The authors would like to thank Steven Gray, Matthew Rioux, Nanda Regmi, and Lacey Stearman of UT Southwestern for a productive discussion of the challenge posed by juvenile rats for intrathecal injection. This work was partly supported by funding from Jaguar Gene Therapy (to JLFK).

This study was approved by the Emory University Institutional Animal Care and Use Committee (IACUC). Sprague-Dawley rats (28-30 days of age, mass in the range of about 90-135 g, males and females) were used in the present study.
1. Preparation of the vector
<ol>
	<li>Thaw the AAV9 vector (see Table of Materials) on ice at the beginning of the procedure.</li>
	<li>Centrifuge the microcentrifuge tube containing the vector briefly in a table-top centrifuge to ensure that all of the liquid is in the bottom of the tube.</li>
	<li>Gently flick the microcentrifuge tube to ensure that the solution is well-mixed.</li>
</ol>
2. Preparation of the recovery cage
<ol>
	<li>Place a clean cage onto an electric blanket (see Table of Materials) so that only half of the cage is in contact with the blanket.</li>
	<li>Set the temperature of the blanket to ~37 &#176;C.</li>
</ol>
3. Preparation of the surgical platform
<ol>
	<li>Warm an isothermal pad (see Table of Materials) to 39 &#176;C in a microwave or a water bath so that the contents become liquid.</li>
	<li>Place the isothermal pad on the surgical platform and cover it with a clean absorbent bench pad.</li>
</ol>
4. Animal preparation
<ol>
	<li>Anesthetize the rats with isoflurane in a clear box (following institutionally approved protocols). Begin the anesthesia induction using 5% isoflurane, and step down 1% per minute until reaching 2%. Hold the animal at 2% for an additional 3 min.</li>
	<li>Move the box holding the animal to a fume hood and open the box. 
	NOTE: This limits the surgeon&#39;s exposure to the anesthetic.</li>
	<li>Remove the hair from the back of the animal using electric hair clippers. 
	NOTE: Alternatively, a depilatory cream or manual razor and shaving cream can be used.</li>
	<li>Place the animal on the surgical platform with its snout in the anesthesia nose cone. 
	NOTE: The animal may begin to regain consciousness while the fur is removed from the surgical site. If this happens, anesthetize it again as described above.</li>
	<li>Apply lubricating eye ointment to each eye to prevent drying of the corneas during the procedure.</li>
	<li>Disinfect the surgical area using three alternating applications of povidone-iodine and isopropanol wipes.</li>
	<li>Inject the analgesic(s) subcutaneously. 
	NOTE: Buprenorphine is generally used at a dose of 0.01-0.05&#160;mg/kg, given every 12 h. Alternatively, a slow-release form of this drug can be given once at 1 mg/kg to provide adequate pain control for 72 h. Consult with the institution&#39;s IACUC for their guidelines regarding pain management.</li>
	<li>Inject 100 &#181;L of 1% lidocaine subcutaneously above the L2 to L6 spinous processes to provide local anesthesia.</li>
	<li>Place a roll of paper towel or a tube of 1.5 cm in diameter under the animal, just rostral to the hips. This helps flex the spine, making it easier to insert the needle between the two laminae.</li>
	<li>Place a fenestrated drape (see Table of Materials) on the animal, centering the fenestration over the lumbar spine.</li>
</ol>
5. Exposing the lumbar spine
<ol>
	<li>Confirm the depth of anesthesia by pinching each of the animal&#39;s paws and looking for the absence of a withdrawal response.</li>
	<li>Using a #11 scalpel blade, create an incision of approximately 3 cm long in the skin down the midline from L2 to L6.</li>
	<li>Loosen the skin from the muscle by inserting a sterile curved pair of surgical scissors between the muscle and the skin and then opening the tips.</li>
	<li>Remove the fascia covering the L2-L5 spinous processes.</li>
</ol>
6. Loading of the syringe
<ol>
	<li>Pipette 25-35 &#181;L of the vector (to achieve the desired dose) into the cap of a sterile microcentrifuge tube.</li>
