatal Mice at JoVE.com

Intravenous Injections in Neonatal Mice

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 ...

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57.4K Views.  Ohio State University. 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. 