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="Brain injection sites diagram; mouse brain perfusion results at 15 min, 60 min, 12 hours." data-altupdatedat="1747911167000" 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="Neonatal mouse injection site diagram and chimeric mouse pup model for experimental gene editing." data-altupdatedat="1747911167000" 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.

<ol>
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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

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.

Watch this Scientific Journal Video about Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice at JoVE.com

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

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

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 ...

delivery-therapeutic-agents-through-intracerebroventricular-icv

Research

JoVE Journal

Medicine

59.9K Views.  University of Missouri. Dit artikel legt uit twee zeer verschillende methoden voor injectie: 1) in de hersenen (intracerebroventriculaire) en 2) systemische (intraveneus) op de therapeutische middelen te introduceren in het centrale zenuwstelsel van neonatale muizen.

Video: Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Neuroscience

Common Carotid Artery Repair After Injection in a Mouse Model 

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

Author Spotlight: Advancing Glioblastoma Treatment Through Intraarterial Delivery Strategies for Oncolytic Viruses and MicroRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Cancer Research

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

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

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.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

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.

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 ...

Biology

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

Performing Intracerebroventricular Injection in Mice Using a Free-Hand Approach

Free-Hand Intracerebroventricular Injections in Mice

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter&#39;s hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.

Author Spotlight: Insights, Innovations, and Future Avenues in Reproductive Neurocontrol 

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.

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

A Comprehensive Protocol for Delivering Intrathecal Injections in Neonatal Mice

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

Author Spotlight: Intrathecal Injection &#8211; An Efficient and Reliable Delivery Method to Test the Efficacy of Gene Editing in Neonatal Mouse Brains

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

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

Catheter Based Intravenous Injection of Experimental Agents: A Technique for Delivering Experimental Agents Intravenously Through Jugular Vein in Mouse Model

Source: Bornert, A. et al. <a href="https://www.jove.com/v/62515">In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow.</a> J. Vis. Exp. (2021)
In this video, we describe a method for implanting a catheter into the jugular vein of a mouse model through which experimental agents such as drugs and tracer dyes can be delivered intravenously in vivo. Such an arrangement when connected to other systems can also be used for monitoring hemodynamics and blood sampling.

Source: Bornert, A. et al. In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow. J. Vis. Exp. (2021)
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Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

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