	<li>Draw the entire volume into the insulin syringe. 
	NOTE: Take care not to draw up air during this process.</li>
</ol>
7. Performing the injection
<ol>
	<li>Identify the L5 and L4 spinous processes. 
	NOTE: L6 sits directly between the two iliac crests, and its spinous process should be easy to identify by probing with a blunt instrument. The instrument can then be gently run up the back to find the boundaries of the L5 and L4 processes.</li>
	<li>Place one hand so that the thumb rests gently on the animal&#39;s tail and one leg. Use a finger to steady the syringe.</li>
	<li>Position the needle of the syringe so that it is to the left of the L5 spinous process and lined up with its caudal end. Position the syringe so that it is about 30&#176; off midline and 30&#176; up from the plane of the table. 
	NOTE: It may be helpful to use a surgical microscope to better identify landmarks and position the tip of the needle.</li>
	<li>Advance the syringe needle forward about 8 mm, over the top of the L5 lamina and then under the L4 lamina into the lumbar cistern until the bone is hit. Correct placement will result in a twitch of the leg and/or tail that can be seen or felt by the thumb resting on the leg/tail. If there is no twitch, remove the needle and attempt the procedure from the left side. If there is still no twitch, repeat the procedure between L4/L3 and L3/L2 as necessary.</li>
	<li>Depress the plunger slowly for about 5 s. 
	NOTE: There may be a twitch in the leg or tail during the injection.</li>
	<li>Hold the syringe in place for about 30 s after fully depressing the plunger to allow the pressure to equilibrate and minimize reflux of the injectate when the needle is withdrawn.</li>
	<li>Slowly remove the needle.</li>
</ol>
8. Closing of the incision
<ol>
	<li>Approximate the edges of the incision.</li>
	<li>Beginning at one end of the wound, use a 4-0 suture (see Table of Materials) or surgical staples to close the incision.</li>
</ol>
9. Recovery and monitoring
<ol>
	<li>Place the animal in the prewarmed cage.</li>
	<li>Check the animal at least every 15 min until it is fully ambulatory. 
	NOTE: This should take between 15 min and 45 min.</li>
	<li>For the next three days, perform wellness checks at least daily. Provide analgesics for the first 2 days following surgery or as required by the IACUC.</li>
	<li>One week after surgery, remove the sutures or staples.</li>
</ol>
10. Follow-up procedure
NOTE: To determine the accuracy of the injection technique, inject trypan blue dye as described above and then immediately euthanize the animal (following institutionally approved protocols) and perform a laminectomy to visualize the result.
<ol>
	<li>While the animal remains under anesthesia, euthanize it by administering a lethal dose of pentobarbital via intraperitoneal injection at a dose of 150 mg/kg.</li>
	<li>Once respiration and cardiac activity cease, open the chest cavity to ensure death.&#160;Extend the surgical incision up the back to the neck.</li>
	<li>Make an incision 4 cm long into the muscle parallel to the spine on both sides of the spinous processes, keeping as close as possible to the processes.</li>
	<li>Using fine forceps or scissors, remove the muscle from in between the spinous processes.</li>
	<li>Remove the spinous processes from L6 up to the lower-thoracic spine using a rongeur (see Table of Materials). Avoid twisting motions, as this may damage the rongeurs.</li>
	<li>Insert the lower tip of the rongeur under the L5 lamina and remove the bone overlying the spinal cord by taking several &#34;bites&#34; out of it. 
	NOTE: Pulling back on the L6 spinous process can make it easier to insert the tip of the rongeur. Care must be taken to prevent damage to the spinal cord.</li>
	<li>Continue expanding the laminectomy at least four laminae rostrally. Inspect the interior surface of the laminae for signs of dye, which can indicate a failed injection.</li>
</ol>

