This work was funded by a grant from the National Institute of Neurological Disorders and Stroke R01 R01NS103481 and the Shriners Hospital for Pediatric Research grants SHC 84051 and SHC 86000 and the Department of Defense (SC140089).

All of the following surgical and animal care procedures have been approved by the Animal Care and Use Committee of Temple University.
1. Pre-surgical preparations
<ol>
	<li>Prepare pulled glass needles for viral injection a few days before surgery using 3.5 nanoliter glass capillary pipettes designed for nanoliter injectors. Pull each pipette on a two-step needle puller according to the manufacturer&#8217;s instructions to create two needle templates.</li>
	<li>Refine the tip of the needle ...

Genetic manipulation of neurons in the brain and spinal cord has served to highlight sensory, motor and autonomic pathways via fluorescent tracing and to explore regrowth potential of neuronal tracts after injury27,28,29,30,31,32,33. Direct injection of a retrogradely transportable viral vec...

Viral vectors are important biological tools that can introduce genetic material into cells in order to compensate for defective genes, upregulate important growth proteins or manufacture marker proteins that highlight the structure and synaptic connections of their targets. This article focuses on direct injection of a highly efficient retrogradely transportable lentiviral vector into the rat spinal cord in order to highlight major motor pathways with fluorescent tracing.&#160; This method is also highly appropriate for axonal regeneration and regrowth studies to introduce proteins of interest into diverse populations of neurons and has been used to silence neurons for functional mapping studies1,2.
Many of the anatomical details of spinal motor pathways were elucidated through direct injection studies with classical tracers such as BDA and fluoro-gold3,4,5,6,7,8. These tracers are considered gold standard but may have certain disadvantages such as uptake by damaged axons, or axons in passage in the white matter surrounding an injection site9,10,11. This could lead to incorrect interpretations of pathway connectivity and may be a drawback in regeneration studies where dye absorption by damaged or severed axons could be mistaken for regenerating fibers during later analysis12.
Lentiviral vectors are popular in gene therapy studies, as they provide stable, long-term expression in neuronal populations13,14,15,16,17,18,19. However, traditionally packaged lentiviral vectors can have limited retrograde transport and may trigger immune system response when used in vivo4,20,21. A highly-efficient retrograde transport vector termed HiRet has been produced by Kato et al. by modifying the viral envelope with a rabies virus glycoprotein to create a hybrid vector that improves retrograde transport22,23.
Retrograde tracing introduces a vector into the synaptic space of a target neuron, allowing it to be taken up by that cell&#8217;s axon and transported to the cell body. Successful transport of HiRet has been demonstrated from neuronal synapses into the brains of mice and primates23,24 and from the muscle into motor neurons22. This protocol demonstrates injection into the lumbar spinal cord, specifically targeting the synaptic terminals of propriospinal interneurons and brainstem neurons. PNs receive connections from many different spinal pathways and can thus be utilized to target a diverse population of neurons in the spinal cord and brainstem. Labeled neurons in this study represent circuits innervating motor neuron pools relating to hindlimb motor function. Robust labeling is seen in the spinal cord and brainstem, including high fidelity details of dendritic arbors and axon terminals. We have also used this method in previous studies within the cervical spinal cord to label propriospinal and brainstem reticulospinal pathways25.
This protocol demonstrates injection of a viral vector into the lumbar spinal cord of a rat. As seen in Movie 1, the incision is targeted by identifying the L1 vertebra located at the last rib. This is used as a caudal landmark for a 3-4 cm incision that exposes musculature over the L1-L4 spinal cord. Laminectomies of the dorsal aspects of the T11-T13 vertebrae are performed and a beveled glass needle is directed 0.8 mm lateral from the midline and lowered 1.5 mm deep into the gray matter to inject virus.

