Mesenchymal Stem Cells Enhance Nerve Regeneration in a Rat Sciatic Nerve Repair and Hindlimb Transplant Model methods
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Syngeneic Hindlimb Transplantation
Building on the results from the sciatic nerve transection groups, we sought to determine if a similar effect could be detected in animals undergoing hindlimb transplantation. A syngeneic transplant model (Lew to Lew) was first used to isolate the effect of MSCs independent of an alloimmune response. Animals underwent electromyography (EMG), histomorphometry and gait analysis outcomes testing similar to that performed in the nerve transection/repair groups. In electrophysiological testing, rats receiving syngeneic hindlimb transplants had recovery curves with shapes similar to those receiving sciatic nerve repairs. There was improvement in all groups over all time points. Mean normalized CMAP amplitudes at 16 weeks in the tacrolimus-only control, local MSC treatment, and systemic MSC treatment groups were 0.49 ± 0.1, 0.69 ± 0.19, and 0.58 ±...
Methods
Bone marrow-derived MSCs (BM-MSCs) were harvested from the femurs and tibias of euthanized adult Lewis (LEW) and Brown Norway (BN) rats (4-6 weeks old). BM-MSCs were isolated based on their inherent plastic adherence when grown in culture media consisting of DMEM/F12 with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin and 1% Fungizone (unless otherwise mentioned, all products obtained from Gibco, Life Technologies, Carlsbad, California). After 90% confluence, cells were stained with Sytox-Blue (Life Sciences, Carlsbad, California), and for CD29-FITC, CD90-PE, CD45-PerCP, and CD11b/c-AlexaFluor645 (BioLegend, San Diego, California). Labeled cells were sorted based on being CD29 and CD90 double positive and Sytox-Blue, CD45, and CD11b/c triple negative. Immunophenotypic characterization was performed by flow cytometric analysis for the aforementioned cell surf...
Surgical Models
All sciatic nerve transection and repair procedures were standardized and performed by one single highly experienced microsurgeon under aseptic conditions with the aid of an operating microscope (Leica M525 F40 surgical microscope). Of note, the microsurgeon was blinded with regard to the groupings of the rats. All animals were anesthetized with isoflurane using a VetEquip anesthesia machine (VetEquip Inc). The animal was placed in a right lateral position. A gluteal skin incision was made from the sciatic notch to a point proximal to the knee joint. The gluteal muscles were separated to expose the sciatic nerve from the sciatic notch to the point of bifurcation. The nerve was sharply transected at 1 cm proximal to the knee and re-approximated with four interrupted epineurial 10-0 nylon suture using microscopic visualization. All animals in the local injection groups received 5...
Orthotopic Hindlimb Transplantation
Orthotopic hindlimb transplants were standardized and performed from LEW to LEW rats (syngeneic transplant) or BN to LEW rats (allogeneic transplant) by a single highly, experienced microsurgeon under aseptic conditions with the aid of an operating microscope (Leica M525 F40 surgical microscope). Of note, the microsurgeon was blinded with regard to the groupings of the rats. In brief, the femoral nerve, artery, and vein were isolated and divided ensuring adequate length for subsequent anastomoses. The remaining thigh muscle groups as well as the sciatic nerve were transected to expose the mid-portion of the femur. A transverse osteotomy was performed through the femur to complete allograft harvest. The recipient animal was prepared in a similar fashion. Transplantation of the allograft was performed beginning with osteosynthesis of the femur. The femoral vein and then femoral artery w...