Lumbar Puncture Delivery of AAV9 in Juvenile Rats for CNS Gene Therapy

<ol>
	<li>Wurster, C., Petri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+spinal+muscular+atrophy+research.">Progress in spinal muscular atrophy research.</a> Curr Opin Neurol. 35 (5), 693-698 (2022).</li><li>Wu, K. Y., 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=Retinitis+pigmentosa:+Novel+therapeutic+targets+and+drug+development.">Retinitis pigmentosa: Novel therapeutic targets and drug development.</a> Pharmaceutics. 15 (2), 685 (2023).</li><li>Larkin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+FDA-approved+gene+therapy+for+hemophilia.">First FDA-approved gene therapy for hemophilia.</a> JAMA. 329 (1), 14 (2023).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nadofaragene+firadenovec:+First+approval.">Nadofaragene firadenovec: First approval.</a> Drugs. 83 (4), 353-357 (2023).</li><li>Taghian, T., 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+safe+and+reliable+technique+for+CNS+delivery+of+AAV+vectors+in+the+cisterna+magna.">A safe and reliable technique for CNS delivery of AAV vectors in the cisterna magna.</a> Mol Ther. 28 (2), 411-421 (2020).</li><li>Donsante, A., 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=Intracerebroventricular+delivery+of+self-complementary+adeno-associated+virus+serotype+9+to+the+adult+rat+brain.">Intracerebroventricular delivery of self-complementary adeno-associated virus serotype 9 to the adult rat brain.</a> Gene Ther. 23 (5), 401-407 (2016).</li><li>Pellot, J. E., Jesus, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboccipital+puncture.+[Updated+2022+Jul+25].">Suboccipital puncture. [Updated 2022 Jul 25].</a> StatPearls [Internet]. , (2022).</li><li>Elliger, S. S., Elliger, C. A., Aguilar, C. P., Raju, N. R., Watson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+lysosomal+storage+in+brains+of+MPS+vii+mice+treated+by+intrathecal+administration+of+an+adeno-associated+virus+vector.">Elimination of lysosomal storage in brains of MPS vii mice treated by intrathecal administration of an adeno-associated virus vector.</a> Gene Ther. 6 (6), 1175-1178 (1999).</li><li>De La Calle, J. L., Paino, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+direct+lumbar+puncture+in+rats.">A procedure for direct lumbar puncture in rats.</a> Brain Res Bull. 59 (3), 245-250 (2002).</li><li>O'connor, D. M., Lutomski, C., Jarrold, M. F., Boulis, N. M., Donsante, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lot-to-lot+variation+in+adeno-associated+virus+serotype+9+(AAV9)+preparations.">Lot-to-lot variation in adeno-associated virus serotype 9 (AAV9) preparations.</a> Hum Gene Ther Methods. 30 (6), 214-225 (2019).</li><li>Manfredsson, F. P., Rising, A. C., Mandel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV9:+A+potential+blood-brain+barrier+buster.">AAV9: A potential blood-brain barrier buster.</a> Mol Ther. 17 (3), 403-405 (2009).</li><li>Hudry, E., Vandenberghe, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+AAV+gene+transfer+to+the+nervous+system:+A+clinical+reality.">Therapeutic AAV gene transfer to the nervous system: A clinical reality.</a> Neuron. 101 (5), 839-862 (2019).</li><li>Georg-Fries, B., Biederlack, S., Wolf, J., Zur Hausen, H. <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+proteins,+helper+dependence,+and+seroepidemiology+of+a+new+human+parvovirus.">Analysis of proteins, helper dependence, and seroepidemiology of a new human parvovirus.</a> Virology. 134 (1), 64-71 (1984).</li><li>Schulz, M., 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=Binding+and+neutralizing+anti-aav+antibodies:+Detection+and+implications+for+rAAV-mediated+gene+therapy.">Binding and neutralizing anti-aav antibodies: Detection and implications for rAAV-mediated gene therapy.</a> Mol Ther. 31 (3), 616-630 (2023).</li><li>Gray, S. J., Nagabhushan Kalburgi, S., Mccown, T. J., Samulski, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+CNS+gene+delivery+and+evasion+of+anti-aav-neutralizing+antibodies+by+intrathecal+aav+administration+in+non-human+primates.">Global CNS gene delivery and evasion of anti-aav-neutralizing antibodies by intrathecal aav administration in non-human primates.</a> Gene Ther. 20 (4), 450-459 (2013).</li><li>Meyer, K., 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=Improving+single+injection+CSF+delivery+of+AAV9-mediated+gene+therapy+for+sma:+A+dose-response+study+in+mice+and+non-human+primates.">Improving single injection CSF delivery of AAV9-mediated gene therapy for sma: A dose-response study in mice and non-human primates.</a> Mol Ther. 23 (3), 477-487 (2015).</li><li>Hinderer, C., 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=Evaluation+of+intrathecal+routes+of+administration+for+adeno-associated+viral+vectors+in+large+animals.">Evaluation of intrathecal routes of administration for adeno-associated viral vectors in large animals.</a> Hum Gene Ther. 29 (1), 15-24 (2018).</li><li>Wang, Y. F., 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=Cerebrospinal+fluid+leakage+and+headache+after+lumbar+puncture:+A+prospective+non-invasive+imaging+study.">Cerebrospinal fluid leakage and headache after lumbar puncture: A prospective non-invasive imaging study.</a> Brain. 138, 1492-1498 (2015).</li><li>Hordeaux, 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=Adeno-associated+virus-induced+dorsal+root+ganglion+pathology).">Adeno-associated virus-induced dorsal root ganglion pathology).</a> Hum Gene Ther. 31 (15-16), 808-818 (2020).</li><li>Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., Noble-Haeusslein, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Prog Neurobiol. 106-107, 1-16 (2013).</li><li>Fang, H., 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=Comparison+of+adeno-associated+virus+serotypes+and+delivery+methods+for+cardiac+gene+transfer.">Comparison of adeno-associated virus serotypes and delivery methods for cardiac gene transfer.</a> Hum Gene Ther Methods. 23 (4), 234-241 (2012).</li></ol>