<table>
	<tbody>
		<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 IP injection, potential anesthetic booster shots, and antibiotic injections.</td>
		</tr>
		<tr>
			<td>10mL Syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309604</td>
			<td>For injecting saline into the animal, post-surgery.</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, and surgeon&#39;s hands during surgery, as well as all other minor maintainances of sterility.</td>
		</tr>
		<tr>
			<td>Anesthetic (Ketamine/Xylazine Solution)</td>
			<td>Zoetis</td>
			<td>240048</td>
			<td>For keeping the animal in the correct plane of consciousness during surgery.</td>
		</tr>
		<tr>
			<td>Antibiotic (Cefazolin)</td>
			<td>West-Ward Pharmaceuticals</td>
			<td>NPC 0143-9924-90</td>
			<td>To be injected subcutaneously to prevent infection post-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>Bonewax</td>
			<td>Fine Science Tools</td>
			<td>19009-00</td>
			<td>To seal up bone in the case of bone bleeding.</td>
		</tr>
		<tr>
			<td>Cauterizer</td>
			<td>Fine Science Tools</td>
			<td>18010-00</td>
			<td>To seal any arteries or veins severed during surgery to prevent excessive blood loss.</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>Gelfoam</td>
			<td>Pfizer</td>
			<td>H68079</td>
			<td>To seal up bone in the case of bone bleeding.</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>Hair Clippers</td>
			<td>Oster</td>
			<td>111038-060-000</td>
			<td>For clearing the surgical site of hair.</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Roboz</td>
			<td>RS-7231</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>34155</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>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>Ophthalamic Ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>RAC 0119</td>
			<td>To protect the animal&#39;s eyes during surgery.</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>Rimadyl Tablets</td>
			<td>Bio Serv</td>
			<td>MP275-050</td>
			<td>For pain management post-surgery.</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>Stainless Steal Wound Clips</td>
			<td>CellPoint</td>
			<td>201-1000</td>
			<td>To bind the skin of the surgical wound during closing.</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>T/Pump Heat Therapy Water Pump</td>
			<td>Gaymar</td>
			<td>TP500C</td>
			<td>To pump warm water into the water convection warming pad.</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>
	</tbody>
</table>

direct injection of a lentiviral vector highlights multiple motor pathways in the rat spinal cord

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

Successful injection and transport of the viral vector should result in transduction of a robust population of unilateral neurons in the spinal cord and in certain brainstem nuclei. Figure 1 demonstrates stereotypical labeling of neurons and axons in the thoracic spinal cord and in the pontine reticular formation of the brainstem at four weeks post-injection. Significant GFP expression is seen in neurons in the gray matter of the thoracic spinal cord on the side ipsilater...

Watch this Scientific Journal Video about Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord at JoVE.com

