This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.
Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.
Spinal cord injury repair is a complex and challenging problem that will require a combinatorial treatment strategy involving, for example, the use of cells and a biomaterial to provide a favorable microenvironment for transplanted cell function and axon regeneration at the site of injury. Hemisection1,2,3,4,5,6,7,8,9 and complete transection10,11,12,13,14,15,16,17,18,19,20,21,22 models are frequently used to assess the effects of biomaterial-based bridging therapies. The advantage of using a hemisection model is that it provides more stability for the bridging construct compared to complete transection. However, in hemisection models, it is difficult to prove axon regeneration as an outcome of the applied therapeutic method due to the presence of spared tissue. The complete transection model is the most rigorous method to demonstrate axon regeneration.
Various natural and synthetic materials have been studied for use as an injectable gel, a pre-formed gel placed in contusion or hemisection models, or as a structured conduit into hemisection or complete transection models (detailed in the reviews23,24,25). In situ gelling of an injectable matrix/SC mixture creates a more permissive interface between the transplant and the host cord for axon crossing26,27 compared to pre-gelled matrix/SC implants5,18,19,28. In situ gelling allowed the matrix to contour around the irregular host interfaces whereas a more rigid and structured conduit or a less moldable pre-formed gel could not. A structured conduit often provides contact guidance and implant stability in contrast to an injectable matrix. The protocols presented here describe a surgical procedure that takes advantage of both an injectable basement membrane matrix (e.g., matrigel, see the Table of Materials, referred to as injectable matrix here) and a structured conduit to evaluate axon regeneration in the most rigorous spinal cord injury model.
Electrospun poly-vinylidenedifluoride-trifluoroethylene (PVDF-TrFE) aligned fibrous hollow conduits are used in our experimental approach. PVDF-TrFE is a piezoelectric polymer that generates a transient charge when mechanically deformed and has been shown to promote neurite extension and axon regeneration both in vitro29,30 and in vivo31. Electrospinning is a common scaffold fabrication method that can rapidly produce reliable fibrous scaffolds using a variety of polymers with controllable properties such as fiber alignment, fiber diameter, and thickness of the scaffold for neural and other applications32,33,34.
Numerous studies of rat SCs transplanted into spinal cord injury sites have demonstrated treatment efficacy5,9,18,19,20,21,26. These transplants are neuroprotective for tissue surrounding the lesion, reduce lesion cavity size, and promote axon regeneration into the lesion/transplant site and myelination of the regenerated axons. Human SCs can be autologously transplanted, an advantage when compared to most other neural-related cells24. After a peripheral nerve biopsy, SCs can be isolated and purified and will proliferate to the desired amount for transplantation into humans. Autologous SC transplantation for spinal cord injured patients has been proven to be safe in Iran35,36,37,38, China39,40, and the United States41,42. SCs are known to secrete numerous neurotrophic factors and extracellular matrix proteins important for axon growth and to play an essential role in axon regeneration after peripheral nerve injury. Our goal here is to describe methods which can investigate conduit designs to improve the outcome of SC transplantation in a complete rat spinal cord transection model.
Female adult Fischer rats (180 – 200 g body weight) are housed according to NIH and USDA guidelines. The institutional Animal Care and Use Committee (IACUC) of the University of Miami approved all animal procedures.
1. Pre-Transplantation Preparation
2. Complete Transection at Thoracic Level 8 (T8)
3. Conduit insertion
4. Schwann Cell/ Injectable Matrix (see Table of Materials) Preparation and Injection
5. Wound Closure and Postoperative Care
The goal of using this surgical technique is to evaluate the use of a structured conduit and injectable matrix that maximizes SC function after transplantation into completed transected spinal cords. Three weeks after transplantation, the animals are perfused with 4% paraformaldehyde and the spinal columns are grossly dissected and fixed in the same fixative for another 24 h. The spinal cord is then dissected and the samples for cryostat sagittal sections are placed into a 30% sucrose solution for cryoprotection. 1-mm thick cross sections isolated from the middle of the SC bridge of another set of dissected spinal cords are placed into glutaraldehyde fixative to process for plastic sections. The samples are subjected to a schedule for electron microscopic preparation as detailed by Bates et al.47. Cryostat sagittal sections were immunostained with primary antibody against GFP, glial fibrillary acidic protein (GFAP), heavy chain neurofilament (RT97), and medium chain neurofilament (NF) followed by secondary antibodies including goat-anti-chicken-488, goat-anti-rabbit-546, goat-anti-mouse-647, and goat-anti-rabbit 647, respectively. GFP-SCs were distributed evenly along and within the conduit (Figure 3A; inner walls indicated by yellow lines). Axon regeneration is observed (Figure 3B) and is closely associated with the presence of GFP-SCs (Figure 3A and Figure 3C) indicating that this technique is successful in providing a SC bridge within a structured conduit and in promoting axon regeneration along the bridge between the rostral and caudal stumps. Blood vessels and myelinated axons were also found in the center of the SC bridge (Figure 3D). More details of the effect of SCs with electrospun PVDF-TrFE fibrous conduits on axon regeneration can be found in our recent published work31.