Dr. Donsante is an inventor on a pending patent regarding CSF administration of AAV9 vectors.

A wide variety of diseases affect the CNS. Providing a functional copy of the relevant gene via a viral vector is an attractive treatment strategy for those that are recessive and monogenic in nature, such as spinal muscular atrophy. However, the blood-brain barrier (BBB) excludes most gene therapy vectors given intravenously11. Those that can cross the BBB, such as AAV9, must be given in high doses to overcome the vector loss due to peripheral transduction12. The age is al...

The promise of viral-mediated gene therapies has finally been realized in recent years with the FDA approval of treatments for spinal muscular atrophy, retinal dystrophy, factor IX hemophilia, cancer, and more1,2,3,4. Countless other therapeutics are currently in development. Gene therapy aims to deliver a therapeutic gene to a patient&#39;s cells. The products of this new gene can replace the missing activity from a deficient endogenous gene, inhibit a toxic gene, kill cancerous cells, or provide some other beneficial function.
For diseases affecting the central nervous system (CNS), delivering the gene therapy vector directly to the target tissue is often desirable. Non-systemic approaches provide two benefits: they minimize off-target side effects that may be caused by peripheral transduction, and they greatly reduce the amount of vector needed to achieve adequate levels of transduction in the target tissue5.
There are a variety of approaches to delivering gene therapy vectors to the CNS. Intraparenchymal injection, the injection of a vector directly into the spinal cord or brain tissue, can be used for delivery to a defined region. However, for many diseases, broad transduction of the CNS is desired. This can be accomplished by delivering a vector to the cerebrospinal fluid (CSF)5, the fluid that flows in and around the brain and spinal cord. There are three primary ways to deliver vectors to the CSF. The most invasive approach is intracerebroventricular delivery, which involves drilling a burr hole through the skull and advancing a needle through the brain into the lateral ventricles. This yields transduction throughout the brain. However, the procedure may cause intracranial hemorrhage, and the approach generally produces only limited transduction of the spinal cord6. Injection into the cisterna magna at the base of the skull is less invasive, but carries the risk of damage to the brainstem. While often used in animal research5, injection into the cisterna magna is no longer used routinely in the clinic7. Lumbar puncture is the least invasive approach to access the CSF. This involves placing a needle between two lumbar vertebrae and into the lumbar cistern.
Lumbar puncture for vector delivery is routinely performed in adult rats and mice and in neonatal mice8,9. The authors of this study recently performed lumbar punctures in juvenile rats (28-30 days of age) to deliver adeno-associated virus serotype 9 (AAV9) vectors. In adult rats, a neonatal lumbar puncture needle was placed vertically between the L3 and L4 vertebrae9. Proper placement results in a tail flick and CSF flowing up into the needle reservoir. In juvenile rats, though, neither of these read-outs could be achieved. The authors then attempted to adapt an adult mouse procedure using a 27 G insulin syringe inserted at an angle between L5 and L610. In adult mice, which are typically smaller than P28 rats, this does not produce a tail flick, but incorrect needle placement is evident by the backflow of the injectate. In juvenile rats, however, this approach uniformly led to the injectate being delivered epidurally, likely resulting from different elasticity between adult mice and juvenile rats of the tissue layers surrounding the spinal cord. Catheter approaches were evaluated next. Specifically, a catheter was introduced through an incision in the dura of the lumbar cistern and up to the mid-thoracic spinal cord; however, this approach led to substantial reflux of the injectate back out of the incision site during delivery. Attempts to place the catheter into the intrathecal space percutaneously using a guide needle were also unsuccessful. Due to the narrowness of the interlaminar width, the catheter would likely hit the rostral lamina and fail to advance.
Here, a method is described to achieve successful and reproducible solution delivery via a lumbar puncture in the juvenile rat. This approach can be used for viral vectors, and likely also for cells, pharmaceuticals, and other therapeutics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#181;L filtered pipette tips</td>
			<td>MidSci</td>
			<td>PR-200RK-FL</td>
			<td>Pipetting virus</td>
		</tr>
		<tr>
			<td>AAV9-GFP</td>
			<td>Vector Builder</td>
