This work was funded by the ALS Association, Karyopharm Therapeutics, Inc., Meira GTx, and a charitable donation for ALS research from Thomas Lawson, for which we are grateful. We thank Elysse Orchard and Donna Burney for advice and training. JAS was supported by the Lied Foundation and National Institutes of Health grant GM103418.

All procedures followed the appropriate NIH guidelines. All of the procedures followed animal protocols that were approved by the Animal Care and Use Committee at LSU Health Sciences Center in Shreveport. Female Sprague-Dawley rats at 6 weeks of age were used for this procedure.
1. Determine the doses/volumes of vector needed
<ol>
	<li>Weigh the rats to be injected and determine the amount of viral vector needed for each animal based on the titer (concentration) of the vector preparation. Use vector doses that have been successful for efficient expression in the rat CNS previously (see Jackson et al.6). 
	NOTE: For AAV9 TDP-43 or AAV9 GFP, a typical dose is between 3 x 1013 - 1 x 1014 vector genomes/kg. The young adult rats at 6 weeks of age weigh about 150 g.</li>
	<li>Make equivalent doses per kg in all animals for systematic comparisons. Ensure the volume does not exceed standard advised injection volumes (250 - 500 &#181;L for a 250 g rat). A typical injection volume is 200 &#956;l to a young adult rat.</li>
</ol>
2. Prepare work station
<ol>
	<li>Gather all required supplies: one paraffin film square (about 5 x 5 cm) per animal, one alcohol preparation pad per animal, gauze pads (2 - 3 per animal), pipet tips, and a micropipette.</li>
	<li>Prepare the working surface by cleaning the surface with 70% ethanol. Place a heating pad down with the temperature set to low (~37 &#176;C). Place a bench pad on top of the heating pad.</li>
	<li>Prepare the gas anesthesia (isoflurane). Ensure adequate oxygen and gas anesthesia for the procedure. Clean the induction chamber and anesthesia mask with 70% ethanol, and place the anesthesia mask on the bench pad.</li>
</ol>
3. Prepare the vector
<ol>
	<li>Thaw the vector at room temperature, and label one sterile microcentrifuge tube for each animal.</li>
	<li>Pipet the volume of AAV determined in step 1.1 into the appropriate tube and bring the volume up to the desired injection volume (here, 200 &#956;l) with lactated Ringer&#39;s solution or saline.</li>
	<li>Pulse vortex the sample for 1 - 2 s and then pulse centrifuge for 5 s to bring the sample to the bottom of the tube.</li>
	<li>Pipet the total injection volume for the rat out of the tube and onto the square of paraffin film.</li>
	<li>Place a 30 gauge needle on a 1 mL syringe.</li>
	<li>Place the needle with the bevel facing down into the vector on the paraffin film. Pull back on the syringe plunger until all the virus is in the needle and syringe. Be careful to avoid drawing air bubbles into the needle, which becomes easier with practice.</li>
</ol>
4. Prepare the rat
<ol>
	<li>Turn the oxygen up to 1 L/min and set the isoflurane anesthesia to 5%. Ensure that the gas anesthesia is flowing to the induction chamber by checking all connecting hoses and stopcock orientations.</li>
	<li>Place the rat in the gas anesthesia induction chamber about 3 min, until the rat is unresponsive.</li>
	<li>Ensure adequate depth of anesthesia by pinching the toe. There should be no movement in the foot or leg after the toe pinch.</li>
	<li>Reduce the isoflurane anesthesia to 2% for maintenance of anesthesia and adjust the stopcock so that the anesthesia is flowing to the anesthesia mask.</li>
	<li>Place the rat&#39;s nose in the anesthesia mask and adjust the rat so that it is lying on its side.</li>
	<li>Identify the lateral tail vein. 
	NOTE: The lateral tail veins lie at approximately 10 and 2 o&#39;clock with the dorsal top of the tail as 12 o&#39;clock. With the rat lying on its side, the lateral tail vein should be facing up.</li>