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

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

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

Viral vectors can introduce genetic material into cells to compensate for defective genes, to up-regulate important growth proteins, or to manufacture markers that can trace neural circuits. Introducing viral vectors into the nervous system is challenging due to innate biological barriers. Direct injection into spinal cord tissue bypasses these barriers, allowing for access to cell bodies or synapses.Correctly targeting the L1 to L4 spinal levels can be difficult. It's important to use landmarks to verify the target and remember that these segments line to the T11 to T13 vertebrae. On the day of the procedure, plug the injector into the micro-pump and place the micro-pump into a micromanipulator with a vernier scale.Use a syringe with a flexible needle to carefully load a colored dye into one of the glass needles and insert the glass needle into the injector. Then confirm that the needle is seeded correctly into the washers, and that the injector cap is screwed on tight, and that the steel injector needle is extended approximately three-quarters of the length of the glass needle. Before beginning the procedure, thaw at least one microliter, plus a small volume of extra virus per injection from freezer storage, and confirm a lack of response of toe pinch in an anesthetized rat.Shave along the dorsal midline from the hips to the inferior angle of the scapulae of the animal, pulling the skin taut for an easier and more precise shave, and disinfect the exposed skin with sequential 5%iodine and 70%ethanol scrubs. Apply ophthalmic ointment to both eyes. Then place the animal on a sterile cloth and place gauze underneath the bladder to collect any urine.To identify the site of the skin incision, press the fingers gently at the last rib to locate the L1 vertebra. Using this vertebra as a landmark and holding the skin taut by gentle spreading while pressing firmly with the scalpel, use a number 10 surgical blade to make a three to four-centimeter skinned incision ending just inferior to L1 to expose the muscle. Use forceps and scissors to feel for the spinous processes.Make small rostral cuts to allow room to grab securely onto an upper process with rat tooth forceps. And make two long, deep cuts as close to the processes as possible. After securing the lateral muscles in place, clear the muscle around the processes to determine the shape of their heads.Then visualize the shapes of the heads of the spinous processes in order to identify the laminectomy site. The T11 and the joining T12 and T13 processes will be the site of the laminectomy. Once the target area has been correctly identified, gently spread the vertebrae to reveal the intervertebral ligaments, and use rongeurs to take small bites of the spinous processes and the dorsal aspect of the vertebrae.Clear the bone away from the midline so that the midline blood vessel can be observed, leaving a window that clearly reveals the spinal cord tissue and is free of debris. Then fasten stabilizing forceps to the spinous processes rostral and caudal to the laminectomy window to secure the animal in a spinal holder. And raise the abdomen of the animal to negate the effect of breathing movements.To load the virus into the injector, pipette approximately five microliters of the thawed stock onto a piece of Parafilm, and position the glass needles so that the tip is inside the drop. Use the micro-pump to withdraw up to four microliters of virus at a rate of 20 to 100 nanoliters per second, and set the controller to inject before releasing a small amount of virus from the needle tip to ensure that it is not blocked. Wipe off any excess virus with a laboratory wipe and position the micromanipulators so that the vernier scale is visible.Place the needle at the midline of the spinal cord, and use the vernier scale on the micromanipulator to direct the needle laterally 0.8 millimeters. Then lower the needle to the spinal chord until it is indenting, but not puncturing, the dura, and use a quick twisting motion to puncture the dura with the needle until the needle tip has sunk to a depth of 1.5 millimeters. Once the needle is in place, program the injector to deliver the virus at a rate of 400 nanoliters per minute, and confirm that virus is entering the spinal chord by observing the progress of the dye front.Once the injection is finished, allow the needle to rest in the spinal cord for two to five minutes to facilitate the diffusion of the virus, before slowly withdrawing the needle for positioning at the next injection site. After the last injection, remove the animal from the spinal holder to allow the removal of any devices used to spread the lateral muscle. Then close the muscle with a 4.0 chromic catgut suture and staple the skin with nine-millimeter wound clips.Four weeks after a successful injection, significant GFP expression is observed in the neurons within the gray matter of the thoracic spinal cord on the side, ipsilateral to the injection. In the white matter, GFP expression is observed in the axons in the ipsilateral cord, especially in areas typical to the propriospinal axons. A higher magnification of the gray matter neurons reveals an example of a typical signal expression in neuronal cell bodies and dendrites.GFP expression in neurons can also be observed in brainstem nuclei, such as within the pontine reticular formation. The key factors for a successful spinal cord injection procedure are correct vertebral targeting and ensuring that virus is entering the tissue If a rostral spinal cord injury is performed prior to this procedure, direct injection can be used to trace neurons that reestablish connections around the injury.

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8.4K 次观看.  Temple University. 病毒载体可以将遗传物质引入细胞，以补偿有缺陷的基因，提高重要的生长蛋白，或制造可以追踪神经回路的标记物。由于与生俱来的生物学障碍，将病毒载体引入神经系统是具有挑战性的。直接注射到脊髓组织中绕过这些屏障，允许进入细胞体或突触。正确定位 L1 到 L4 脊髓水平可能很困难。使用地标来验证目标并记住这些段线到 T11 到 T13 椎骨非常重要。在手术当天?...