This work was funded by a fellowship grant from Shriners Hospitals for Children SHC-84706.

All of the following surgical and animal care procedures have been approved by the Animal Care and Use Committee of Temple University. The protocol described is a survival surgery, and the animals were eventually euthanized by intraperitoneal injection of 100 mg/kg sodium pentobarbital at the completion of their time points.
1. Pre-surgical preparation
<ol>
	<li>Prepare at least two pulled glass needles for viral injection using 3.5 nL glass capillary pipettes; one needle for the DREADD and ...

<ol>
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The authors have nothing to disclose.

Inducible genetic modulation of brain activity with injectable chemogenetic modifiers is a powerful tool in studying the various mechanisms that underlie the recovery from SCI. The accuracy of the targeting for the inducible G-protein-coupled receptors (DREADDs) is further increased when considering that fluorescence tracing validates the anatomical precision in histology. This paper discusses a reliable method for exploring whether or not inhibiting or stimulating select neuronal pathways (with either excitatory or inhi...

While SCI is a relatively rare occurrence in the pediatric population, it is particularly traumatic and causes a permanent disability requiring immense logistical foresight. Furthermore, a higher proportion of pediatric SCIs is classified as cervical and complete compared to the adult population1,2. A hallmark across mammalian species is that neonates recover notably better from SCI than adults, and this offers an opportunity to assess the driving mechanisms for recovery in younger populations3,4,5. Despite this, there are fewer multimodal studies tackling neonate and infant rodent research, partly due to the added difficulty of accurately targeting select populations of neurons in the much tighter anatomical landmarks of younger animals6. This article focuses on the direct injection of highly efficient anterograde and retrograde adeno-associated vectors into the rat spinal cord to modulate major motor pathways with the application of Cre-dependent-DREADDs, expanding the reach of multimodal regeneration studies.
Viral vectors are important biological tools with a breadth of applications, including the introduction of genetic material to substitute for target genes, upregulate growth proteins, and trace the anatomical landscape of the CNS7,8,9. Many of the anatomical details of spinal motor pathways have been studied using classical tracers, i.e., biotinylated dextran amine. While traditional tracers have been instrumental in unearthing neuroanatomy, they are not without their disadvantages: they indiscriminately label pathways even if correctly injected, and studies have found that they are taken up by damaged axons10,11,12. Consequently, this could lead to incorrect interpretations in regeneration studies where severed axons could be mistaken for regenerating fibers.
The following method utilizes the two-viral vector system recently popularized in modulation studies, with two different viral vectors in two separate areas of the same neuron13,14. The first is a vector that locally infects the cell bodies of projection neurons. The other is a retrograde vector being transported from the axon terminals of the projection neurons (Figure 1). The retrograde vector carries Cre recombinase, and the local vector incorporates the &#34;Cre-On&#34; double-floxed sequence in which a fluorescent protein (mCherry) is encoded. The native transgene expressing both hM3Dq and mCherry is inverted relative to the promoter and is flanked by two LoxP sites (Figure 2). Thus, mCherry is only expressed in the doubly transduced projection neurons where Cre recombinase induces a recombination event between the LoxP sites, flipping the orientation of the transgene into the appropriate reading frame and allowing the expression of both the DREADD and the fluorescent protein. Once the viral transgene is in the correct orientation, and when applicable, the DREADDs can transiently induce neuromodulation through a separately injected ligand, i.e., clozapine-N-oxide. The protocol was designed to authenticate inducible neuromodulation research in neonates, wherein DREADDS are injected to modulate the CSTs selectively. The two-viral system acts as an insurance policy, ensuring that every DREADD-positive&#160;cell is traceable under fluorescence with high fidelity to validate the injections.