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Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
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- Group comparisons of gait indices, stride metrics, or recovery curves
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Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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Evidence quotes (4)
Building on the results from the sciatic nerve transection groups, we sought to determine if a similar effect could be detected in animals undergoing hindlimb transplantation. A syngeneic transplant model (Lew to Lew) was first used to isolate the effect of MSCs independent of an alloimmune response. Animals underwent electromyography (EMG), histomorphometry and gait analysis outcomes testing similar to that performed in the nerve transection/repair groups. In electrophysiological testing, rats receiving syngeneic hindlimb transplants had recovery curves with shapes similar to those receiving sciatic nerve repairs. There was improvement in all groups over all time points. Mean normalized CMAP amplitudes at 16 weeks in the tacrolimus-only control, local MSC treatment, and systemic MSC treatment groups were 0.49 ± 0.1, 0.69 ± 0.19, and 0.58 ± 0.26, respectively ( ). Nerve histomorphometry data failed to demonstrate any significant differences in axonal counts and density for local and systemic MSC treatment groups when compared to controls ( ). Additionally, there were no statistical significant differences in axon diameter, fiber...
Bone marrow-derived MSCs (BM-MSCs) were harvested from the femurs and tibias of euthanized adult Lewis (LEW) and Brown Norway (BN) rats (4-6 weeks old). BM-MSCs were isolated based on their inherent plastic adherence when grown in culture media consisting of DMEM/F12 with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin and 1% Fungizone (unless otherwise mentioned, all products obtained from Gibco, Life Technologies, Carlsbad, California). After 90% confluence, cells were stained with Sytox-Blue (Life Sciences, Carlsbad, California), and for CD29-FITC, CD90-PE, CD45-PerCP, and CD11b/c-AlexaFluor645 (BioLegend, San Diego, California). Labeled cells were sorted based on being CD29 and CD90 double positive and Sytox-Blue, CD45, and CD11b/c triple negative. Immunophenotypic characterization was performed by flow cytometric analysis for the aforementioned cell surface markers plus RT1A and RT1B (rat major histocompatibility complex antigens). The culture-grown BM-MSCs were tested for their ability to differentiate into adipocytes, chondrocytes, and osteoblasts using the StemPro Adipogenesis Differentiation Kit (A10070-01), Chondrogenesis Differentiation Kit (...
All sciatic nerve transection and repair procedures were standardized and performed by one single highly experienced microsurgeon under aseptic conditions with the aid of an operating microscope (Leica M525 F40 surgical microscope). Of note, the microsurgeon was blinded with regard to the groupings of the rats. All animals were anesthetized with isoflurane using a VetEquip anesthesia machine (VetEquip Inc). The animal was placed in a right lateral position. A gluteal skin incision was made from the sciatic notch to a point proximal to the knee joint. The gluteal muscles were separated to expose the sciatic nerve from the sciatic notch to the point of bifurcation. The nerve was sharply transected at 1 cm proximal to the knee and re-approximated with four interrupted epineurial 10-0 nylon suture using microscopic visualization. All animals in the local injection groups received 5 × 10 4 MSCs (reconstituted to a 5 µl volume) injected into the epineurium of the distal nerve stump using a 33-gauge blunt tip needle and custom made 50 µl plunger syringe. All animals in the systemic groups received 1 × 10 6 MSCs injected into the p...
Orthotopic hindlimb transplants were standardized and performed from LEW to LEW rats (syngeneic transplant) or BN to LEW rats (allogeneic transplant) by a single highly, experienced microsurgeon under aseptic conditions with the aid of an operating microscope (Leica M525 F40 surgical microscope). Of note, the microsurgeon was blinded with regard to the groupings of the rats. In brief, the femoral nerve, artery, and vein were isolated and divided ensuring adequate length for subsequent anastomoses. The remaining thigh muscle groups as well as the sciatic nerve were transected to expose the mid-portion of the femur. A transverse osteotomy was performed through the femur to complete allograft harvest. The recipient animal was prepared in a similar fashion. Transplantation of the allograft was performed beginning with osteosynthesis of the femur. The femoral vein and then femoral artery were anastomosed. The sciatic as well as the femoral nerve were approximated with four interrupted epineurial 10-0 nylon sutures. The sciatic nerve was transected 1 cm proximal to the knee joint in all animals. The ventral and dorsal muscle groups were then repaired with 4-0 polyglactin suture...
Machine-readable layer
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