Other outcome measures can be performed including: quantifying axon regeneration in sagittal sections by the line-transect-method described in our recent work31 and quantifying the number of myelinated axons and vessels in plastic cross sections. Samples prepared for plastic sections can be further sectioned for transmission electron microscopy to quantify the number of unmyelinated axons. The cryoprotected spinal cord samples can also be cross-sectioned instead of sagittally sectioned and immunostained to quantify axon regeneration and the presence of GFP-SCs. Behavioral tests can also be performed on the animals at appropriate intervals while the animals are being maintained.
Figure 1: Window preparation in the conduit. Fold one side along the longitudinal axis of the 5 mm conduit (A). Cut four incisions about 0.4 mm long and at least 1 mm from the openings of the conduit (B). Each incision is about 1 mm apart. By unfolding the conduit, the 4 parallel cuts are observed (C). Cut between the two incisions along the rostral-caudal axis to create a window that can be opened by lifting the flap. Please click here to view a larger version of this figure.
Figure 2: Window closing after SC/DMEM/F12/matrix injection. Windows are opened by folding back each flap after placing the conduit between rostral and caudal stumps (A). Windows are closed after injection (B). Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 3: Confocal fluorescent images of sagittal sections and a bright field image of a plastic cross-section from spinal cords transplanted with GFP-SCs in aligned fibrous PVDF-TrFE conduits. Overview of the SC bridge within the conduits where the inner walls are indicated by the yellow lines and immunostained for GFP and glial fibrillary acidic protein (GFAP) to visualize transplanted SCs and host spinal cord astrocytes, respectively. Regenerated axons were labeled with RT97 (heavy-chain neurofilament) (A,B) and medium-chain neurofilament (NF, C) antibodies. Axons are not as visible in (A) due to their close association with GFP-SCs as is observed in the mid-conduit region in (C) with higher magnification. Axons are not visible in the rostral stump due to the incomplete scan in the depth of the tissue section when imaging by confocal microscopy. Blood vessels (labeled as v in D) and myelinated axons (labeled as MA in D) were both observed in the mid-conduit region in a plastic section. Magnifications and scale bars: A,B (10x, 200 µm); C (20x, 100 µm); D (63x, 25 µm). Please click here to view a larger version of this figure.
The most critical step in creating an effective transection model is severing the spinal cord in one or two cuts. A 2-2.5 mm gap between the rostral and caudal spinal cord stumps should be present at the transection site. The three most likely reasons for such a gap not appearing are (1) the dorsal/ventral roots were not removed properly, (2) the ventral dura was not removed adequately, and/or (3) the animal was not positioned properly on the roll placed beneath her.
To perform an effective conduit insertion between the stumps: the (1) diameter of the conduit should be tailored for the specific species and age of the animal used in the experiment; (2) laminae must be removed laterally enough until the gap between the transverse processes can be visualized; (3) roots must be removed and (4) conduit insertion between the stumps should be accomplished on the first try. If multiple attempts are needed, this may cause edema in the stumps, further complicating the task of conduit insertion between the stumps and causing additional injury.
To perform effective GFP-SC/DMEM/F12/injectable matrix introduction into the conduit, ensure that: (1) the spinal cord is not bleeding before conduit insertion between the stumps. If there is fluid in the conduit after insertion between the stumps, use a small piece of absorption spear to remove it via the pre-cut windows. (2) Ensure that the conduit is slipped onto both spinal cord stumps well and (3) that the dorsal pre-cut windows are open. The injection of 20 µl of the SC/injectable matrix mixture should be more than enough to fill the conduit. Overflow from the pre-cut windows is a good gauge for effective transplantation.
