Allotransplanted neurons used to repair peripheral nerve injury do not elicit overt immunogenicity

Abstract

A major problem hindering the development of autograft alternatives for repairing peripheral nerve injuries is immunogenicity. We have previously shown successful regeneration in transected rat sciatic nerves using conduits filled with allogeneic dorsal root ganglion (DRG) cells without any immunosuppression. In this study, we re-examined the immunogenicity of our DRG neuron implanted conduits as a potential strategy to overcome transplant rejection. A biodegradable NeuraGen® tube was infused with pure DRG neurons or Schwann cells cultured from a rat strain differing from the host rats and used to repair 8 mm gaps in the sciatic nerve. We observed enhanced regeneration with allogeneic cells compared to empty conduits 16 weeks post-surgery, but morphological analyses suggest recovery comparable to the healthy nerves was not achieved. The degree of regeneration was indistinguishable between DRG and Schwann cell allografts although immunogenicity assessments revealed substantially increased presence of Interferon gamma (IFN-γ) in Schwann cell allografts compared to the DRG allografts by two weeks post-surgery. Macrophage infiltration of the regenerated nerve graft in the DRG group 16 weeks post-surgery was below the level of the empty conduit (0.56 fold change from NG; p<0.05) while the Schwann cell group revealed significantly higher counts (1.29 fold change from NG; p<0.001). Major histocompatibility complex I (MHC I) molecules were present in significantly increased levels in the DRG and Schwann cell allograft groups compared to the hollow NG conduit and the Sham healthy nerve. Our results confirmed previous studies that have reported Schwann cells as being immunogenic, likely due to MHC I expression. Nerve gap injuries are difficult to repair; our data suggest that DRG neurons are superior medium to implant inside conduit tubes due to reduced immunogenicity and represent a potential treatment strategy that could be preferable to the current gold standard of autologous nerve transplant.

Figure 1. Surgical Procedures.

(A) After skin incision, an 8 mm length of sciatic nerve was excised. (B) A NeuraGen® nerve guide tube of 2.0 mm ID×1.0 cm length was used to provide a physical guide for axons sprouting from the proximal nerve stump to reach the disconnected nerve segment. (C) The rats were sacrificed at 16 weeks post-PNI and sciatic nerves were harvested by excising 1.0 cm outside the NG tube. (D) The tube (top) is distinguishable from the contralateral healthy tissue (bottom) as an enlarged section of the nerve. The contralateral nerve served as a control for each rat. (E) For histological analysis, frozen sections were cut and examined at five locations: nerve proximal (P) and distal (D) to the tube and proximal (Lp), distal (Ld), and middle (Lm) longitudinal regions inside the tube.

Figure 2. Evaluating the health of cultured neurons.

(A) At the end of antimitotic treatment (day 3 and 4), the DRG cells (MAP-2 immunopositive neurons; green) had extended axons, and non-neuronal cells were essentially nonexistent. (B) Schwann cells demonstrated strong GFAP (red) expression and an elongated shape and dipolar morphology at day 2 and 3. 20×, Scale bar: 50 um. DAPI (blue).

Figure 3. Histological analysis of the sciatic nerve 16 weeks post-PNI.

For anatomical terminology, refer to Fig. 1E . (A) Myelination (MBP, green) of the regenerated axons (MAP2, red). Inset show the structure of individual myelinated axon fascicles, enlarged from 40× image. Axial sections 40×, longitudinal sections 20×. Thickness = 15 um. (B) Expression of laminin (green) was consistent with results from Fig. 3A . DAPI (blue), 20×. Thickness = 15 um.

Figure 4. Histological and quantitative analyses of macrophage presence.

For anatomical terminology, refer to Fig. 1E . (A) ED1 IHC shows macrophage (red) location and number in each group in neuronal structure (NF-200, green). Note Sham, Wistar-DRG, NG, and Wistar Schwann represent macrophage presence in increasing order in all tissue sections. DAPI (blue), 20×. Thickness = 15 um. (B) Quantification and statistical analysis of ED1 immunoreactive cells. Macrophage count reflects immunohistochemistry microscopy analysis in Fig. 4A . ANOVA Bonferroni and Tukey multiple comparison post tests determined significant differences in the mean macrophage count in each group: *P<0.05; **P<0.01; ***P<0.001. Both post tests found the same results. Error bars represent ± standard error of the mean.

Figure 5. MHC I immunoreactivity (red) in neuronal structure (NF-200, green).

For anatomical terminology, refer to Fig. 1E . (A) Increased MHC I immunoreactivity were observed in NG, DRG, and Schwann groups compared to the Sham group. DAPI (blue), 20×. Thickness = 15 um. (B) Axon regeneration estimated by NF-200 expressing axons. Axon density in distal stump was calculated and expressed as standard optical density (SOD) for each group. n = 3, 40× image of 10 um sections.

Figure 6. ELISA assays of IFN-γ in serum 2, 4, 6, 8 and 16 weeks post-surgery, respectively.

IFN-γ levels of the allograft Wistar-Schwann group was higher than the Sham group in serum 2 weeks after surgery (P<0.01). For the NG and Wistar-DRG groups, IFN-γ concentrations in serum were not significantly different compared with that in sham control at all time points following PNI surgery. All measurements were done in triplicates. Data are representative of using n = 5, 6, 6, 5 mice in Sham, NG, Wistar-Schwann and Wistar-DRG groups, respectively.