	<li>Wipe the injection area with the alcohol preparation pad. The injection site is two-thirds down the length of the tail.</li>
</ol>
5. Inject the vector
<ol>
	<li>Firmly hold the tail slightly above the injection area with one finger directly over the lateral tail vein; this will dilate the vein.</li>
	<li>With the bevel facing up, align the needle with the visible tail vein in the same direction.</li>
	<li>Pierce the skin over the tail vein while taking great care to avoid the other hand holding the tail and pressing on the tail vein.</li>
	<li>Release pressure from the tail vein to allow for normal blood flow.</li>
	<li>Move the needle into the vein. To ensure that the needle has punctured the tail vein, pull back slightly on the plunger. If the needle is in the vein, blood will flow into the needle.
	<ol>
		<li>Reposition the needle as needed. Then, to stabilize the needle, move the hand above the injection site to below the injection site and hold the end of the syringe against the tail.</li>
	</ol>
	</li>
	<li>Slowly inject the vector into the tail (approximately 20 &#181;L/s). 
	NOTE: The vein may lose color as the vector flows through it. Signs that the solution was injected outside of the vein include blebbing, blanching of the skin, or resistance when injecting the vector. With practice, extra-vascular exposure can be minimized.</li>
	<li>After the vector has been administered, remove the needle from the rat and press a gauze pad against the injection site to help stem the bleeding for 30 - 60 s.</li>
</ol>
6. Clean-up
<ol>
	<li>Turn off the isoflurane anesthesia and oxygen. Monitor the rat until it regains consciousness, and then return it to its cage.</li>
	<li>Carefully place all needles in the sharps container. Dispose of all other disposable materials that are potentially contaminated with AAV into an appropriate biohazard container. Clean the working stations with 70% ethanol.</li>
</ol>
7. Evaluate hindlimb escape reflex
NOTE: Hindlimb deficits usually arise 2 - 6 weeks after TDP-43 gene transfer, depending on the vector dose.
<ol>
	<li>On a flat surface clear of debris, with two fingers grab the rat&#39;s tail approximately 2 cm from the body. Lift the rat gently until the forelimbs are hanging while viewing the ventral underside of the rat.
	<ol>
		<li>Perform the procedure weekly throughout the experimental time-course after gene transfer (e.g., up to 12 weeks after gene transfer). 
		NOTE: Also score forelimb deficits in this manner, but hindlimb deficits are more likely to occur in this model, depending on the vector dose used.</li>
	</ol>
	</li>
	<li>Observe the hindlimbs for 10 s. Note if one or both hindlimbs clench towards the midline during the 10 s.</li>
	<li>Repeat this test for a total of three times with 30 s between trials. If one or both hindlimbs are clenched for each of three trials, the rat is showing motor dysfunction. Record the data (manually in notes) as either unilateral or bilateral hindlimb deficit present if there is consistent clenching across all three trials.</li>
</ol>
8. Evaluate motor function on the rotarod
<ol>
	<li>To set up the rotarod, place a bench pad beneath the rotarod and set acceleration to 4 - 40 revolutions per minute (rpm) over 2 min (the rate increases by ~0.3 rpm/s). For a 12 week time-course, run the subjects at 2, 4, 8, and 12 weeks after gene transfer.</li>
	<li>Allow the rat to acclimate to the testing room for 20 min.</li>
	<li>To train the rat to use the rotarod for the first time, first start the rotarod at a set speed of 4 rpm, then place the rat on the rotarod. Allow the rat to walk for one full revolution at 4 rpm, then start the acceleration and allow the rat to walk on the rotarod until it falls off. Continue training until the rat can consistently walk for at least 40 s. A healthy, young adult rat will be readily trained in this manner.