This method also helps to bridge the gap in neonatal research. Pediatric SCI presents its challenges, and research analyzing regeneration, sprouting, or plasticity should emphasize the differences between neonates and adults3,15,16,17. By optimizing the surgical procedure and performing prior anatomical studies with Nissl staining, the coordinates for both the cranial and spinal injections were validated. The aim was to provide a method for dual injections into a neonatal rat with increased fidelity and survivability.
For the current model, the anterograde vector was injected into the cell bodies of the somatomotor cortex using bregma as reference18,19. In terms of the spinal injections, the retrograde vector was injected into laminae V-VII, where the CST axon terminals reside20,21. There are many fundamental questions underlying how certain lesion models affect younger animals differently, and how the subsequent recovery diverges from an older animal. This study demonstrates a robust means of studying cervical injuries and the recoverability of forelimb function in neonatal rodents. In contrast, the majority of previous studies have addressed recovery locomotion following lumbar or thoracic injuries5,22,23,24. By pairing the double-viral vector with the novel injection technique described here, this protocol helps mitigate certain issues (i.e., survivability) that may plague neonatal rodent investigations. This method is robust, practical, and versatile: slight variations in the technique will allow for targeting different pathways, i.e., ventral CST, dorsal CST, and the ascending dorsal pathways.
For this system, one locally acting virus (e.g., AAV2) is injected in the region of the neuronal cell bodies of interest. A second retrogradely transported virus that controls the expression of the local virus is injected at the axon terminals for that neuronal population. Thus, by definition, only corticospinal neurons are labeled. The retroAAV-Cre virus was chosen with a constitutively active CMV promoter as the shuttle plasmid is used to generate several AAV serotypes for Cre-dependent expression in several cell types. For cortical injections, AAV2 was chosen with the transgene driven by the synapsin-1 promotor to limit any expression to neurons. Because the 2-viral system relies more on the origin and termination of the neuronal population of interest, several different promoters could be used, if they can drive the expression of the genes of interest within the neuronal population of interest. For example, the excitatory neuronal promoter, CamKII, could be substituted for the synapsin-1. In addition to the use of these AAV serotypes, retrograde transport into immature, and to a much lesser extent, adult corticospinal motor neurons can also be achieved using the high retrograde transportable lentivirus (HiRet)25. HiRet lentiviruses use a chimeric Rabies/VSV glycoprotein to target uptake at the synapse for retrograde transport. Combined with a Tet-On promoter, this 2-viral system supports inducible expression in a retrograde-dependent fashion26,27.
