Name: Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice (Video) | JoVE Journal
Uploaded: 2017-07-13T12:30:00.000+00:00
Duration: 8 min 32 s
Description: Learn subpial AAV9 delivery in adult mice to achieve potent spinal transgene expression across gray and white matter. This technique advances research on spinal neurodegenerative disorders like ALS.

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5 minutes

This study was supported by the SANPORC and ALSA Foundation grant (Martin Marsala); the National Sustainability Programme, project number LO1609 (Czech Ministry of Education, Youth and Sports); and RVO: 67985904 (Stefan Juhas and Jana Juhasova).

These studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee of the University of California, San Diego and were in compliance with the Association for Assessment of Laboratory Animal Care guidelines for animal use. All studies were performed in such a manner as to minimize group size and animal suffering.
1. General Animal and Surgical Preparation
<ol>
	<li>Before starting the surgical procedure, thaw the virus (AAV9-UBI-GFP; 5 &#181;L aliquots)8. Prepare a 5% dextran (10,000 MW) solution by mixing dextran powder in distilled water. Mix the virus solution with 5% dextran solution 1:1 to a final dextran concentration of 2.5%.
	<ol>
		<li>Store the virus solution on ice (4 &#176;C).</li>
	</ol>
	</li>
	<li>Use adult C57BL/6J mice (male and female, 20-30 g). Anesthetize the mice using 5% isoflurane (in O2, 1 L/min) and maintain them at 2-3% inhaled isoflurane (in O2, 1 L/min) by nose cone during surgery, depending on the breathing rate and paw pinch response.</li>
	<li>Shave the back of the animals with shaving clippers and clean the skin with 2% chlorhexidine.</li>
	<li>In chronic recovery studies follow a strict sterile technique.</li>
	<li>If lumbar subpial injections are to be performed, cut the skin overlaying the Th8-L1 vertebrae with a scalpel and detach the paravertebral muscle from Th10-12 spinal vertebrae using scissors.
	<ol>
		<li>Mount the animal into a standard stereotaxic frame using mouse spinal clamps.</li>
		<li>Shave the both sides of the lamina of the Th10-12 vertebrae using a dental drill (drill bit: 0.9 mm, speed: 20,000 rpm) until cracks appear.</li>
		<li>Remove cracked bone fragments with forceps and expose the dorsal surface of the lumbar spinal cord.</li>
		<li>Cut open the dura about 1 cm using a 30 G stainless steel needle and forceps.</li>
	</ol>
	</li>
	<li>If cervical subpial injections are to be performed, incise the dorsal neck skin 1.5-2 cm using scissors and expose the C1-C2 segments.
	<ol>
		<li>Remove the atlanto-occipital membrane of the cisterna magna using a 23G stainless steel needle and forceps.</li>
		<li>Clean the incision site of any tissue and bone debris using cotton swabs.</li>
		<li>Cut open the dura about 2-3 mm using a 30 G stainless steel needle and forceps.</li>
	</ol>
	</li>
</ol>
2. Opening the Pial Membrane and Inserting the Subpial Needle for AAV9 Delivery
<ol>
	<li>Mount the 34 G pia-penetrating needle into the Z-arm of an XYZ manipulator using a glass capillary holder (Figure 1A and B). 
	NOTE: To manufacture the pia-penetrating needle, the original beveled tip of the 34G needle is sharpened using a glass capillary beveller with diamond abrasive plate - coarse (5.0 &#181;m to 50 &#181;m tip sizes) and grinding angle of 15 - 20&#176;. The tip of the needle (1 mm length, measured from the tip) is then gently bent to about 90&#176; (Figure 1B, left insert).</li>
	<li>Using a surgical dissecting scope, set to 8-10 X magnification, penetrate the pia with the pia-penetrating needle by about 1 mm (Figure 1C) using the X-arm.
	<ol>
		<li>Keep the angle of the penetrating needle to the tissue surface at 5-10&#176;.</li>
	</ol>
	</li>
	<li>After the pia opening, remove the pia-penetrating needle horizontally from the subpial space (Figure 1 D) using the X-arm. 
	NOTE: Remember the penetrated site by a landmark, such as a blood vessel.</li>
	<li>Load a blunt 36G injection needle with AAV9-UBI-GFP virus using a 50-&#181;L microsyringe connected to the injection needle with PE-10 or PE-20 tubing.</li>
	<li>Mount the needle into the Z-arm of a second XYZ manipulator (Figure 1A and B) using a glass capillary holder (Figure 1B, right insert). 
	NOTE: To manufacture the subpial AAV9 injection needle, the blunt tip of a 36 G needle is polished using a glass capillary beveller with diamond abrasive plate - coarse (5.0 &#181;m to 50 &#181;m tip sizes) to remove the sharp edges. The tip of the needle (2-3 mm length, measured from the tip) is then gently bent to about 90&#176;. The pia-penetrating and subpial injection needles are inserted into 1-2 cm-long 20G slave stainless steel tubing (10 mm from the end of the needle) and glued with epoxy. The use of 20 G (0.91 mm-diameter) tubing is required for a secure attachment to the glass capillary holder.</li>
