We appreciate the assistance of the NHGRI mouse facility staff, the NCI Molecular Pathology Lab, and especially Andrew Warner. R.J.C. is supported by the Intramural Research Program of the NHGRI through 1ZIAHG200318-16 and this work was partially funded by the National Center for Advancing Translational Sciences (NCATS). Figure 2C was created with BioRender.

This protocol and the accompanying video are for the retrobulbar injection space of postnatal, juvenile, and runted adult mice by retrobulbar sinus injection; the protocol has been approved by the National Human Genome Research Institute&#39;s Institutional Animal Care and Use Committee (ACUC) under protocol number G-03-4. Other institutions may have different requirements and restrictions, and this protocol may need to be modified for approval in your institute. Obtain approval from your institution&#39;s ACUC before performing this or any other animal procedure.
1. Pre-injection preparation
<ol>
	<li>Dilute the AAV to the desired injection volume and concentration with sterile phosphate-buffered saline (PBS) in a sterile 1.5 mL microcentrifuge tube for each injection. Add an extra 50% volume for each injection to allow for accurate filling of the single-use sterile syringe. 
	NOTE: Here we are diluting an AAV8-CAG-eGFP reporter to deliver a dose of 1 &#215; 1013viral genomes per kg of body mass (vg/kg) in a volume of 50 &#956;L. The amount of AAV in the 50 &#956;L to be injected is calculated using the weight of the animal at the time of injection. In this video, AAV is being injected via the retrobulbar route to replicate systemic delivery in humans. Other gene therapy vectors (i.e., lentivirus, adenovirus), RNA therapies, and small molecules can be delivered systemically using this procedure.</li>
	<li>Ensure that the table-top laboratory animal anesthesia system (LAAS) is properly set up and functioning correctly in accordance with the manufacturer&#39;s instructions. 
	NOTE: If an alternative anesthesia method is used, ensure that anethesia is prepared prior to beginning injections. The retrobulbar injection procedure should be compatible with most anesthesias (e.g., an injectable chemical restraint like ketamine and xylazine).</li>
	<li>Prior to filling the single-use sterile syringe (here, an insulin syringe, 31 G, 8 mm long, 3/10 mL capacity), move the plunger up and down several times to ensure the plunger can be depressed smoothly. Then, fill the syringe to the desired volume, ensuring there are no air bubbles. 
	NOTE: Volumes up to 150 &#181;L can be injected; our lab typically injects a volume of 50 &#181;L.</li>
</ol>
2. Mouse sedation via administration of isoflurane gas with a laboratory animal anesthesia system (LAAS)
<ol>
	<li>Ensure that gas is only flowing to the induction chamber. Close the two-way stopcock to the non-rebreathing (NRB) circuit.</li>
	<li>Turn on the green oxygen flow knob on the front of the flow meter so that there is a flow rate of 1 L/min.</li>
	<li>Turn isoflurane to &#8804;4% by depressing the lever at the top of the vaporizer and turning the dial to the desired concentration.</li>
	<li>Place the mouse in the clear induction chamber. Watch the animal&#39;s breathing and movement carefully. Once the animal is recumbent, turn the vaporizer knob down to 2-2.5% of isoflurane.</li>