Retrograde viruses insert vectors into the synaptic space of a target neuron, allowing it to be taken up by that cell&#39;s axon and transported to the cell body. While lentiviral vectors have previously had tremendous success, providing long-term expression in gene therapy studies, this method pivoted towards adeno-associated viral vectors for a few simple reasons26,28: AAV is more economical, similarly effective, and presents less of a logistical burden, given that it has a lower biosafety level designation29,30,31,32. While AAV2, the most used serotype, demonstrates robust transfection of CST axons, future researchers may note that AAV1 offers some versatility as it labels transynaptically, thus putting forth several possible iterations in future studies33. The final adaptation is to encode the retrograde virus with Cre-recombinase so that multiple anterograde vectors can be introduced simultaneously, thereby reducing unnecessary in-house virus waste and maximizing the likelihood of the DREADDs expressing in the correct orientation.
Ultimately, this protocol demonstrates simultaneous injection into the cortex and cervical spine, specifically targeting the cell bodies and the axon terminals of the corticospinal tract, respectively. High-fidelity transfection is seen in the cerebral cortex and spinal cord. While the protocol described was perfected for Sprague Dawley rats 5 days of age, it is suitable for postnatal days 4-10 with minor adjustments to anesthesia and&#160;stereotactic coordinates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#11 scalpel blades</td>
			<td>Roboz</td>
			<td>RS-9801-11</td>
			<td>For use with the scalpel.</td>
		</tr>
		<tr>
			<td>#10 Scalpel Blades</td>
			<td>Roboz</td>
			<td>RS-9801-10</td>
			<td>For use with the scalpel.</td>
		</tr>
		<tr>
			<td>1 mL Syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309659</td>
			<td>For anesthetic SC injection and fluid bolus</td>
		</tr>
		<tr>
			<td>4.0 silk suture</td>
			<td>Ethicon</td>
			<td>771-683G</td>
			<td>For skin closure</td>
		</tr>
		<tr>
			<td>4.0 Chromic Catgut Suture</td>
			<td>DemeTECH</td>
			<td>NN374-16</td>
			<td>To re-bind muscle during closing.</td>
		</tr>
		<tr>
			<td>48000 Micropipette Beveler</td>
			<td>World Precision Instruments</td>
			<td>32416</td>
			<td>Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles.</td>
		</tr>
		<tr>
			<td>5% Iodine Solution</td>
			<td>Purdue Products L.P.</td>
			<td>L01020-08</td>
			<td>For use in sterilzation of the surgical site.</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For sterilization of newly prepared glass needles, animal models during surgical preparation</td>
		</tr>
		<tr>
			<td>Ketamine (Ketaset)</td>
			<td>Zoetis</td>
			<td>240048</td>
			<td>For keeping the animal in the correct plane of consciousness during surgery.</td>
		</tr>
		<tr>
			<td>Bead Sterilizer</td>
			<td>CellPoint</td>
			<td>5-1450</td>
			<td>To heat sterilize surgical instruments.</td>
		</tr>
		<tr>
			<td>Digital Scale</td>
			<td>Okaus</td>
			<td>REV.005</td>
			<td>For weighing the animal during surgical preparation.</td>
		</tr>
		<tr>
			<td>Flexible Needle Attachment</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>For cleaning glass needles and loading red oil into glass needles.</td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes</td>
			<td>World Precision Instruments</td>
			<td>4878</td>
			<td>For pulled glass needles - should be designed for nanoliter injectors.</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Roboz</td>
			<td>RS-7231</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Medium Point Curved Forceps</td>
			<td>Roboz</td>
			<td>RS-5136</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Micromanipulator with a Vernier Scale</td>
			<td>Kanetec</td>
			<td>N/A</td>
			<td>For precise targeting during surgery.</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td>For cutting glass whisps off of freshly pulled glass capillary tubes.</td>
		</tr>
		<tr>
			<td>Lab Standard Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td>51600</td>
			<td>To hold the neonatal sterotaxic holder in place</td>
		</tr>
		<tr>
			<td colspan="2">Lab Standard with Mouse &#38; Neonates Adaptor</td>
			<td>51615</td>
			<td>For neonatal skull fixation during cranial surgery and spinal injections</td>
		</tr>
		<tr>
			<td>Microscope with Light and Vernier Scale Ocular</td>
			<td>Leitz Wetzlar</td>
			<td>N/A</td>
