We would like to thank Ionis Pharmaceuticals for supplying the ASOs described in the article.

All in vivo procedures were performed under Biogen Institutional Animal Use and Care Committee (IACUC) approved protocols which follow the guidelines set forth by the United States National Institutes of Health guide for the care and use of laboratory animals.
1. Material and instrument preparation
<ol>
	<li>Prepare the special guide cannulas.
	<ol>
		<li>Use a rotary tool with cut-off wheel (or a sharp saw) to cut off the two ends of a 19 G needle, resulting in a ~1.5&#8722;2 cm long guide ...

<ol>
	<li>Abbott, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+CNS+barriers:+evolution,+differentiation,+and+modulation.">Dynamics of CNS barriers: evolution, differentiation, and modulation.</a> Cellular and Molecular Neurobiology. 25 (1), 5-23 (2005).</li><li>Greene, C., Campbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junction+modulation+of+the+blood+brain+barrier:+CNS+delivery+of+small+molecules.">Tight junction modulation of the blood brain barrier: CNS delivery of small molecules.</a> Tissue Barriers. 4 (1), e1138017 (2016).</li><li>Daneman, R., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+barriers+in+health+and+disease.">Brain barriers in health and disease.</a> Neurobiology of Disease. 107, 1-3 (2017).</li><li>Ballabh, P., Braun, A., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+an+overview:+structure,+regulation,+and+clinical+implications.">The blood-brain barrier: an overview: structure, regulation, and clinical implications.</a> Neurobiology of Disease. 16 (1), 1-13 (2004).</li><li>Cardoso, F. L., Brites, D., Brito, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+the+blood-brain+barrier:+molecular+anatomy+and+possible+investigation+approaches.">Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches.</a> Brain Research Reviews. 64 (2), 328-363 (2010).</li><li>Larsen, J. M., Martin, D. R., Byrne, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+delta+opioid+analgesia+and+binding+by+antisense+oligodeoxynucleotides+to+a+delta+opioid+receptor.">Selective loss of delta opioid analgesia and binding by antisense oligodeoxynucleotides to a delta opioid receptor.</a> Neuron. 12 (4), 805-810 (1994).</li><li>Wahlestedt, 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=Antisense+oligodeoxynucleotides+to+NMDA-R1+receptor+channel+protect+cortical+neurons+from+excitotoxicity+and+reduce+focal+ischaemic+infarctions.">Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions.</a> Nature. 363 (6426), 260-263 (1993).</li><li>Wahlestedt, C., Pich, E. M., Koob, G. F., Yee, F., Heilig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+anxiety+and+neuropeptide+Y-Y1+receptors+by+antisense+oligodeoxynucleotides.">Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides.</a> Science. 259 (5094), 528-531 (1993).</li><li>Mazur, 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=Development+of+a+simple,+rapid,+and+robust+intrathecal+catheterization+method+in+the+rat.">Development of a simple, rapid, and robust intrathecal catheterization method in the rat.</a> Journal of Neuroscience Methods. 280, 36-46 (2017).</li><li>Wolf, D. 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=Dynamic+dual-isotope+molecular+imaging+elucidates+principles+for+optimizing+intrathecal+drug+delivery.">Dynamic dual-isotope molecular imaging elucidates principles for optimizing intrathecal drug delivery.</a> Journal of Clinical Investigation Insight. 1 (2), e85311 (2016).</li><li>Becker, L. 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=Therapeutic+reduction+of+ataxin-2+extends+lifespan+and+reduces+pathology+in+TDP-43+mice.">Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.</a> Nature. 544 (7650), 367-371 (2017).</li><li>Zhang, X., Hamblin, M. H., Yin, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long+noncoding+RNA+Malat1:+Its+physiological+and+pathophysiological+functions.">The long noncoding RNA Malat1: Its physiological and pathophysiological functions.</a> RNA Biology. 14 (12), 1705-1714 (2017).</li><li>Crooke, S. T., Witztum, J. L., Bennett, C. F., Baker, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Targeted+Therapeutics.">RNA-Targeted Therapeutics.</a> Cell Metabolism. 27 (4), 714-739 (2018).</li><li>DeVos, S. L., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+intraventricular+delivery+of+drugs+to+the+rodent+central+nervous+system.">Direct intraventricular delivery of drugs to the rodent central nervous system.</a> Journal of Visualized Experiments. (75), e50326 (2013).</li><li>McCampbell, 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=Antisense+oligonucleotides+extend+survival+and+reverse+decrement+in+muscle+response+in+ALS+models.">Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.</a> Journal of Clinical Investigation. 128 (8), 3558-3567 (2018).</li><li>Schoch, K. M., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+Oligonucleotides:+Translation+from+Mouse+Models+to+Human+Neurodegenerative+Diseases.">Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases.</a> Neuron. 94 (6), 1056-1070 (2017).</li><li>Lane, R. 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=Translating+Antisense+Technology+into+a+Treatment+for+Huntington's+Disease.">Translating Antisense Technology into a Treatment for Huntington's Disease.</a> Methods in Molecular Biology. 1780, 497-523 (2018).</li><li>Wurster, C. D., Ludolph, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+oligonucleotides+in+neurological+disorders.">Antisense oligonucleotides in neurological disorders.</a> Therapeutic Advances in Neurological Disorders. 11, (2018).</li><li>Hach&#233;, 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=Intrathecal+Injections+in+Children+With+Spinal+Muscular+Atrophy:+Nusinersen+Clinical+Trial+Experience.">Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience.</a> Journal of Child Neurology. 31 (7), 899-906 (2016).</li><li>Goodkey, K., Aslesh, T., Maruyama, R., Yokota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nusinersen+in+the+Treatment+of+Spinal+Muscular+Atrophy.">Nusinersen in the Treatment of Spinal Muscular Atrophy.</a> Methods in Molecular Biology. 1828, 69-76 (2018).</li><li>Wurster, C. D., Ludolph, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nusinersen+for+spinal+muscular+atrophy.">Nusinersen for spinal muscular atrophy.</a> Therapeutic Advances in Neurological Disorders. 11, (2018).</li></ol>