	<ol>
		<li>In the case of a rat with a noticeable motor impairment, which may prevent the rat from achieving a baseline latency to fall of 40 s, provide the rat 3 - 5 training attempts before beginning rotarod scoring.</li>
	</ol>
	</li>
	<li>To begin rotarod testing, place the rat on the rotarod and start acceleration of the rotarod. Note the time at which the rat falls from the rotarod; this is the latency to fall. If the rat remains on the rotarod after 120 s, cap the session at this point, remove the rat from the rotarod and record a fall latency of 120 s.</li>
	<li>Repeat the testing two more times for a total of three trials. To minimize the effect of fatigue, space the trials at least 5 min apart. Average the scores of the three trials. An unimpaired adult rat typically has an average latency to fall of &#62; 40 s.</li>
	<li>Place the rat back in the cage, and clean the rotarod and surrounding areas with 70% ethanol.</li>
</ol>
9. Quantification of GFP expression in the cerebellum
<ol>
	<li>Anesthetize the rats and perfuse with phosphate buffered saline (PBS) and then 4% paraformaldehyde. Dissect the brain and spinal cord and soak the tissues in the fixative overnight and then move them to a 30% sucrose solution. After equilibration, cut 50 &#956;m coronal sections on a sliding microtome with freezing stage4,5,6.</li>
	<li>Choose 3-6 sections of the cerebellum that are evenly spaced in the vermis, the central lobe of the cerebellum. Fluorescently label the GFP to maximize the signal. Use the primary antibody for GFP at 1:500 and use the Alexa-488 conjugated secondary antibody at a dilution of 1:3004,5,6. 
	NOTE: See references 4,5,6 for more detail on the sampling, sectioning, and staining procedures.</li>
	<li>Photomicrograph each section in the defined region of interest with a low magnification lens using a microscope. Use a 2.5X objective (N.A. 0.12) and filter set 10, 450-490/515-565 excitation/emission). Keep the camera settings (exposure times, e.g., 2 s) the same across samples to be analyzed. Save as a .TIF file.</li>
	<li>Open the photomicrograph in the widely available Scion image program; two windows will open. Close the window indicated as &#34;Indexed Color&#34;.</li>
	<li>Choose &#34;Options&#34;, then &#34;Density Slice&#34;; a range of shades will be indicated in red on the Look-Up Table (LUT) window. In the window, use the cursor to fill in the specific GFP labelling only and stop when the non-specific background begins to be picked up in the image. 
	NOTE: This will highlight the fluorescent area on the photomicrograph to be quantified. The settings should be optimized to specifically capture all of the visibly fluorescent cells. With practice, this method can precisely highlight the specific fluorescence.</li>
	<li>Before measuring fluorescent area, first ensure that the measurements made are in the correct units (pixels). To do this, click &#34;Analyze&#34;, then &#34;Set Scale&#34;; the fourth line should say &#34;Units&#34;. From the drop-down list, choose pixels to measure in pixels, then click &#34;OK&#34;. Toggle the analysis options to &#34;Include Interior Holes&#34; to ensure the inclusion of the entire cell body in the analysis.</li>
	<li>To measure the fluorescent area, click &#34;Analyze&#34; and then click &#34;Measure&#34;. To see this value, click &#34;Analyze&#34; then click &#34;Show Results&#34;; a results window will appear. The fluorescent area measurement will be under the heading labeled &#34;Area&#34;.</li>
	<li>To ensure standardization across the measurements, do not change the highlighted values in the LUT window.</li>
	<li>Once all measurements have been recorded, average the values for each animal (from 3 - 6 sections per animal). Use an appropriate statistical test to compare the transduced areas between the groups.6</li>
</ol>