	<li>Open the two-way stopcock to the NRB circuit attached to the face mask and close the gas flow to the induction box.</li>
	<li>Remove the animal and place it in the NRB circuit&#39;s face mask.</li>
	<li>Turn down the isoflurane concentration setting to 1.5%-1.75%, as determined by reaction to stimuli (e.g., toe-pinch or paw-squeeze).</li>
	<li>Always continuously monitor the mouse&#39;s breathing and mucous membrane color (if possible). If the animal&#39;s breathing becomes labored or the mucous membrane color is not pink, turn down the anesthetic concentration.</li>
	<li>Keep the animal warm throughout the entire procedure. Use a handwarmer wrapped in a paper towel that is placed underneath the underpad and located directly underneath the mouse.</li>
	<li>Turn off the oxygen and the vaporizer after completion of the procedure.</li>
</ol>
3. Injection of the animal
<ol>
	<li>If right-handed, inject the mouse&#39;s right eye and position the mouse on its left side with the snout pointing towards the right hand. If left-handed, inject the mouse&#39;s left eye and position the mouse on its right side with the snout pointing towards the left hand.</li>
	<li>Apply one or two drops of ophthalmic anesthetic to the eyeball, which is to be injected. Then, remove any excess ophthalmic anesthetic solution using a sterile absorbent gauze pad. Apply gentle pressure with fingertips to the skin dorsal and ventral to the eye to partially protrude the mouse&#39;s eyeball from the socket (Figure 2A,B). 
	NOTE: Be careful not to apply excessive pressure to surrounding cervical vessels when protruding the eye as this will impede blood flow and injection. In addition, applying pressure to the trachea could prevent the mouse from breathing. Ensure the mouse can breathe throughout the procedure.</li>
	<li>Keep the needle in a bevel-down position at an angle of approximately 30&#176; and placed into the medial canthus (Figure 2C). 
	NOTE: The depth of the needle placement to reach the retrobulbar sinus will vary based on the size of the animal. Having the needle in the bevel-down position during the injection decreases the risk of ocular damage. Be careful not to place the needle too deep and puncture the eye socket. The injection should take less than a minute.</li>
	<li>Slowly and smoothly apply pressure to the syringe plunger to deliver the injectate. This will reduce the chance of extravasation.</li>
	<li>Remove the needle slowly and smoothly.</li>
	<li>Remove the face mask from the mouse to allow for recovery from anesthesia.</li>
</ol>
4. Post injection
<ol>
	<li>Turn off the oxygen and the vaporizer after completion of the procedure.</li>
	<li>Use sterile gauze to remove blood if residual bleeding occurs.</li>
	<li>Ensure the mouse is in a warm area (approximately 37 &#176;C), but not excessively hot, to prevent hypothermia during recovery from anesthesia.</li>
	<li>Observe the mouse in isolation until completely recovered before returning the mouse to the cage and rack. 
	NOTE: Isolating the mouse during recovery prevents cage mates from injuring the sedated mouse during recovery.</li>
</ol>