The limitations of this procedure are: (1) no restoration of the dura, (2) no control over the movement of the conduit/SC transplant after completion of the surgery, and (3) no alternative method if there is leakage while injecting the SC/injectable matrix mixture.
The advantage of this procedure is the combination of the use of both a structured conduit with an injectable matrix. Any conduit made with a material of choice that is pliable to be inserted between the stumps can be used in this surgical procedure. Any injectable matrix of choice can also be used in this procedure; a temperature-sensitive gel is preferable due to its ability to gel in situ and contour to the shape of the stump creating a seamless interface. Conduits with a more complex interior structure can be used instead of a hollow interior. Drugs, growth factors, small molecules, or any cell type can be incorporated into the structured conduit or the injectable matrix or both to enhance transplant survival, provide neural protection immediately after injury, reduce inflammation, promote axon regeneration, and promote angiogenesis.
The authors have nothing to disclose.
We would like to thank the Viral Vector and Animal Cores at the Miami Project to Cure Paralysis for producing the lenti-GFP-virus and providing animal care, respectively, and the Histology and Imaging Cores for the use of the cryostat, confocal microscope, and fluorescent microscope with Stereo Investigator. Funding was provided by NINDS (09923), DOD (W81XWH-14-1-0482), and NSF (DMR-1006510). M.B. Bunge is the Christine E Lynn Distinguished Professor of Neuroscience.
Cryogenic vials | ThermoFisher Scientific | 5000-0020 | |
10 cm Petri dish | VWR | 25382-428 | |
Dulbecco's modified Eagle's medium: nutrient mixture F-12 | ThermoFisher Scientific | 11039-021 | "DMEM/F12" in protocol. |
Penicillin-streptomycin | ThermoFisher Scientific | 15140-122 | "Pen/Strep" in protcol. |
Fetal bovine serum | Hyclone | SH300-70-03 | "FBS" in protocol. |
Pituitary extract | Biomedical Technologies | BT-215 | |
Forskolin | Sigma-Aldrich | F6886 | |
Heregulin | R&D Systems | 396-HB/CF | |
Poly L-lysine | Sigma-Aldrich | P2636 | "PLL" in protocol. |
Dulbecco's modified Eagle's medium | ThermoFisher Scientific | 11965-092 | "DMEM" in protocol. |
Hank's balanced salt solution | ThermoFisher Scientific | 14170-112 | "HBSS" in protocol. |
Tryspin-EDTA | ThermoFisher Scientific | 15400-054 | |
Female Fischer rat (160-180g) | Envigo | ||
Vannas scissor, straight | FST | 15018-10 | |
Ketamine | Vedco Inc | 5098976106 | 100 mg/ml |
Xylazine | Lloyd Inc | AnaSed | 20 mg/ml |
Gentamycin | APP Pharmaceuticals | NDC 63323-010-02 | Can be any brand of choice. |
Micro Spatula | FST | 10089-11 | Can be any brand of choice. |
Curved scissors with blunt end | FST | 14017-18 | Can be any brand of choice. |
Blunt forceps | FST | 11006-12 | Can be any brand of choice. |
rongeur | FST | 16121-14 | Can be any brand of choice. |
Angled spring scissors | FST | 15006-09 | Can be any brand of choice. |
Absorption triangles | FST | 18105-03 | Can be any brand of choice. |
Gelfoam | Henry Schein | 9083300 | "Compressed foam" in protocol. |
#10 blades | Sklar | 06-3010 | Can be any brand of choice. |
Matrigel | Corning | 354234 | "Injectable matrix" in protocol. |
Chicken anti-green fluorescent protein antibody | Millipore | AB16901 | |
Mouse RT97 hybridoma antibody | DSHB | RT97 | |
Rabbit anti-neurofilament antibody | Encor Biotechnology, Inc | PRCA-NF-H | |
Polyclonal Rabbit anti-Glial Fibrillary Acidic Protein antibody | Dako | Z033401 | |
Alexa Fluor 488 goat anti-chicken IgG (H+L) | ThermoFisher Scientific | A-11039 | |
Alexa Fluor 546 goat anti-rabbit IgG (H+L) | ThermoFisher Scientific | A-11035 | |
Alexa Fluor 647 goat anti-rabbit IgG (H+L) | ThermoFisher Scientific | A-21244 | |
Alexa Fluor 647 goat anti-mouse IgG (H+L) | ThermoFisher Scientific | A-21236 | |
Confocal Microscopy | Nikon | clsi |