<ol>
	<li>Foust, K., Nurre, E., Montgomery, C., Hernandez, A., Chan, C., Kaspar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol. Ther. 17 (7), 1187-1196 (2009).</li><li>Gombash Lampe, S., Kaspar, B., Foust, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injections+in+neonatal+mice.">Intravenous injections in neonatal mice.</a> J. Vis. Exp. (93), e52037 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansive+gene+transfer+in+the+rat+CNS+rapidly+produces+amyotrophic+lateral+sclerosis+relevant+sequelae+when+TDP-43+is+overexpressed.">Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed.</a> Mol. Ther. 18 (12), 2064-2074 (2010).</li><li>Dayton, R., 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=Selective+forelimb+impairment+in+rats+expressing+a+pathological+TDP-43+25+kDa+C-terminal+fragment+to+mimic+amyotrophic+lateral+sclerosis.">Selective forelimb impairment in rats expressing a pathological TDP-43 25 kDa C-terminal fragment to mimic amyotrophic lateral sclerosis.</a> Mol. Ther. 21 (7), 1324-1334 (2013).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV9+supports+wide-scale+transduction+of+the+CNS+and+TDP-43+disease+modeling+in+adult+rats.">AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats.</a> Mol. Ther. Methods Clin. Dev. 2, 15036 (2015).</li><li>Jackson, 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=Preservation+of+forelimb+function+by+UPF1+gene+therapy+in+a+rat+model+of+TDP-43-induced+paralysis.">Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced paralysis.</a> Gene Therapy. 22 (1), 20-28 (2015).</li><li>Jackson, K., Dayton, R., Deverman, B., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+targeting,+better+efficiency+for+wide-scale+neuronal+transduction+with+the+synapsin+promoter+and+AAV-PHP.B.">Better targeting, better efficiency for wide-scale neuronal transduction with the synapsin promoter and AAV-PHP.B.</a> Front. Mol. Neurosci. 9, 116 (2016).</li><li>Jackson, 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=Severe+respiratory+changes+at+end+stage+in+a+FUS-induced+disease+state+in+adult+rats.">Severe respiratory changes at end stage in a FUS-induced disease state in adult rats.</a> BMC Neuroscience. 17 (1), 69 (2016).</li><li>Xu, L., 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=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+vector+&amp;#34;magic+bullet&amp;#34;:+targeted+expression+in+the+central+nervous+system+after+peripheral+delivery+using+the+synapsin+promoter.">Gene vector &amp;#34;magic bullet&amp;#34;: targeted expression in the central nervous system after peripheral delivery using the synapsin promoter.</a> Expert Opin Ther Targets. 20 (10), 1153-1154 (2016).</li><li>van Hulst, R., Klein, J., Lachmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+embolism:+pathophysiology+and+treatment.">Gas embolism: pathophysiology and treatment.</a> Clin. Physiol. Funct. Imaging. 23 (5), 237-246 (2003).</li></ol>