The authors are all employees of Biogen, Inc. or Ionis Pharmaceuticals. The authors receive the antisense oligonucleotides described in the article from Ionis Pharmaceuticals.

The present article shows a powerful method to deliver therapeutic agents directly into the rat CNS. In theory, a similar technique can be also performed in mice, though due to the smaller size, the method can be more challenging. Therefore, our group performs intracerebroventricular (ICV) injections in mice for CNS drug delivery, which reach the same goals through a different route of administration. That method has been described in another study16.
The advantage of t...

The vascular system of the central nervous system (CNS) has evolved as a critical regulator of homeostasis, controlling traffic of molecules, supplying nutrients and getting rid of waste. This system is also the first line of defense from attacks of external pathogens, thanks to a dense distribution of tight junctions along the walls of the endothelial cells. These tight junctions make up one aspect of the blood brain barrier (BBB). While the BBB allows the transport of molecules required to fulfill nutrient and energy demands (e.g., ions, glucose), it also selectively limits the passage of pathogens as well as toxic chemicals1,2,3.
Ironically, the same protective function of the BBB that limits passage of pathogens and toxic chemicals also is the major obstacle to our ability to easily access the CNS with therapeutic treatments after systemic administration to the organism2,4,5. This role of the BBB has prompted the development of a plethora of new drug distribution technologies and approaches6.
One way to overcome this obstacle is to inject the drugs directly into the cerebrospinal fluid (CSF) that continuously perfuses both the brain and spinal cord7,8,9,10. In this article, we describe a method to successfully deliver agents into the lumbar intrathecal space by placing the internal end of the catheter completely in the cauda equina space of the rat spine. A description of this procedure was previously published by Mazur et al. elsewhere11.
The protocol is very effective and produces a greater than 90% success rate of antisense oligonucleotide (ASO) delivery to the CNS when assessed by quantitative polymerase chain reaction (qPCR) analysis of target gene knockdown8. The procedure causes minimal discomfort to the animals, as 100% of the rats survive the surgery and show minimal swelling around the surgical wound and no signs of distress (e.g., hyperactivity, dehydration, circling, loss of balance, decreased food intake, and dehydration) during post-op observation. Another advantage of the method described here is that it does not require expensive equipment, nor any special tools.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3M Steri-Drape Small Drape with Adhesive Aperture</td>
			<td>3M</td>
			<td></td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Decon Laboratories, Inc</td>
			<td></td>
			<td>8416-160Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swab sticks</td>
			<td>Dynarex</td>
			<td></td>
			<td>NO 1204</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://punchout.fishersci.com/shop/products/bd-general-use-syringes-5/22124967?crossRef=BD%20302830#?keyword=BD+302830">BD General Use Syringes 1 mL Luer-Lok tip</a></td>
			<td>BD</td>
			<td>1ml TB Luer-Lok tip</td>
			<td>BD 302830</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing</td>
			<td>BD</td>
			<td>Polyethylene tubing PE50 Diameter 0.023 in</td>
			<td>BD 427400 (10ft, Fischer Scientific 22-204008) or 427401 (100ft, Fischer Scientific 14-170-12P)</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing</td>
			<td>BD</td>
			<td>Polyethylene tubing PE10 Diameter 0.011 in</td>
			<td>BD 427410 (10ft, Fischer Scientific 14-170-11B) or 4274011 (100ft, Fischer Scientific 14-170-12B)</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing Adapters</td>