			<td>Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles.</td>
		</tr>
		<tr>
			<td>MicroSyringe Pump Controller</td>
			<td>World Precision Instruments</td>
			<td>62403</td>
			<td>To control the rate of injection.</td>
		</tr>
		<tr>
			<td>Nanoliter 2000 Pump Head Injector</td>
			<td>World Precision Instruments</td>
			<td>500150</td>
			<td>To load and inject virus in a controlled fashion.</td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td>To heat and pull apart glass capillary tubes to form glass needles.</td>
		</tr>
		<tr>
			<td>pAAV-CMV-scCre</td>
			<td>Wu lab&#160;</td>
			<td></td>
			<td>Cre plasmid</td>
		</tr>
		<tr>
			<td>pAAV-hSyn-DIO-hM3Dq-mCherry (plasmid #44361)</td>
			<td>Bryan Roth&#8217;s lab through Addgene</td>
			<td></td>
			<td>DREADD plasmid</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>To assist with loading virus into the nanoinjector.</td>
		</tr>
		<tr>
			<td>PrecisionGlide Needles (25G x 5/8)</td>
			<td>Becton, Dickinson and Company</td>
			<td>305122</td>
			<td>For use with the 1mL and 10 mL syringes to allow injection of the animal model.</td>
		</tr>
		<tr>
			<td>Rat Tooth Forceps</td>
			<td>Roboz</td>
			<td>RS-5152</td>
			<td>For griping spinous processes.</td>
		</tr>
		<tr>
			<td>Red Oil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To provide a front for visualization of virus entering tissue during injection.</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Roboz</td>
			<td>RS-6510</td>
			<td>To hold open the surgical wound.</td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Roboz</td>
			<td>RS-8300</td>
			<td>To remove muscle from the spinal column during surgery.</td>
		</tr>
		<tr>
			<td>Scalpel Blade Handle</td>
			<td>Roboz</td>
			<td>RS-9843</td>
			<td>To slice open skin and fat pad of animal model during surgery.</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-5980</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Staple Removing Forceps</td>
			<td>Kent Scientific</td>
			<td>INS750347</td>
			<td>To remove the staples, should they be applied incorrectly.</td>
		</tr>
		<tr>
			<td>Sterile Cloth</td>
			<td>Phenix Research Products</td>
			<td>BP-989</td>
			<td>To provide a sterile surface for the operation.</td>
		</tr>
		<tr>
			<td>Sterile Cotton-Tipped Applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td>To soak up blood in the surgical wound while maintaining sterility.</td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>Covidien</td>
			<td>2146</td>
			<td>To clean the surgical area and surgical tools while maintaining sterility.</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Baxter Healthcare Corporation</td>
			<td>281324</td>
			<td>For use in blood clearing, and for replacing fluids post-surgery.</td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For use by the surgeon to maintain sterile field during surgery.</td>
		</tr>
		<tr>
			<td>Surgical Heating Pad</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For maintaining the body temperature of the animal model during surgery.</td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For enhanced visualization of the surgical wound.</td>
		</tr>
		<tr>
			<td>Surgical Stapler</td>
			<td>Kent Scientific</td>
			<td>INS750546</td>
			<td>To apply the staples.</td>
		</tr>
		<tr>
			<td>Water Convection Warming Pad</td>
			<td>Baxter Healthcare Corporation</td>
			<td>L1K018</td>
			<td>For use in the post-operational recovery area to maintain the body temperature of the unconscious animal.</td>
		</tr>
		<tr>
			<td>Weighted Hooks</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To hold open the surgical wound.</td>
		</tr>
		<tr>
			<td>Liquid bandage</td>
			<td>NewSkin</td>
			<td>985838</td>
			<td>To apply along sutures following surgery and encourage wound healing</td>
		</tr>
		<tr>
			<td>Wire Cage Lamp</td>
			<td>ZooMed</td>
			<td>LF10EC</td>
			<td>To help animals recover from anesthesia and retain warm body temperature naturally</td>
		</tr>
	</tbody>
</table>