The authors have nothing to disclose.

A variety of delivery routes have been tested for AAV vectors: intra-parenchymal, intra-cerebroventricular, intra-thecal, intravenous, intranasal, intra-muscular, etc. Each route may have specific advantages as well as drawbacks. Tail vein administration of AAV to adult rats is a reliable method for achieving consistent transduction of the CNS. The peripheral injection method avoids the introduction of an injection cannula into the CNS tissues and is therefore minimally invasive. The efficient CNS expression that can be ...

Recombinant adeno-associated virus (AAV) vectors are indispensable tools for CNS research because they are so efficient for transducing neurons in vivo. AAV vectors are very versatile for studying different transgenes and protein isoforms, different tissues, different host species, and different routes of administration. For instance, AAV can be administered to mice by a peripheral, relatively non-invasive, intravenous injection to transduce neurons throughout the CNS as first described in Foust et al. and Duque et al. (see JOVE paper by Gombash et al. (2014)).1,2,3 This gene delivery approach is used in rats to efficiently express either green fluorescent protein (GFP) or the ALS related protein, transactive response DNA-binding protein of 43 kDa (TDP-43) in the CNS.4,5,6,7,8,9 Working in rats is significant because the rat&#39;s physiologic and metabolic parameters are closer to humans as compared to mice and there are behavioral and toxicological assays designed specifically for rats. Furthermore, more transgenic rat lines are becoming available that can be utilized in AAV gene transfer studies.
Methods are detailed for expansive CNS gene transfer in the rat, and rapid, reliable quantification of the outcomes. Wide-scale CNS transduction is used to mimic the symptomatology of ALS in rats by expressing TDP-43 throughout the spinal cord. The method is tail vein injections of the TDP-43 vector to young adult rats as used in Jackson et al.6,8,9 After several weeks, TDP-43-induced motor deficits are scored by two methods: escape reflex and rotarod as used in Dayton et al. and Jackson et al.5,6,7,9 For the control GFP vector, in post-mortem analysis, the fluorescent area of the cerebellum is calculated as an index of transduction efficiency as used in Jackson et al.6,8 The analysis of cerebellum has proven to be a rapid and reliable index of the degree of CNS transduction after peripheral gene delivery and should be applicable to a variety of approaches attempting peripheral-to-central gene transfer.

<table>
	<tbody>
		<tr>
			<td>Scale</td>
			<td>Pelouze</td>
			<td>SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene bucket</td>
			<td>Thermo Scientific</td>
			<td>12014000</td>
			<td>4000 mL</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#39;s Solution</td>
			<td>Baxter Healthcare</td>
			<td>0338-0114-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortexer</td>
			<td>Fisher Scientific</td>
			<td>128101</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>22624415</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory film</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>1-mL syringe</td>
			<td>BD</td>
			<td>309626</td>
			<td>Comes with a 25g needle attached. 
			Replace with 27-30g needle for injection</td>
		</tr>
		<tr>
			<td>30g needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>66794-013-25</td>
			<td>Isoflurane USP 250 mL</td>
		</tr>
		<tr>
			<td>Anesthesia mask</td>
			<td>Kopf Instruments</td>
			<td>906</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Henry Schein</td>
			<td>100-2524</td>
			<td>2&#34; x 2&#34; (5cm x 5cm)</td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>USA Scientific</td>
			<td>1111-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipet</td>
			<td>Gilson</td>
			<td>4642080</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas anesthesia vaporizer</td>
			<td>SurgiVet</td>
			<td>4214322</td>
			<td>Classic T3</td>
		</tr>
		<tr>
			<td>Gas anesthesia scavenger</td>
			<td>SurgiVet</td>
			<td>32373B10</td>
			<td>Enviro-PURE</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sears</td>
			<td>17280</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench pad</td>
			<td>VWR</td>
			<td>56617-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotarod</td>
			<td>Panlab/Harvard Apparatus</td>
			<td>76-0772</td>
			<td>4 rats/ 4 mice</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2401</td>
			<td>140 proof</td>
		</tr>
		<tr>
			<td>70% Isopropanol</td>
			<td>VWR</td>
			<td>89108-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Scion Image</td>
			<td>Scion Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Tag Polyclonal Antibody</td>
			<td>Invitrogen</td>
			<td>A11122</td>
			<td>Primary Antibody - 1:500 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11070</td>
			<td>Secondary Antibody - 1:300 dilution</td>
		</tr>
		<tr>
			<td>Axioskop 40 microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methods and tips for intravenous administration of adeno-associated virus to rats and evaluation of central nervous system transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

TDP-43 induced motor impairments should arise within 2 - 6 weeks depending on the dose of the AAV TDP-43 used (Figure 1). Unsuccessful tail vein injections will result in partial or no impairments; the AAV must reach the bloodstream to produce the effect. A successful injection of AAV GFP will result in GFP expression throughout the brain and spinal cord in a vector-dose dependent manner. When using the strong recombinant promoter to drive expression, the cyt...

Watch this Scientific Journal Video about Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction at JoVE.com

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

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.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

methods-tips-for-intravenous-administration-adeno-associated-virus-to

Research

JoVE Journal

Neuroscience

11.4K Views.  Louisiana State University Health Sciences Center. 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. 

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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

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 ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

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. ...