	<li>By manipulating the X, Y, and Z arms of the second manipulator, position the tip of the AAV9 injection needle into the pia-penetrated site and then advance it about 2-3 mm into the subpial space through the previous pial membrane opening using the X-arm ( Figure 1E and F). 
	NOTE: 1) The AAV9-UBI-GFP is prepared according to previously reported protocols9,10, and the final titers are adjusted to 1.2 x 1013 genome copies per mL (gc/mL). 2) There is no need to mark the site of the pial opening because it is readily identifiable (Figure 1D).</li>
	<li>Inject the AAV9-UBI-GFP (1.5, 3, or 5 &#181;L) into the subpial space using a 50 &#181;L microsyringe (see Table 1 for experimental groups).</li>
	<li>Remove the injection needle from the subpial space after the AAV9-UBI-GFP injection is complete.</li>
	<li>Close the muscle and skin using 4.0 monofilament suture and surgical clips. 
	NOTE: There is no need to seal the open vertebra.</li>
	<li>Allow the animals to recover on a heating pad.</li>
	<li>For pain control inject Buprenorphine 0.05 mg/kg/sc &#160;every 12 h for 2-3 days post-surgery.</li>
</ol>
3. Perfusion-fixation, Tissue Cryoprotection, and Immunofluorescence Staining
<ol>
	<li>At a predetermined time point after the subpial AAV9 injections, deeply anesthetize the mice with euthanasia solution (see the Table of Materials, 0.3 mL) and transcardially perfuse them with 20 mL of heparinized saline followed by 20 mL of 4% paraformaldehyde in PBS.</li>
	<li>Dissect the spinal cords and brains using a bone rongeur and post-fix them in 4% formaldehyde in PBS overnight at 4 &#176;C.</li>
	<li>Cryoprotect the spinal cords and brains with 30% sucrose in PBS for a minimum of 5-7 days.</li>
	<li>Cut coronal, transverse, or longitudinal/horizontal frozen sections (30 &#181;m-thick) on a cryostat and store them in PBS at 4 &#176;C.</li>
</ol>
4. Immunofluorescence Staining of Spinal Cord and Brain Sections (See the Table of Materials)
<ol>
	<li>Incubate free-floating sections in primary antibodies overnight.</li>
	<li>After incubation with primary antibodies, wash the sections three times in PBS and incubate with fluorescence-conjugated donkey anti-rabbit, donkey anti-chicken, and donkey anti-goat secondary antibodies.</li>
	<li>Mount the sections on microscopy slides, dry them at room temperature, and cover them with an anti-fade medium.</li>
	<li>Capture images using epifluorescence fluorescence microscope (objectives: 10X, NA-0.3; 20X, NA-0.8; and 63X, NA-1.4).</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+%22Nat+Biotechnol%22%5BJournal%5D)">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat Biotechnol. 27 (1), 59-65 (2009).</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 (7), 1187-1196 (2009).</li>
<li>Gray, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+differences+of+intravascular+AAV9+delivery+to+neurons+and+glia%3A+a+comparative+study+of+adult+mice+and+nonhuman+primates%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates.</a> Mol Ther. 19 (6), 1058-1069 (2011).</li>
<li>Meyer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+single+injection+CSF+delivery+of+AAV9-mediated+gene+therapy+for+SMA%3A+a+dose-response+study+in+mice+and+nonhuman+primates%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates.</a> Mol Ther. 23 (3), 477-487 (2015).</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 (12), 2148-2159 (2013).</li>
<li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+fidelity+of+intrathecal+delivery+of+self-complementary+AAV9-survival+motor+neuron+1+for+spinal+muscular+atrophy%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.</a> Hum Gene Ther. 25 (7), 619-630 (2014).</li>
<li>Bell, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+neuron+transduction+after+intracisternal+delivery+of+AAV9+in+a+cynomolgus+macaque%5BTitle%5D)+AND+%22Hum+Gene+Ther+Methods%22%5BJournal%5D)">Motor neuron transduction after intracisternal delivery of AAV9 in a cynomolgus macaque.</a> Hum Gene Ther Methods. 26 (2), 43-44 (2015).</li>
<li>Miyanohara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potent+spinal+parenchymal+AAV9-mediated+gene+delivery+by+subpial+injection+in+adult+rats+and+pigs%5BTitle%5D)+AND+%22Mol+Ther+Methods+Clin+Dev%22%5BJournal%5D)">Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.</a> Mol Ther Methods Clin Dev. 3, 16046(2016).</li>
<li>Xu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+gene+knockdown+in+rat+dorsal+root+ganglia+mediated+by+self-complementary+adeno-associated+virus+serotype+5+following+intrathecal+delivery%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.</a> PLoS One. 7 (3), 32581(2012).</li>
<li>Xiao, X., Li, J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> J Virol. 72 (3), 2224-2232 (1998).</li>
</ol>