			<td>BD</td>
			<td>23 gauge intramedic luer stub adaper</td>
			<td>BD 427565 or Fisher Scientific 14-826-19E</td>
			<td>120V 1.2A</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Single-use Needles 30G</td>
			<td>BD</td>
			<td></td>
			<td>BD 305128</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine Sustained Release-lab</td>
			<td>ZooPharm</td>
			<td>Prescription required</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene oxide sterilizer</td>
			<td>Andersen Sterilizer INC.</td>
			<td>AN 74i, gas sterilizer</td>
			<td>AN 74i</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>BD</td>
			<td>19G x 1 WT (1.1 mm x 25mm) needle</td>
			<td>BD 305186</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 100ul</td>
			<td>Hamilton company</td>
			<td>Hamilton syringe 100ul</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>STERILIZER MODELNO FST 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra veterranery product</td>
			<td></td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Pocket Pro Pet Trimmer</td>
			<td>Braintree Scientific</td>
			<td></td>
			<td>CLP-9931 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone scrub</td>
			<td>PDI</td>
			<td></td>
			<td>S48050</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>Sodium Chloride 0.9% Intravenous Infusion BP 50ml</td>
			<td>FE1306G</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>disposable scalpel</td>
			<td>No. 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal heating pad</td>
			<td>K&#38;H Manufacturing</td>
			<td></td>
			<td>Model # 1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylet Wire</td>
			<td>McMaster-Carr</td>
			<td>1749T14</td>
			<td>LH-36233780</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery Towel drape</td>
			<td>Dynarex</td>
			<td></td>
			<td>4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors and forceps</td>
			<td>FST and Fisher Scientific</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td>4-0 or 5-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool to make the Guide cannular</td>
			<td>Grainger</td>
			<td>Rotary tool (Dremel)</td>
			<td>14H446 (Mfr: EZ456)</td>
			<td>1.5&#8221; diameter, Pk5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>EZ lock cut off Wheel</td>
			<td>1PKX5 (Mfr: 3000-1/24)</td>
			<td>1.5&#8221;, Pk2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Grinding Wheel, Aluminum Oxide</td>
			<td>38EY44 (Mfr: EZ541GR)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>EZ lock Mandrel</td>
			<td>1PKX8 (Mfr: EZ402-01)</td>
			<td>1.5&#8221; diameter</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Diamond wheel floor Tile</td>
			<td>3DRN4 (Mfr: EZ545)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alternative source for pre-made and sterilized materials for this procedure</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dosing catheter system</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-GCA</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Catheters</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-INC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylet Wire</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-STY</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the method described here, we injected two groups of adult female rats (250&#8722;300 g; n = 10/group) with either a single bolus of phosphate-buffered saline PBS or 300 &#181;g of ASO targeting the long non-coding (linc) RNA Malat1; in our lab we routinely use the Malat1 ASO as a tool compound, because Malat1 is expressed ubiquitously and at high levels in all tissues14, including brain and spinal cord. The Malat1 ASO works via an RNaseH1-mediated mechanism<...

Watch this Scientific Journal Video about Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System at JoVE.com

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

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 ...