Effective Systemic Delivery in Mice Using Retrobulbar Injection Technique

<ol>
	<li>Perlman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+human+disease:+An+evolutionary+perspective.">Mouse models of human disease: An evolutionary perspective.</a> Evol Med Public Health. 2016 (1), 170-176 (2016).</li><li>Samelson-Jones, B. J., George, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+gene+therapy+for+hemophilia.">Adeno-associated virus gene therapy for hemophilia.</a> Annu Rev Med. 74, 231-247 (2023).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrobulbar+injections+in+mice.">Retrobulbar injections in mice.</a> Lab Anim (NY). 40 (5), 155-160 (2011).</li><li>Chandler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+gene+therapy+for+methylmalonic+acidemia+using+the+novel+adeno-associated+viral+vector+44.9.">Systemic gene therapy for methylmalonic acidemia using the novel adeno-associated viral vector 44.9.</a> Mol Ther Methods Clin Dev. 27, 61-72 (2022).</li><li>Chandler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+aav9+gene+therapy+improves+the+lifespan+of+mice+with+niemann-pick+disease,+type+c1.">Systemic aav9 gene therapy improves the lifespan of mice with niemann-pick disease, type c1.</a> Hum Mol Genet. 26 (1), 52-64 (2017).</li><li>Venturoni, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+advantage+of+corrected+hepatocytes+in+a+juvenile+model+of+methylmalonic+acidemia+following+liver+directed+adeno-associated+viral+mediated+nuclease-free+genome+editing.">Growth advantage of corrected hepatocytes in a juvenile model of methylmalonic acidemia following liver directed adeno-associated viral mediated nuclease-free genome editing.</a> Mol Genet Metab. 137 (1-2), 1-8 (2022).</li><li>Ilyinskii, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immtor+nanoparticles+enhance+aav+transgene+expression+after+initial+and+repeat+dosing+in+a+mouse+model+of+methylmalonic+acidemia.">Immtor nanoparticles enhance aav transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.</a> Mol Ther Methods Clin Dev. 22, 279-292 (2021).</li><li>Davidson, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+systemic+aav+gene+therapy+with+a+neurotrophic+capsid+in+niemann-pick+disease+type+c1+mice.">Improved systemic aav gene therapy with a neurotrophic capsid in niemann-pick disease type c1 mice.</a> Life Sci Alliance. 4 (10), e202101040 (2021).</li><li>Chandler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+gene+therapy+using+an+aav44.9+vector+rescues+a+neonatal+lethal+mouse+model+of+propionic+acidemia.">Systemic gene therapy using an aav44.9 vector rescues a neonatal lethal mouse model of propionic acidemia.</a> Mol Ther Methods Clin Dev. 30, 181-190 (2023).</li><li>Rocha-Ferreira, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neonatal+rodent+model+of+retroorbital+vein+injection.">A neonatal rodent model of retroorbital vein injection.</a> J Vis Exp. (204), e65386 (2024).</li><li>Bohnert, B. N., Artunc, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+nephrotic+syndrome+in+mice+by+retrobulbar+injection+of+doxorubicin+and+prevention+of+volume+retention+by+sustained+release+aprotinin.">Induction of nephrotic syndrome in mice by retrobulbar injection of doxorubicin and prevention of volume retention by sustained release aprotinin.</a> J Vis Exp. (135), e57642 (2018).</li><li>Bohnert, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrobulbar+sinus+injection+of+doxorubicin+is+more+efficient+than+lateral+tail+vein+injection+at+inducing+experimental+nephrotic+syndrome+in+mice:+A+pilot+study.">Retrobulbar sinus injection of doxorubicin is more efficient than lateral tail vein injection at inducing experimental nephrotic syndrome in mice: A pilot study.</a> Lab Anim. 53 (6), 564-576 (2019).</li><li>Meijer, M. K., Spruijt, B. M., Van Zutphen, L. F., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+restraint+and+injection+methods+on+heart+rate+and+body+temperature+in+mice.">Effect of restraint and injection methods on heart rate and body temperature in mice.</a> Lab Anim. 40 (4), 382-391 (2006).</li><li>Nohara, M., Tohei, A., Sato, T., Amao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+response+to+restraint+stress+by+salivary+corticosterone+levels+in+adult+male+mice.">Evaluation of response to restraint stress by salivary corticosterone levels in adult male mice.</a> J Vet Med Sci. 78 (5), 775-780 (2016).</li><li>Schoch, A., Thorey, I. S., Engert, J., Winter, G., Emrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+routes+of+antibody+administration+in+pharmacokinetic+studies.">Comparison of the lateral tail vein and the retro-orbital venous sinus routes of antibody administration in pharmacokinetic studies.</a> Lab Anim (NY). 43 (3), 95-99 (2014).</li><li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+routes+of+intravenous+drug+delivery+in+a+transgenic+mouse+model.">Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model.</a> Lab Anim (NY). 37 (1), 26-32 (2008).</li><li>Zincarelli, C., Soltys, S., Rengo, G., Rabinowitz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+aav+serotypes+1-9+mediated+gene+expression+and+tropism+in+mice+after+systemic+injection.">Analysis of aav serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.</a> Mol Ther. 16 (6), 1073-1080 (2008).</li></ol>

The authors have nothing relevant to this publication to disclose.