Martin Marsala is a co-founder of Neurgain Technologies, Inc. (San Diego, USA).

The current study describes a technique of subpial vector (AAV9) delivery in adult mice. As demonstrated in the accompanying video, this approach and technique can effectively be used, provided that the required instruments and pia-penetrating needle and subpial injection needle are properly manufactured, according to the established and tested specifications.
Critical Technical Variables in Performing a Consistent and Safe Subpial Injection in Mice: 
As demonstrated, a s...

The use of AAV vectors to treat a variety of spinal cord and CNS neurodegenerative disorders is becoming a well-accepted platform to effectively upregulate or silence the expression of gene(s) of interest. One of the key limitations to the more effective utilization of this technology to treat CNS/spinal cord disorders is the limited ability to deliver AAV vector(s) to the deep brain or spinal cord parenchyma in adult mammals.
It was demonstrated, for example, that the systemic delivery of AAV9 in adult rodents, cats, or non-human primates is only moderately effective at inducing transgene expression in neurons in the brain and spinal cord1,2,3. The more effective intrathecal delivery of AAV9 vectors has also been shown to lead to only limited transgene expression in anatomically-defined pools of neurons. More specifically, it has been demonstrated that cisternal or lumbo-sacral intrathecal AAV9 delivery in non-human primates, pigs, or rodents leads to a high level of transgene expression in spinal &#945;-motoneurons and segmental dorsal root ganglion neurons. However, minimal or no expression in spinal interneurons or ascending or descending axons in the white matter is seen4,5,6,7. Collectively, these data show that a highly effective biological-anatomical barrier exists, which prevents the diffusion of intrathecally delivered AAV into deeper spinal parenchyma.
In a previous study using adult rats and pigs, a novel subpial vector delivery technique was developed8. Using this approach, highly potent and multi-segmental transgene expression was demonstrated after a single-bolus subpial AAV9 delivery. Intense GFP expression was consistently seen in neurons, glial cells, and descending/ascending axons through the injected spinal segments. This study demonstrated for the first time that the pia mater represents the primary barrier limiting effective AAV9 diffusion into the spinal parenchyma from the intrathecal space. While this previously developed technique and subpial injection device is relatively easy to use in large rodents (like rats) or adult pigs, the system is not suitable for use in small animals, such as adult mice. Because of the high number of available transgenic mouse models of a variety of neurodegenerative disorders, there is a clear need for the development of an effective spinal-parenchymal vector delivery technique in mice. The availability of such a technique would permit the study of the effect of specific gene silencing (e.g., using shRNA) or upregulation using cell-non-specific (e.g., cytomegalovirus-CMV or Ubiquitin) or cell-specific (e.g., synapsin or glial fibrillary acidic protein (GFAP)) promoters during early postnatal development or under diseased conditions.
Accordingly, in the present study, we have developed and validated a miniature subpial vector delivery system that can effectively be used in adult mice. Similarly, as in previous rat and pig studies, this work demonstrates potent transgene expression throughout the spinal parenchyma after a single-bolus subpial AAV9 delivery in mice. The simplicity of this approach, the very good tolerability of injected mice to subpial AAV9 delivery, and the high potency of transgene expression in the spinal parenchyma suggest that this technique can effectively be implemented in any laboratory setting and used in experiments targeting spinal gene expression.