This protocol allows the direct delivery of therapeutics into the rat's central nervous system to evaluate the pharmacokinetics, pharmacodynamics, and efficacy of novel therapeutics in rat disease models. This procedure is safe, effective, and does not require expensive equipment or surgical tools. Demonstrating the procedure will be Yi Chen and Yi Luo, research scientists from my laboratory.To prepare the special guide cannulas, use a rotary tool with a cut off wheel to cut off both ends of a 19 gauge needle to obtain an approximately one and a half to two centimeter long guide cannula. Then use the grinding wheel of the rotary tool to smooth both ends. To prepare the catheter wire assembly, cut one eight centimeter long piece of 011 inch diameter PE10 tubing to serve as the intrathecal catheter for each animal.Use an ethanol-resistant marker pen to make a mark two centimeters from one end of each piece of tubing. For each animal, insert an 11 centimeter long stylet wire cut from polytetrafluoroethylene coated stainless steel wire into the lumen of the eight centimeter PE10 catheter. To prepare the delivery catheter assembly, cut a piece of five to 10 centimeter 023 inch diameter PE50 catheter and insert a 23 gauge tubing adapter into one end of the catheter.Then insert a 30 gauge needle with a hub cut off into an approximately 0.5 centimeter piece of PE10 tubing and connect the PE10 tubing to the other end of the catheter. To prepare a guide cannula-needle assembly, place a guide cannula over the end of a 23 gauge needle. Before beginning the surgery, place a heating pad on the surgery table and cover the pad with a sterile drape sheet.Place a 50mL conical centrifuge tube onto the sheet and confirm a lack of response to toe pinch in the anesthetized experimental rat. After weighing the animal, shave the back from the tail to the caudal thoracic spine and place the shaved rat onto the sterile sheet in the prone position. Position the 50mL tube under the abdomen to flex the spine in the lumbar region, and apply ophthalmic ointment to the eyes.Next, subcutaneously inject one milligram per kilogram of sustained release Buprenorphine and clean the exposed skin with sequential povidone and alcohol scrubs. Drape the animal with a fenestrated sterile transparent sheet. For placement of the catheter and injection of the compound, identify the two natural pits between the muscles above the shaved pelvis, and with one hand holding the pits, use the other hand to gently press and feel the spine from the caudal to rostral direction to locate the first major indentation between the vertebrae.After identifying the intervertebral space between the S1 and L6 vertebrae, move slightly rostrally to identify the intervertebral space between the L5 and L6 vertebrae. Use a scalpel to make a no more than two centimeter long incision in skin and muscle capsule at this site along the midline from the rostral to caudal direction so that the injection site will be at the center of the incision. Use dissection scissors to dissect away the connective tissue until the muscle layer can be visualized.Make a one centimeter incision in the muscle capsule immediately lateral to the dorsal spinal process of the L6 lumbar vertebra. Position the guide cannula needle assembly near the anterior aspect of the sixth lumbar vertebra and push the assembly into the intervertebral space along the anterior aspect of the sixth vertebra so that the end of the needle penetrates the spinal canal. Use blunt forceps to locate the dorsal spinal process of the L6 lumbar vertebra then push the needle along the anterior aspect of the sixth vertebra into the invertebrata space.Push the guide cannula in place along the needle, remove the needle, and insert the catheter wire assembly into the guide cannula. Angling the catheter at an approximately 45 degree angle to the spinal canal, force the end of the catheter approximately 0.3 centimeters into the canal. Remove the stylet wire approximately 2.5 centimeters from the intrathecal tip of the catheter and advance the catheter into the spinal canal until the two centimeter marking on the catheter is just visible below the muscle.When the catheter is in place, remove the guide cannula to leave the catheter and stylet wire in place. Advance the catheter into the spinal canal until the two centimeter mark is at the entrance of the canal and completely withdraw the stylet wire. Cerebral spinal fluid may be visualized entering the implanted catheter.Then connect the delivery catheter assembly to the distal end of the implanted catheter via the 30 gauge needle end. Next, load 60 microliters of sterile saline into a 100 microliter syringe and load a bolus of 30 microliters of the test compound into a second syringe. Connect the saline syringe to the tubing adapter end of the delivery catheter assembly and flush 20 microliters of sterile saline into the intrathecal space.When all of the saline has been flushed, replace the saline syringe with the bolus loaded syringe and inject 30 microliters of the test compound into the intrathecal space over a period of 30 seconds. When all of the bolus has been delivered, replace the bolus loaded syringe with another saline loaded syringe and flush the catheter with an additional 40 microliters of saline. When the second volume of saline has been delivered, detach the delivery catheter assembly from the implanted catheter and use a pair of very hot heat-sterilized dissection forceps to clamp down on the tubing to aseptically cut and heat-seal the implanted catheter.Use absorbable monofilament sutures to secure the heat-sealed catheter to the connective tissue and then use nonabsorbable sutures to close the skin. Then use gauze and saline to wash any blood from the skin and place the rat in a heated incubator with monitoring until full recompency. In this representative experiment, very good knockdown was obtained in all of the regions collected after single bolus antisense oligonucleotide delivery as demonstrated.However, some degree of regional variability was measured with the spinal cord exhibiting the highest percentage of knockdown. After a successful injection of the antisense oligonucleotide, or other therapeutics, into an appropriate model, one can evaluate the pharmacokinetics, pharmacodynamics, and efficacy of the therapeutics.

intrathecal-delivery-antisense-oligonucleotides-rat-central-nervous

Research

JoVE Journal

Neuroscience

17.8K Views.  Biogen, Inc.. This protocol allows the direct delivery of therapeutics into the rat's central nervous system to evaluate the pharmacokinetics, pharmacodynamics, and efficacy of novel therapeutics in rat disease models. This procedure is safe, effective, and does not require expensive equipment or surgical tools. Demonstrating the procedure will be Yi Chen and Yi Luo, research scientists from my laboratory.To prepare the special guide cannulas, use a rotary tool with a cut off wheel to cut off both e...