			<td>P200624-1005ynr</td>
			<td>AAV9 vector expressing GFP</td>
		</tr>
		<tr>
			<td>Absorbable Suture with Needle Coated Vicryl Polyglactin 910 FS-2 3/8 Circle Reverse Cutting Needle Size 4 - 0 Braided</td>
			<td>McKesson</td>
			<td>J422H</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>Bench pad</td>
			<td>VWR</td>
			<td>56616-031</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Braintree Scientific Isothermal Pads, 8&#39;&#39; x 8&#39;&#39;</td>
			<td>Fisher Scientific</td>
			<td>50-195-4664</td>
			<td>Maintains body temperature</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>McKesson</td>
			<td>1013922</td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Buprenorphine-ER (1 mg/mL)</td>
			<td>Zoopharma</td>
			<td></td>
			<td>Extended-release analgesic</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>19-365-409</td>
			<td>Blood removal</td>
		</tr>
		<tr>
			<td>Drape, Mouse, Clear Plastic, 12&#34; x 12&#34;, with Adhesive Fenestration</td>
			<td>Steris</td>
			<td>1212CPSTF</td>
			<td>Surgical drape</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Forceps</td>
		</tr>
		<tr>
			<td>Electric Blanket</td>
			<td>CVS Health</td>
			<td></td>
			<td>CVS Health Series 500 Extra Long Heating Pad</td>
		</tr>
		<tr>
			<td>Eppendorf Research plus, 1-channel pipette, variable, 20&#8211;200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td>Pipetting virus</td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14059-11</td>
			<td>Curved surgical scissors</td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16121-14</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>Halsey Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td>Needle driver</td>
		</tr>
		<tr>
			<td>Insulin Syringes with Ultra-Fine Needle 12.7 mm x 30 G 3/10 mL/cc</td>
			<td>BD</td>
			<td>328431</td>
			<td>Syringe</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>McKesson</td>
			<td>803250</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Isopropanol wipes</td>
			<td>Fisher Scientific</td>
			<td>22-031-350</td>
			<td>Skin disinfection</td>
		</tr>
		<tr>
			<td>Lidocaine, 1%</td>
			<td>McKesson</td>
			<td>239935</td>
			<td>Local anesthesia</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-137</td>
			<td>Loading the syringe</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Fisher Scientific</td>
			<td>50-118-0481</td>
			<td>Skin disinfection</td>
		</tr>
		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td>Scalpel blade holder</td>
		</tr>
		<tr>
			<td>Sure-Seal Induction Chamber</td>
			<td>Braintree Scientific</td>
			<td>EZ-17</td>
			<td>Anesthesia box</td>
		</tr>
		<tr>
			<td>Surgical Blade Miltex Carbon Steel No. 11 Sterile Disposable Individually Wrapped</td>
			<td>McKesson</td>
			<td>4-111</td>
			<td>#11 Scalpel blade</td>
		</tr>
		<tr>
			<td>SYSTANE NIGHTTIME Eye Ointment</td>
			<td>Alcon</td>
			<td></td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>VWR</td>
			<td>97063-702</td>
			<td>Injection</td>
		</tr>
	</tbody>
</table>

intrathecal vector delivery in juvenile rats via lumbar cistern injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

To determine the accuracy of the injection technique, a dye, trypan blue, was used as a surrogate for the therapeutic. This dye readily binds to proteins, so it generally stays within the structure into which it was injected. This means the dye may not accurately predict the post-injection distribution of the therapeutic; it is simply used to reveal the accuracy of the injection. When successfully introduced into the lumbar cistern, trypan blue binds to the dura mater, staining the perimeter of the spinal cord blue. Howe...

Watch this Scientific Journal Video about Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats at JoVE.com

Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

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

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

intrathecal-vector-delivery-juvenile-rats-via-lumbar-cistern

Research

JoVE Journal

Neuroscience

866 Views.  Emory University. 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. 

Video: Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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 ...

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 ...

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.

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

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. ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

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

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

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.

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...