<table><tbody><tr><td>C57BL/6J Mice</td><td>Jackson Labs</td><td>664</td><td></td></tr><tr><td>Lab Standard Stereotaxic for Mice</td><td>Harvard Apparatus</td><td>72-9568</td><td></td></tr><tr><td>Mouse Spinal Adaptor</td><td>Harvard Apparatus</td><td>72-4811</td><td></td></tr><tr><td>XYZ Manipulator</td><td>Stoelting</td><td>51604</td><td></td></tr><tr><td>Manual Infusion Pump</td><td>Stoelting</td><td>51218</td><td></td></tr><tr><td>34G Beveled Nanofill Needle</td><td>World Precision Instruments</td><td>NF34BV-2</td><td></td></tr><tr><td>36G Blunt Nanofill needle</td><td>World Precision Instruments</td><td>NF-36BL-2</td><td></td></tr><tr><td>Fluriso, Isoflurane</td><td>MWI Veterinary Supply</td><td>502017</td><td></td></tr><tr><td>Chlorhexidine Solution</td><td>MWI Veterinary Supply</td><td>501027</td><td></td></tr><tr><td>20G Stainless Steel Needle</td><td>Becton-Dickinson</td><td>305175</td><td></td></tr><tr><td>23G Stainless Steel Needle</td><td>Becton-Dickinson</td><td>305145</td><td></td></tr><tr><td>30G Stainless Steel Needle</td><td>Becton-Dickinson</td><td>305128</td><td></td></tr><tr><td>Cotton Tipped Applicator</td><td>MWI Veterinary Supply</td><td>27426</td><td></td></tr><tr><td>Glass Capillary Beveller&amp;nbsp;</td><td>Narishige International</td><td>SM-25B</td><td></td></tr><tr><td>Slide Microscope Superfrost</td><td>Leica Microsystems</td><td>M80</td><td></td></tr><tr><td>50 &amp;mu;L Microsyringe&amp;nbsp;</td><td>Hamilton</td><td>81242</td><td></td></tr><tr><td>BD Intramedic PE-20 Tubing</td><td>Becton, Dickinson</td><td>427406</td><td></td></tr><tr><td>BD Intramedic PE-10 Tubing</td><td>Becton, Dickinson</td><td>427401</td><td></td></tr><tr><td>4-0 monofilament suture</td><td>VetOne</td><td>V1D397</td><td></td></tr><tr><td>Glass Capillary Beveller&amp;nbsp;</td><td>Narishige</td><td>Pipet Micro Grinder EG-40&amp;nbsp;</td><td></td></tr><tr><td>5 min Epoxy (Epoxy Clear)</td><td>Devcon</td><td>14310</td><td></td></tr><tr><td>Euthanasia Solution</td><td>MWI Veterinary Supply</td><td>11168</td><td></td></tr><tr><td>Heparin Inj 1,000 U/mL</td><td>MWI Veterinary Supply</td><td>54254</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Anti NeuN Antibody</td><td>EMD-Millipore</td><td>ABN78</td><td>Primary Rabbit Polyclonal Antibody, 1:1000</td></tr><tr><td>Anti-Choline Acetyltransferase (CHAT) Antibody</td><td>EMD-Millipore</td><td>AB144P</td><td>Primary Goat Polyclonal Antibody, 1:100</td></tr><tr><td>Anti GFP Antibody</td><td>Aves Labs</td><td>GFP-1020</td><td>Primary Chicken Polyclonal Antibody, 1:1000</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td><td>ThermoFisher Scientific</td><td>A21207</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>&amp;nbsp;Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 680</td><td>ThermoFisher Scientific</td><td>A10043</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>Donkey anti-Chicken IgY Secondary Antibody, Alexa Fluor 488</td><td>Jackson Immunoresearch Labs</td><td>703-545-155</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>Donkey Anti-Goat IgG H&amp;amp;L (Alexa Fluor 647</td><td>Abcam</td><td>ab150131</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>Slide Microscope Superfrost</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Fisher Scientific</td><td>P36930</td><td></td></tr><tr><td>Epifluorescence Microscope</td><td>Zeiss</td><td>Zeiss AxioImager M2</td><td></td></tr><tr><td>Fluorescence Confocal Microscope</td><td>Olympus</td><td>Olympus FV1000</td><td></td></tr><tr><td>Dextran</td><td>Polysciences, Inc</td><td>19411</td><td></td></tr><tr><td>AAV9-UBC-GFP</td><td>UCSD Viral Vector Core Laboratory</td><td></td><td></td></tr></tbody></table>

