Summary

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

Published: March 15, 2019
doi:

Summary

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.

Abstract

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.

Introduction

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.  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’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.

Protocol

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 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’s instructions to create two needle templates. Refine the tip of the needle …

Representative Results

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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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).

Materials

#10 Scalpel Blades Roboz RS-9801-10 For use with the scalpel.
1 mL Syringes Becton, Dickinson and Company 309659 For anesthetic IP injection, potential anesthetic booster shots, and antibiotic injections.
10mL Syringes Becton, Dickinson and Company 309604 For injecting saline into the animal, post-surgery.
4.0 Chromic Catgut Suture DemeTECH NN374-16 To re-bind muscle during closing.
48000 Micropipette Beveler World Precision Instruments 32416 Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles.
5% Iodine Solution Purdue Products L.P. L01020-08 For use in sterilzation of the surgical site.
70% Ethanol N/A N/A For sterilization of newly prepared glass needles, animal models during surgical preparation, and surgeon's hands during surgery, as well as all other minor maintainances of sterility.
Anesthetic (Ketamine/Xylazine Solution) Zoetis 240048 For keeping the animal in the correct plane of consciousness during surgery.
Antibiotic (Cefazolin) West-Ward Pharmaceuticals NPC 0143-9924-90 To be injected subcutaneously to prevent infection post-surgery.
Bead Sterilizer CellPoint 5-1450 To heat sterilize surgical instruments.
Bonewax Fine Science Tools 19009-00 To seal up bone in the case of bone bleeding.
Cauterizer Fine Science Tools 18010-00 To seal any arteries or veins severed during surgery to prevent excessive blood loss.
Digital Scale Okaus REV.005 For weighing the animal during surgical preparation.
Flexible Needle Attachment World Precision Instruments MF34G-5 For cleaning glass needles and loading red oil into glass needles.
Gelfoam Pfizer H68079 To seal up bone in the case of bone bleeding.
Glass Capillary Tubes World Precision Instruments 4878 For pulled glass needles – should be designed for nanoliter injectors.
Hair Clippers Oster 111038-060-000 For clearing the surgical site of hair.
Hemostats Roboz RS-7231 For general use in surgery.
Kimwipes Kimtech 34155 For general use in surgery.
Medium Point Curved Forceps Roboz RS-5136 For general use in surgery.
Micromanipulator with a Vernier Scale Kanetec N/A For precise targeting during surgery.
Microscissors Roboz RS-5621 For cutting glass whisps off of freshly pulled glass capillary tubes.
Microscope with Light and Vernier Scale Ocular Leitz Wetzlar N/A Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles.
MicroSyringe Pump Controller World Precision Instruments 62403 To control the rate of injection.
Nanoliter 2000 Pump Head Injector World Precision Instruments 500150 To load and inject virus in a controlled fashion.
Needle Puller Narishige PC-100 To heat and pull apart glass capillary tubes to form glass needles.
Ophthalamic Ointment Dechra Veterinary Products RAC 0119 To protect the animal's eyes during surgery.
Parafilm Bemis PM-996 To assist with loading virus into the nanoinjector.
PrecisionGlide Needles (25G x 5/8) Becton, Dickinson and Company 305122 For use with the 1mL and 10 mL syringes to allow injection of the animal model.
Rat Tooth Forceps Roboz RS-5152 For griping spinous processes.
Red Oil N/A N/A To provide a front for visualization of virus entering tissue during injection.
Retractors Roboz RS-6510 To hold open the surgical wound.
Rimadyl Tablets Bio Serv MP275-050 For pain management post-surgery.
Rongeurs Roboz RS-8300 To remove muscle from the spinal column during surgery.
Scalpel Blade Handle Roboz RS-9843 To slice open skin and fat pad of animal model during surgery.
Scissors Roboz RS-5980 For general use in surgery.
Stainless Steal Wound Clips CellPoint 201-1000 To bind the skin of the surgical wound during closing.
Staple Removing Forceps Kent Scientific INS750347 To remove the staples, should they be applied incorrectly.
Sterile Cloth Phenix Research Products BP-989 To provide a sterile surface for the operation.
Sterile Cotton-Tipped Applicators Puritan 806-WC To soak up blood in the surgical wound while maintaining sterility.
Sterile Gauze Covidien 2146 To clean the surgical area and surgical tools while maintaining sterility.
Sterile Saline Baxter Healthcare Corporation 281324 For use in blood clearing, and for replacing fluids post-surgery.
Surgical Gloves N/A N/A For use by the surgeon to maintain sterile field during surgery.
Surgical Heating Pad N/A N/A For maintaining the body temperature of the animal model during surgery.
Surgical Microscope N/A N/A For enhanced visualization of the surgical wound.
Surgical Stapler Kent Scientific INS750546 To apply the staples.
T/Pump Heat Therapy Water Pump Gaymar TP500C To pump warm water into the water convection warming pad.
Water Convection Warming Pad Baxter Healthcare Corporation L1K018 For use in the post-operational recovery area to maintain the body temperature of the unconscious animal.
Weighted Hooks N/A N/A To hold open the surgical wound.

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Cite This Article
Keefe, K. M., Junker, I. P., Sheikh, I. S., Campion, T. J., Smith, G. M. Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord. J. Vis. Exp. (145), e59160, doi:10.3791/59160 (2019).

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