targeting the corticospinal tract in neonatal rats with a double-viral vector using combined brain and spine surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

Successful injection and transport of the viral vector should result in the transduction of unilateral neurons in the spinal cord and the motor cortex. Figure 4 demonstrates the labeling of layer V CST neurons in the motor cortex of a brain coronal section expressing Cre-dependent-DREADDs-mCherry co-injected with a contralateral spine injection of rCre. The sections were stained with dsRed antibody.
<img alt="Figure 1" class="xfigi...

Watch this Scientific Journal Video about Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery at JoVE.com

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

This is the first protocol demonstrating the successful co-infection of the corticospinal tract in neonatal rats. It provides an accurate blueprint to investigate how individual subpopulations of cells in young mammals contribute to the recovery from spinal cord injury. Like other double viral vector techniques, this method allows the user to accurately transduce specific subpopulations of neurons, in turn, allowing researchers to identify the effectiveness of various chemogenetic techniques in promoting recovery.We would expect researchers that are unfamiliar with this technique to struggle with locating the proper spinal anatomy. And so we encourage you to continually count the vertebral segments often and always refer back to your landmarks. After confirming the depth of anesthesia in a neonatal Sprague Dawley pup, identify the area where the incision will be made by pressing the forceps in the midline on the top of the scalp, feeling for the sagittal suture.Holding the skin taut to ensure a clean, straight, and precise incision, make a one-centimeter incision along the sagittal suture plane starting immediately above the I line. Next, identify the bregma by gently probing the forceps along the surface of the skull, paying close attention to the suture lines and an indentation caused by the forceps running along the parietal and frontal bones. Maintain the opening by applying weighted hooks on the side of interest.Clear the aponeurosis using a combination of cotton tips and micro scissors to maximize the exposure of the bregma for increased accuracy. Ensure that the coronal suture is in clear view far enough laterally to provide a rough template for subsequent injections. Using the micro scissors, cut out an approximately three-by-two-millimeter section of the left frontal skull bone immediately adjacent to the bregma.Clear any debris, cerebrospinal fluid, and blood with cotton tips. To inject the virus, place the needle in place and program the injector to inject one microliter at a rate of 250 nanoliters per minute. Upon completion of the injection, allow the needle to rest in the cortex for three minutes.Repeat the injections at the other coordinates for a total of three injections along the cortex in relation to the bregma, all at a depth of 0.6 millimeters. After completing the injections, suture the scalp and transfer the animal to the other operating table for cervical laminectomy. Palpate the base of the skull using fingers to identify the incision site.Hold the skin taut by applying tension with the thumb and index finger. Using a number 11 blade, begin the incision at the midline one to two millimeters posterior to the skull base and extend the incision one centimeter posterior to expose the superficial muscle. Using the sharp point of the blade, gently make a series of cuts along the midline of the superficial muscle to expose the spinal cavity and deep spinal muscles.Then use a pair of forceps to spread open the muscle and visualize the surgical window. Once the deep spinal muscles have been exposed, insert retractors into the surgical window and retract the surgical window to seven to eight millimeters in width, allowing an unobstructed view of the spine. Identify the second cervical vertebra by its prominent spinous process that projects dorsally and encapsulates a large dome-shaped muscle.Using the flat edge of a bone scraper, gently scrape away the deep spinal muscle to the sides to expose the C3 to C7 vertebrae. Start medial and scrape laterally along the direction parallel to the vertebra laminae to ensure proper exposure, allowing clear distinction of the vertebral laminae. Control any bleeding with cotton tips.Using C2 as a guide when counting the vertebral levels, carefully cut the lateral edges of the cartilaginous laminae at C6 and C7 using a pair of micro scissors. Using a pair of fine forceps, carefully remove the dissected portion of the lamina to expose the spinal cord, ensuring that the spinal window is large enough to accommodate the desired injection site. Set the animal up in the stereotaxic cranial holder and place a rolled up piece of gauze under the animal's trunk to elevate its hind quarters.Before beginning injections, clear the spinal window of any blood or cerebrospinal fluid by gently applying cotton tips and Sugi triangles to the area, taking care not to insult the spinal cord. Further, create a barrier around the perimeter of the window by placing a small piece of absorbable cotton in the lateral portions of the spinal window to prevent occlusion of the injection site by continuous bleeding or CSF leakage. For direct injections into the spinal cord, use the tip of the glass injection needle to approximate the midline of the spinal cord by identifying the spinal artery.Next, place the needle just posterior to the C5 lamina at the approximate midline and use this as the reference point. Then, using the micro manipulator, move the needle laterally to the right by 0.3 millimeters and lower the needle until it just touches the surface of the spinal cord. From this depth, plunge the needle.0.6 millimeters into the spinal cord. Inject one microliter of the retro adeno-associated virus two carrying the Cre recombinase gene at 250 nanoliters per minute. Then wait for two minutes for the virus to diffuse into the spinal cord before slowly withdrawing the needle.The labeling of layer five corticospinal tract neurons in the motor cortex of a brain coronal section expressing Cre-dependent DREADDs mCherry co-injected with a contralateral spine injection of retro Cre is shown here. Being able to target specific neuronal populations will help to delineate the nuances driving recovery and provide more accuracy for treating the underlying spinal cord injury in the developing nervous system. This method paved the way for us to investigate the viability of using excitatory DREADDs as a means for treating spinal cord injury in juvenile rats.

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Research

JoVE Journal

Neuroscience

1.9K visualizações.  Temple University. Este é o primeiro protocolo que demonstra a coinfecção bem-sucedida do trato corticoespinhal em ratos neonatais. Ele fornece um modelo preciso para investigar como subpopulações individuais de células em mamíferos jovens contribuem para a recuperação da lesão da medula espinhal. Como outras técnicas de vetor viral duplo, este método permite que o usuário transduzir com precisão subpopulações específicas de neurônios, por sua vez, permitindo que os pesquisadores identifiquem ...