subpial adeno-associated virus 9 (aav9) vector delivery in adult mice

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Potent Transgene Expression in Subpially AAV9-injected Segments: 
The analysis of transgene (GFP) expression in spinal cord sections at 14 days after AAV9 delivery showed AAV9-dose dependent GFP expression throughout the spinal parenchyma. First, two bilateral 3 µL injections of AAV9-UBI-GFP injected into the upper lumbar subpial space were associated with the near-complete infection of the white and gray matter in the whole lumbar spinal cord, extending to the upper t...

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

The overall goal of this subpial AAV9 delivery technique is to achieve a potent spinal parenchymal transgene expression throughout the entire length of spinal chord in adult mice. This method can facilitate studies targeted to manipulate spinal gene expression and help to understand the mechanism of several spinal neurodegenerative disorders including amyotrophic lateral sclerosis, spinal traumatic injury, or chronic pain. The main advantage of this technique is that highly potent transgene expression is achieved through the spinal gray and white matter in multiple spinal segments, which are adjacent to subpial AAV9 injected segment.Visual demonstration of this method is critical because the dura-opening step and placement of subpial needle are difficult to learn without visual demonstration. Demonstrating the procedure will be Takahiro Tadokoro, a post-doctorate fellow in our laboratory. Begin by properly anesthetizing the animal for surgery, isoflurane is used here.Ensure the absence of a paw pinch response before proceeding, then cover the eyes with ophthalmic ointment. Next, shave the back of the animal with clippers and clean the skin with 2%chlorhexidine. If lumbar subpial injections are to be performed, cut the skin overlying the T8 to L1 vertebrae with a scalpel and then use scissors to detach the paravertebral muscle from the T10 to T12 spinal vertebrae.Mount the animal into a standard stereotaxic frame, using mouse spinal clamps. Next, use a dental drill to shave both sides of the lamina of the T10 to T12 vertebrae until cracks appear then use forceps to remove cracked bone fragments and expose the dorsal surface of the lumbar spinal chord. The dura is now visible, use a 30 gauge stainless steel needle to make a one to two millimeter incision into the dura.It is important to carefully cut open the dura to avoid injury to the underlying pia membrane. Now, grasp the edge of the cut dura with forceps and extend the dura opening for up to five millimeters. If cervical subpial injections are to be performed, immobilize the head of the mouse by placing the head into a stereotaxic frame using ear bars and use scissors to make a 1.5 to two centimeter incision in the dorsal neck skin to expose the C1 and C2 segments and occipital bone.Then, shave a portion of exposed occipital bone using a dental drill and remove the bone fragments using forceps. Then, use a 23 gauge stainless steel needle and forceps to remove the atlanto occipital membrane of the cisterna magna. Use cotton swabs to clean the incision site of any tissue and bone debris, then cut open the dura, making a two to three millimeter incision.Mount the 34 gauge pia-penetrating needle and 36 gauge injection needle on to the Z arms of two separate X, Y, Z manipulators using glass capillary holders. Next, use a 50 microliter microsyringe connected with PE10 or PE20 tubing to load AAV9-UBI-GFP virus. After the virus is loaded, connect the end of the tubing to the injection needle.Next, while looking through a surgical dissecting scope set to eight to ten X magnification, use the X arm to lower the pia-penetrating needle into the pia to a depth of one millimeter. Keep the angle of the penetrating needle at five to 10 degrees relative to the tissue surface. After opening the pia, use the X arm to remove the pia penetrating needle from the subpial space.Note the penetrated site using a landmark such as a blood vessel. Move the X, Y, and Z arms of the second manipulator to position the tip of the AAV9 virus loaded injection needle into the pia penetrated site. Then, after inserting the tip of the injection needle about 0.5 millimeters in to the subpial space, use the Z arm to lift the pia about 0.3 millimeters, then use the X arm to continue advancing the needle horizontally into the subpial space.Advance the needle until it enters the subpial space at a depth of about two to three millimeters, then use the 50 microliter microsyringe to inject the AAV9-UBI-GFP virus into the subpial space. Remove the injection needle from the subpial space after the AAV9-UBI-GFP injection is complete. Close the muscle and skin using foromonofilament suture and surgical clips.Allow the animal to recover on a heating pad before housing in a clean cage. Two bilateral injections of AAV9-UBI-GFP were delivered into the upper lumbar subpial space and the animals were profusion fixed 14 days after AAV9 delivery. Intense GFP expression is seen in the gray matter located within the dotted area and white matter outside the dotted area.This extends from the lumbar to the upper thoracic segments in animals injected with three plus three microliters of AAV9. Animals injected with a lower volume of AAV9 have less intense expression of GFP. The following images depict potent retrograde and anterograde AAV9-UBI-GFP mediated GFP expression in brain motor and sensory centers.This low-power image clearly shows the presence of intense GFP positivity in the cervical spinal cord, medulla oblongata, cerebellum, and motor cortex. This high-power image taken from a sagittal brain section shows the presence of GFP florescence in neurons in the reticular formation, nucleus ruber, and axons of the spinocerebellar tract. This lower-power image taken from a coronal brain section demonstrates the presence of GFP florescence in pyramidal neurons in the motor cortex and in the terminals of the spinothalamic tract in areas of the reticular thalamic nuclei.Finally, this high-power image demonstrates an intense GFP expression in pyramidal neurons in the motor cortex. Once mastered, this technique can be completed in 30 to 40 minute. The use of this technique by researchers in the field of neuroscience will be an effective tool to study the role of specific genes in the evolution of spinal degenerative disorders.It will also help to explore potential treatment efficacy by suppressing mutated genes known to be linked with progressive spinal neuronal degeneration as in the familiar form of amyotrophic lateral sclerosis.