While retrobulbar injection is a reliable method to deliver small molecules, proteins, and genomic therapies, practicing the technique with a dye is necessary to ensure that reliable and replicable systemic delivery is achieved. The use of a dye is highly recommended for practicing retrobulbar injections in mice before using this delivery route in experiments. Dyes can be visually checked in the mouse tissues to ensure consistent systemic delivery.
In our demonstration of the retrobulbar injection technique, isoflurane gas was used to anesthetize the mice prior to the procedure. Other forms of anesthesia can be used prior to the procedure, but it is important to ensure that the mouse does not recover from the sedation before the injection is completed. Fortunately, the actual injection usually takes less than a minute and the time for which the mouse needs to be fully anesthetized is brief. The mouse should be completely sedated during the injection, and anesthesia should be readministered if the mouse becomes conscious prior to the injection. Since there are risks associated with using anesthesia, the duration of time the mouse is sedated should be minimized. We have not had issues using isoflurane to sedate small sick mice with methylmalonic and propionic acidemia. However, some mouse models may be more sensitive to sedation and certain anesthesia. This potential problem should be considered prior to attempting to use sedation in a study. Lastly, the use of sedation in combination with the retrobulbar injection greatly reduces the apparent distress the mouse exhibits during the injection process in comparison to TVI where sedation is not commonly used.
We have not observed any postinjection-related issues, although infection is a potential risk with any injection. To reduce the chance of infection a sterile single-use disposable syringe and sterile PBS to dilute the purified AAV are used. All mice in our animal facility are checked daily for signs of potential health issues and receive veterinary care to address any health issues when warranted.
The alternative to retrobulbar sinus injection and the more widely used method of systemic delivery to juvenile and adult mice is TVI. TVI and retrobulbar sinus injection result in a similar biodistribution in the case of small molecules and antibodies, and by extrapolation, the same would be expected for viral vectors15,16. However, no examples comparing the systemic delivery of gene therapy vectors by TVI and retrobulbar sinus injection could be found in the literature. In our opinion, retrobulbar sinus injections are easier to perform in mice with a decreased growth phenotype and/or early lethality.
TVI is often considered to be more analogous to systemic delivery in humans, despite humans having a retrobulbar sinus but not having a tail. In one aspect, the retrobulbar sinus injection is like human systemic delivery in that the injectant enters the upper venous system the same as if an injectant was delivered to a human by a peripherally inserted central catheter (PICC Line) or an intravenous catheter placed in the arm. Conversely, the injectant enters the lower venous system of a mouse after tail vein injection. Unfortunately, neither one of these methods exactly replicates the method(s) used for systemic delivery in humans, but both are effective methods of systemic delivery in mice.

Murine models of genetic diseases are commonly used to demonstrate the efficacy of small molecule, genetic, and cell therapies1. In mice, the most widely used method to replicate systemic delivery to humans is the tail vein injection (TVI), which is typically performed in adult mice at approximately 6-8 weeks of age to ensure the vein is large enough to access. TVI has been successfully used in numerous preclinical proof-of-principle studies of genetic diseases, such as hemophilia, that have supported human clinical trials for gene therapy2. However, many murine models of genetic disease have growth and/or early lethality phenotypes, which prevent them from reaching the age or size of an adult mouse (Figure 1). Treating such mice via TVI can be extremely difficult, if not impossible, depending on the age of lethality and/or the maximum size the animals can achieve.
In contrast, systemic delivery of a therapeutic agent by retrobulbar (frequently and incorrectly referred to as retro-orbital) sinus injection can be done quite readily in mice regardless of age or size3. Retrobulbar injections of adeno-associated virus (AAV) have been used successfully in young growth-retarded murine models of genetic disease, such as methylmalonic acidemia (MMA) and Niemann-Pick type C disease4,5,6,7,8. (This procedure can also be used to inject neonates3,4,9,10; however, this technique is not detailed in this protocol or the accompanying video.) Even highly toxic substances like doxorubicin can be delivered safely by retrobulbar injection11,12. Unlike TVI, mice are anesthetized during retrobulbar sinus injections, which makes the procedure less stressful on the mouse and easier on the operator who does not have to physically restrain the mouse13,14. An additional concern is that TVI frequently use a heat lamp to dilate the tail vein, which could potentially cause dehydration in young mice and could be problematic in murine models of genetic disease that are more suspectable to heat-related stress. Another problem that can arise when using TVI is that the tail vein can be particularly challenging to visualize on highly pigmented mice. However, like TVI, retrobulbar sinus injections result in a wide systemic biodistribution15,16.

