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. 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.
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.
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
2. Anesthesia and surgical site preparation
3. Surgical field and instrument preparation
4. Exposing the vertebral column and identifying the laminectomy site
5. Performing a laminectomy
6. Loading virus and positioning the injector
7. Injecting virus into the spinal cord
8. Wound closure and post-operative care
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 ipsilateral to the injection (Figure 1A, boxed area). A few neurons are also observed on the contralateral side, especially near the midline. In the white matter, GFP expression is observed in axons in the ipsilateral cord (Figure 1A, arrows and arrowheads), especially in areas typical to propriospinal axons (arrowheads). Figure 1A’ shows a higher magnification of the boxed area in A, demonstrating typical expression in neuronal cell bodies and dendrites. GFP expression in neurons can also be observed in brainstem nuclei such as the pontine reticular formation (Figure 1B, higher magnification of the boxed area in Figure 1B’).
Figure 1: Transduction of Neurons in the Spinal Cord and Brainstem. (A) GFP expression in neurons (boxed area), axons of propriospinal neurons (arrowheads) and axons of other tracts (arrows) in the thoracic spinal cord. (A’) Higher magnification of neuronal expression in the boxed area of A. (B) The pontine reticular formation in the brainstem expressing GFP-labeled neurons and dendrites. (B’) Higher magnification of the boxed area in B. Scale bars: (A) = 500 μm; (B) = 1 mm; (A’), (B’) = 50 μm. Please click here to view a larger version of this figure.
Movie 1: Targeting the injection into the lumbar spinal cord of the rat. This video summaries the basics of the targeting and injection of a viral vector into the rat spinal cord. Please click here to view this video. (Right-click to download.)
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 vector into the spinal cord can target neuronal populations via their synaptic connections, making this method an excellent choice for mapping pathways in the central nervous system. The HiRet vector specifically shows selective uptake at the neuronal synapse22,24,25, and previous tracing with similarly structured vectors showed no spread into injured axons or unrelated cells34,35, which is consistent with results featuring the HiRet construct22,23,25. Thus, direct injection of HiRet as a retrograde tracer is an ideal method for exploring interneuronal circuits that undergo plasticity after injury.
There are two critical concepts involved in successful injection of a viral vector into the spinal cord. The first is establishing the correct target with the glass needle, and the second is ensuring adequate flow of the virus from the needle into the tissue. As this protocol involves retrograde tracing, targeting the correct area necessitates a thorough understanding of the synaptic connections of the neuronal population of interest. Propriospinal and reticulospinal neurons connecting to the lumbar spinal cord are targeted here. These neurons are spread throughout the brainstem and cervical and thoracic spinal cord segments, with the majority of PNs localized to laminae V-VII within the gray matter. To ensure transduction of an adequate population of neurons, six viral injections are made over an area spanning four spinal segments. The L1-L4 segments are chosen due to the presence of the central pattern generators, and their known connections to other areas of the spinal cord. Once the correct spinal segment(s) and laminae are known, physical location of these targets via anatomical landmarks is crucial. Atlases showing details of anatomical structure and vertebral shape can be helpful to confirm targeting landmarks8,36. It should be noted that the majority of this protocol remains the same whether one injection or six are made; the difference is only in the number of spinal segments exposed via laminectomy, and the fact that you will need to reload the glass needle with additional virus if injecting more than 4 µL. The protocol can also be easily adapted for the mouse. Due to its smaller size, the volume of virus injected into the cord should be adjusted downward and the measurements needed to hit the correct spinal laminae adjusted. Several videos of similar surgeries on the mouse have previously been published37,38.
The next consideration is adequate diffusion into the tissue. Careful preparation of the needle is necessary to ensure that the aperture is sufficient for the viral suspension to flow outward and that debris does not block the tip, and visualization of the flow from the needle into the spinal cord via a front of colored dye is helpful to confirm that the suspension is penetrating the tissue. Once the suspension has been distributed into the tissue, maintaining the needle within the spinal cord for 2-5 minutes will ensure adequate diffusion.
Successful transduction of a target population can also depend on intrinsic properties of the virus injected such as titer, serotype and infection efficiency. We find a genomic copy titer of at least 1010 GC/mL for HiRet lentivirus to work well for in vivo experimentation. Higher titers or injection volumes might show higher labeling index, but care needs to be taken since virus could diffuse out of the intended area or into the contralateral spinal cord. Consideration should also be given to the type of vector appropriate for labeling cells of interest based on their tropism. Certain viral serotypes may have better infection and transduction rates in different populations of neurons39. Traditional lentiviral vectors are known to infect a broad range of cell types due to the VSV-G envelope protein, however modifications to this vector may influence its tropism40. The HiRet construct modifies the envelope by pseudotyping with a fusion glycoprotein (FuG-B) of rabies virus, which allows for highly-efficient retrograde transport22,23 and is effective in transducing propriospinal and reticulospinal tracts, with neuronal numbers comparable to tract tracing via methods such as fluorogold and microruby4,41 (for details of construction of the HiRet vector see Hirano et al.)22. However, it did not sufficiently label the adult corticospinal tract in these experiments, though it does label the CST in neonates with high efficiency in other studies1. It is possible that receptors necessary for HiRet uptake at the targeted synapses, such as NCAM or p75NTR, are only weakly expressed on adult CST neurons42,43, though this is still being investigated. In any case, this demonstrates the importance of determining whether the vector being used is appropriate for the targeted cells. In this experiment, brain and spinal cord tissue were processed and probed after four weeks to ensure abundant time for amplification and transport from the lumbar cord to all brain areas. HiRet is transported via fast retrograde axonal transport, and thus a shorter experimental period may be appropriate if the distance traveled is lesser, such as if the injection area is in the cervical spinal cord.
Direct injection surgery is a useful tool for introduction of viral vector technology into the spinal cord. Use of HiRet for genetic experimentation is advantageous as it permits stable, long lasting transgene expression, and is non-toxic to neurons. In this experiment, no GFP labeling was seen in neurons that do not make direct synaptic connections to the injection area. This was also true in previous studies in animals with a thoracic contusion injury25. Additionally, HiRet-GFP was unable to label spinal motor neurons or dorsal root ganglion neurons when injected into the transiently demyelinated sciatic nerve (unpublished observations). Together, these data suggest that HiRet does not readily enter through axons and efficiently transduces neurons by uptake of the vector at synapses, providing a more detailed and high-fidelity map of neuronal connections of the targeted population. This is an advantage over other retrogradely transportable viral vectors such as retrograde adeno-associated virus (rAAV-retro), which is known to be taken up by axons in passage44 and makes HiRet especially useful in studies mapping regenerating and reconnecting circuitry in the injured spinal cord. HiRet’s advantages may also allow for specific targeting of neuronal populations for silencing or ablation studies25,44,45,46,47.
The authors have nothing to disclose.
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).
#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. |