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In this video, we demonstrate recombinant adeno associated virus-based gene delivery in the mouse pup. This method helps in studying gene expression in the targeted cells.

Recombinant Adeno Associated Virus Based Gene Delivery: A Technique for Transgene Delivery and Cardiac-Specific Expression in Rodent Model

Source: Tadokoro, T., et al. Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice. J. Vis. Exp. (2017)This video demonstrates a subpial injection technique for gene delivery to the spinal cord in mice. The procedure involves exposing the dura mater, puncturing the pia mater, and injecting a recombinant adeno-associated virus (rAAV) carrying a fluorescent protein-expressing gene into the subpial space. Following injection and recovery, the virus facilitates gene expression in...

Subpial Gene Delivery in the Mouse Spinal Cord to Evaluate Gene Expression

Source: Li, D., et al. Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice. J. Vis. Exp. (2019)This video demonstrates the procedure for the intrathecal injection of adeno-associated viruses (AAV) into the cerebrospinal fluid (CSF) of an awake mouse, covering syringe preparation, spinal alignment, and precise needle insertion. This method validates targeted gene delivery, supporting its potential for CNS therapies.

Intrathecal Delivery of Viruses in a Mouse Model for Treating Central Nervous System Diseases

Source:Pourshafie, N., et al. Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents. J. Vis. Exp. (2018).In this video, a spinal and bulbar muscular atrophy or SBMA-affected mouse undergoes tail vein injection of recombinant AAV (Adeno-associated vectors) vectors carrying a plasmid encoding therapeutic microRNA. The vector produces microRNA that binds to mutant AR (Androgen receptor)-mRNA (messenger ribonucleic acid),...

Injecting Recombinant Adeno-Associated Vectors in a Spinal and Bulbar Muscular Atrophy Mouse Model

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer (≥1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

This report details the localized injection method of AAV vectors via the renal pelvis for effective and safe gene transfer into the mouse kidney.

Effective and Safe Gene Delivery to the Mouse Kidney via Slow Retrograde Renal Pelvis Injection of Adeno-Associated Virus Vectors

Learn subpial AAV9 delivery in adult mice to achieve potent spinal transgene expression across gray and white matter. This technique advances research on spinal neurodegenerative disorders like ALS.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

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