Negative Control FLuc Reporter Lentivirus

The failure of the spinal cord to regenerate after injury is primarily due to growth inhibition caused by the injury-induced inflammatory response, which cannot be alleviated, leading to secondary injury and cell death. Alleviating the inflammatory response by attenuating the inflammatory response and promoting a shift in macrophages toward an anti-inflammatory phenotype, thereby preventing inhibition, is essential to create a microenvironment that favors growth. Inducing the expression of anti-inflammatory factors through viral gene delivery has the potential to modify the host inflammatory response through local delivery. Here, it was shown that bridge implantation reduced antigen-presenting cell infiltration at day 7, while the addition of lentivirus to the bridge induced a transient increase in spinal cord neutrophils at day 7 and macrophages at day 14. Delivery of lentivirus encoding IL-10, an anti-inflammatory factor that inhibits immune cell activation and polarizes the macrophage population toward an anti-inflammatory phenotype, reduced neutrophil infiltration at days 7 and 28. Although the IL-10 lentivirus did not affect macrophage numbers, it skewed the macrophage population toward an anti-inflammatory M2 phenotype and altered macrophage morphology. Furthermore, IL-10 delivery improved motor function, suggesting reduced secondary injury and increased protection. These results suggest that local expression of anti-inflammatory factors such as IL-10 can modulate the inflammatory response after SCI and may be a key component of a combinatorial approach targeting multiple barriers to regeneration and functional recovery.

Axon number and myelination were quantified at day 28 to assess whether IL-10 overexpression was detrimental, as has been reported in the peripheral nervous system. At 28 days post SCI, myelinated (NF200+/MBP+) and unmyelinated (NF200+/MBP-) axons were seen throughout the bridges in animals that received no lentivirus, FLUC-encoding control lentivirus, or IL-10-encoding lentivirus (Figure 1A-D). NF200+ axons were often observed in bundles, as previously reported for multichannel PLG bridges. Empty bridges had approximately 800 neurites/mm² and both lentiviral conditions had approximately 1100 neurites/mm². While the data suggest that the density of regenerating axons tends to increase with lentiviral delivery, these differences were not statistically significant. Similarly, the percentage of axons that were myelinated (22-34%) or that were derived from Schwann cells (15-35%) did not vary between conditions (Figure 1E).

Figure 1. Myelinated axons 4 weeks after injury. (Margul D J, et al., 2016)