<table><tbody><tr><td>AAV8-CAG-eGFP</td><td>Univ. Penn. Vector Core</td><td>Special order</td><td>alternative sources of AAV reporters and alternative AAV reporters are available</td></tr><tr><td>Barrier (Filter) Tips, 20 &amp;mu;L size</td><td>ThermoFisher</td><td>AM12645</td><td>for diluting AAV to disire injection volume and concentration</td></tr><tr><td>Barrier (Filter) Tips, 100 &amp;mu;L size (sterile)</td><td>ThermoFisher</td><td>AM12648</td><td>for diluting AAV to disire injection volume and concentration</td></tr><tr><td>Dual Prodedure Circuit&amp;nbsp;</td><td>VetEquip</td><td>921400</td><td>alternative anesthesia method can be used</td></tr><tr><td>Gilsen PIPETTEMAN Classic P20 pipette</td><td>Gilson</td><td>F123600</td><td>for diluting AAV to disire injection volume and concentration</td></tr><tr><td>Gilsen PIPETTEMAN Classic&amp;nbsp; P100 pipette</td><td>Gilson</td><td>F123615</td><td>for diluting AAV to disire injection volume and concentration</td></tr><tr><td>Hand warmers (HOTHANDS)</td><td>ULINE</td><td>S-1497B</td><td>to keep mouse warm while anesthesized</td></tr><tr><td>Insulin syringes, 31 G,&amp;nbsp; 8 mm length, 3/10 mL capacity</td><td>Becton Dickson</td><td>328438</td><td>used in video; one syringe per injection</td></tr><tr><td>Isoflurane (Fluriso)</td><td>VETONE</td><td>502017</td><td>alternative anesthesia can be used</td></tr><tr><td>Medline Protection Plus Disposable Underpads</td><td>ThermoFisher</td><td>23-666-062</td><td>to place mouse on durring injection</td></tr><tr><td>Oak Ridge Phlebotomy Sharps Container With Transparent Lid</td><td>ThermoFisher</td><td>22-730-434</td><td>for needle disposal</td></tr><tr><td>Phosphate buffered saline PBS, pH 7.4</td><td>Gibco</td><td>10010023</td><td>To dilute AAV to desired concencentration and volume</td></tr><tr><td>Snap Cap Low Retention Microcentrifuge Tubes (sterile)</td><td>ThermoFisher</td><td>3451</td><td>for diluting AAV to disire injection volume and conentration</td></tr><tr><td>Sterile gauze sponge 4&quot;x&quot;4</td><td>Covidien</td><td>3033</td><td></td></tr><tr><td>Trecaine Hydrochloride Ophthalmic (0.5%)</td><td>Ocenanside Pharmaceuticals</td><td>AK102D5DS</td><td>local&amp;nbsp; anesthetic</td></tr><tr><td>Tuberculin syringe with a 27.0 G (or smaller)</td><td>Any</td><td>N/A</td><td>alternative to insulin syringe used in video</td></tr><tr><td>VetEquip&amp;rsquo;s User Guide and Operating Manual for table top and mobile Laboratory Animal Anesthesia System.&amp;nbsp;</td><td>VetEquip</td><td>chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.vetequip.com/pdfs/LAAS%20Manual.pdf</td><td>link to users guide and manual</td></tr></tbody></table>

systemic treatment for postnatal, juvenile, and runted adult mice by retrobulbar sinus injection

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.

