(A-39) Localized Delivery of Indoleamine-2,3-dioxygenase (IDO)-Galectin 3 Fused Enzyme Using Decellularized Rat Sciatic Nerve for Axonal Regeneration in Injured Rat Spinal Cord

Introduction:: Spinal cord injury (SCI) affects over 299,000 patients globally and at present, more than 18,000 new cases arise each year in United State alone. However, there is no treatment for the disease. The presence of neuroinflammation and prominent glial scar formation after SCI hampers the axonal regeneration process. Indoleamine-2,3-dioxygenase (IDO), is a tryptophan-catabolizing enzyme that is upregulated in response to pro-inflammatory cytokines as a feedback mechanism for neutrophils, monocytes, macrophages, and other inflammation-associated cells. In this study, initially IDO enzyme was engineered to have galectin 3 fusion (Gal3), to enhance the tissue localization, mediated by binding of Gal3 to various extracellular matrix, carbohydrates, including glycosaminoglycans, present at site of injury after SCI. The engineered IDO-Gal3 was then incorporated within a decellularized rat sciatic nerve based injectable hydrogel, to slow the release of IDO-Gal3 to surrounding tissue to help resolve persistent neuroinflammatory processes. The combination approach is then studied in moderately contused rat spinal cord injury model, to understand the synergistic effect in promoting axonal regeneration and decreasing microglial and astrocytes mediated neuroinflammation. The localized delivery of IDO-Gal3 from decellularized matrix is a novel approach for combating neuroinflammation and promoting neuronal regeneration in injured rat spinal cord.

Materials and Methods:: IDO was functionalized with Gal3 using our previously published protocol¹. The rat sciatic nerve was isolated and decellularized as per our previously published protocol². In vitro release of IDO-Gal3 from hydrogel was studied, by incorporating IDO-Gal3 in injectable pre-gel solution and solidifying the hydrogel at 37°C. The release of IDO-Gal3 was measured U.V. spectrophotometrically at different time points. Sprague Dawley rats (female, 250-300g) underwent moderately contused SCI at thoracic T9-T11 region (n=6 animals/group), using infinite impactor (150 kDynes force) as per UF IACUC approved protocol. Injury alone, Sham group served as experimental groups for this study. IDO-Gal3 (~2.5 µg) was incorporated into the pregel solution of neutralized digested solution of rat sciatic nerve and then administered using Hamilton syringe at the site of injury after 1 week of injury. The effect of the combinational approach was assessed up to 4 weeks of treatment using perfused paraformaldehyde fixed cryosectioned tissues slices. Immunohistochemistry (IHC) was performed on neuroinflammation markers of astrocytes (GFAP), and microglial cells (CD206, CD86, Arginase-1 and iNOS). The axonal regeneration was measured by staining for β-tubulin III, NF-200 and GAP43. Behavioral outcomes (BBB score and horizontal ladder test) are measured weekly up to 4 weeks of treatment. Results were analyzed using One Way ANOVA, Tukey t test.


Results, Conclusions, and Discussions:: The decellularized rat sciatic nerve based injectable hydrogel was able to release IDO-Gal3 upto 2 days in vitro PBS release media. As shown in Figure 1A, IDO-Gal3+ Hydrogel group animals have lower GFAP expression as compared to injury group, indicating the decrease in astrocytes reactivity. Additionally, IDO-Gal3+Hydrogel treated animals had lower CD86 expression as compared to injured animals, showing a decrease in microglial mediated inflammation (Figure 1B). No significant increase in M2 phenotype (CD206/Arg-1 expression) of macrophages/microglial was seen as compared to injury. Also, IDO-Gal3+ Hydrogel may have increase in neuronal development (increase in β-tubulin III, NF-200 and GAP43 expression) as compared to injury group. IDO-Gal3+Hydrogel have also shown to have better locomotor recovery (improvement in BBB score and horizontal ladder score) as compared to injury group in our preliminary studies. Overall, it was seen that IDO-Gal3 loaded hydrogels were able to decrease the neuroinflammation after SCI and the hydrogel was able to provide pro-regenerative cues for spinal cord regeneration. This study directs the potential of IDO-Gal3 treatment for SCI and vindicates the clinical translation potential to treat SCI in humans.

Acknowledgements (Optional): : NIH-R21NS123
Pruitt Chair Funding

References (Optional): : 1. Farhadi, S.A. et al. Nat.Commun., 2018,9,4943.
2. Bousalis, D. et al., J Biomed Mater Res A., 2022,3,595-611.