Retrobulbar sinus injection has been used successfully to systemically deliver small molecules, antibodies, and adeno-associated virus (AAV)4,5,9,15,16. In Figure 3, the liver of a PBS (vehicle)-treated mouse and an AAV8-treated mouse liver are shown as an example of AAV injection and expression following a retrobulbar injection. AAV8, like many naturally occurring AAV vectors, is liver trophic. Therefore, substantial liver transduction is expected in a mouse that received a systemic dose of 5 × 1012vg/kg17. The large number of hepatocytes expressing methylmalonyl-CoA mutase (MMUT) RNA seen in Figure 3, which is being expressed by the AAV transgene, indicates a successful retroorbital injection.
<img alt="Mouse growth comparison, Propionic Acidemia vs Wildtype, 4 weeks, weight chart." class="xfigimg" src="/files/ftp_upload/66809/66809fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: A growth retarded mouse with propionic acidemia. This is an example of the extreme growth retardation that can occur in murine models of genetic disease. <a href="https://www.jove.com/files/ftp_upload/66809/66809fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mouse eye microinjection process; experimental setup and diagram for intravitreal injection." class="xfigimg" src="/files/ftp_upload/66809/66809fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Images and diagram of retrobulbar sinus injection. (A) Image of finger placement on fur to protrude eyeball (indicated by white arrow). (B) Image of eyeball (indicated by white arrow) protrusion after application of downward pressure on the fur prior to needle placement and injection. (C) Diagram of needle bevel orientation (bevel down relative to eyeball), needle angle (30°), and retrobulbar sinus needle placement. <a href="https://www.jove.com/files/ftp_upload/66809/66809fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Liver cell histology, microscope images showing tissue sections and staining with arrows indicating changes." class="xfigimg" src="/files/ftp_upload/66809/66809fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: RNA in situ hybridization after retrobulbar sinus Injection with AAV8. Images of (A) 10x vehicle-treated liver, (B) 10x AAV8-treated liver, (C) 20x vehicle-treated liver, and (D) 20x AAV8-treated liver stained for MMUT RNA. Mice with methylmalonic acidemia were treated with a dose of 5 × 1012 vg/kg of AAV8-LPS-MMUT or a vehicle control (PBS) at 1 month of age. Liver tissue was collected 1 month post treatment. MMUT RNA stained brown (black arrows indicate areas of positive staining). Liver counterstained with hematoxylin. Scale bars = 100 µm 10x for images, 50 µm for 20x images (B). Abbreviations: AAV = adeno-associated virus; LPS = Liver-specific promoter; MMUT = methylmalonyl-CoA mutase. <a href="https://www.jove.com/files/ftp_upload/66809/66809fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection at JoVE.com

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for ...

systemic-treatment-for-postnatal-juvenile-runted-adult-mice

Research

JoVE Journal

Genetics

863 Views.  National Institutes of Health. Ons onderzoek omvat de ontwikkeling van gentherapieën voor zeldzame genetische aandoeningen, met name met een focus op een groep stofwisselingsstoornissen die bekend staat als de organische acidemieën. Het doel van ons onderzoek is om te bepalen welke benaderingen het meest geschikt zijn om door te gaan naar klinische proeven bij mensen. Meerdere gentherapieën zijn goedgekeurd voor de behandeling van een verscheidenheid aan genetische ziekten, wat tien jaa...

Video: Auteur in de schijnwerpers: Uitdagingen in gentherapie voor organische acidemieën overwinnen door retrobulbaire injectie in muizenmodellen

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Neuroscience

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Medicine

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Biology

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

A Neonatal Rodent Model of Retroorbital Vein Injection

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

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.

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an ...

Compound Administration IV

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound administration in laboratory mice and rats. Protocols may, however, require the use of the less commonly used routes: intracardiac, footpad, and retro-orbital injections. Specialized training is essential for these procedures to be performed successfully. Justification for these routes may need to be provided to gain Institutional Animal Care and Use Committee (IACUC) approval.

Compound Administration in Rodents- Specialized Injections

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound ...

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

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