SCI Recovery Reinvented: Targeting Inflammation through Fused Enzyme Indoleamine-2,3-Dioxygenase and Galectin3 with Pro-Regenerative Hydrogel Delivery

Introduction: : Spinal cord injury (SCI) is a debilitating central nervous system injury that affects more than 300,000 Americans[1]. Chronic inflammation undermines axonal regeneration attempts, as neurons initially spared from injury undergo degeneration through inflammatory secondary injury cascades. In this study, we studied the effect of galectin-3 fused indoleamine 2,3 dioxygenase enzyme (IDO-Gal3) delivered via a decellularized rat peripheral nerve-based hydrogel (iPN) in a thoracic contusion SCI model. Localized delivery of IDO-Gal3 significantly decreased astrocyte and macrophage reactivity, mitigating neuroinflammation at the site of injury. Ex vivo magnetic resonance imaging revealed that the combination of injectable hydrogel and IDO-Gal3 preserved descending corticospinal tracts as compared to IDO-Gal3 or hydrogel alone. Although no statistical difference in overall locomotor recovery was observed, rats treated with IDO-Gal3 in the hydrogel delivery vehicle exhibited more coordinated steps, plantar movement and correct partial paw placement. Overall, this study suggests that the combination of IDO-Gal3 and hydrogel is a promising strategy to curb neuroinflammation and promote axonal regeneration following SCI.

Materials and

Methods: : Indoleamine 2,3 dioxygenase (IDO) was fused with Galectin-3 (Gal3) using a previously established protocol[2]. Sprague Dawley rat sciatic nerves were harvested aseptically and decellularized according to our previously published protocol[3]. The decellularized nerves were digested with pepsin to form an injectable pregel solution which when neutralized and warmed to 37 °C crosslinks into a stable hydrogel (iPN). Sprague Dawley rats (female, 250-300 g) underwent moderate contusive SCI at thoracic T9-T11 (n=6 animals/group), using an Infinite Horizons Impactor (150 kDynes force) as per UF IACUC approved protocol. Injury only, Sham (laminectomy only), IDO-Gal3, Hydrogel (iPN), and IDO-Gal3 within hydrogel (iPN + IDO-Gal3) served as the experimental groups for this study. Full treatment of iPN + IDO-Gal3 is intended to have a synergistic effect in modulating the immune response (IDO-Gal3) while simultaneously providing pro-regenerative cues (iPN). Two different doses of IDO-Gal3 (~2.3 and ~3.9 μg) were incorporated into the pregel solution and administered using a Hamilton syringe at the site of injury 1-week post-injury (within sub-acute phase). MRI and diffusion tension imaging (DTI, n=3/group) were conducted on ex vivo spinal cord tissue isolated after 4 weeks of treatment. Immunohistochemistry (n=5/group) was performed on paraformaldehyde-fixed cryosectioned tissue slices for various neuroinflammation markers: astrocytes (GFAP), microglial/macrophages cells (CD206+, CD86, Arginase-1, and iNOS), and axonal regeneration (β-tubulin III, NF-200, and GAP43). Locomotor recovery was assessed using Basso, Beattie, and Bresnahan's (BBB) score and Horizontal Ladder test measured weekly up to 5 weeks post-injury. Results were analyzed using One Way ANOVA followed by Tukey t test.

Results, Conclusions, and Discussions:: Full treatment animal groups of iPN + IDO-Gal3 showed significantly lower GFAP expression as compared to the injury only group, indicating a decrease in astrocyte reactivity (Figure 1a). An increase in pro-regenerative macrophage populations was seen in groups with higher amounts of IDO-Gal3 (Figure 1b). Additionally, iPN + IDO-Gal3 treated animals had lower CD25 expression as compared to IDO-Gal3 only animals, suggesting an increase in regulatory T cell recruitment (Figure 1c). Although behavioral improvements were observed in the iPN + IDO-Gal3 group, they did not reach statistical significance. We did, however, see an increase of 55.8% in weight-supported steps in our full treatment animal group (iPN + IDO-Gal3) as compared to injury alone (Figure 2). MRI and DTI imaging showed that the higher dose of iPN + IDO-Gal3 (3.9 μg) has better neuronal and axonal sparing as compared to the injury alone, hydrogel alone, and IDO-Gal3 alone group (Figure 3). Overall, our results show that localized IDO-Gal3 delivered via our injectable hydrogel decreases neuroinflammation and promotes axonal regeneration in moderately contused SCI rodent models. This study highlights a novel biomaterial with clinical translation potential for SCI.

Acknowledgements and/or References (Optional):: [1] National Spinal Cord Injury Statistical Center, Traumatic Spinal Cord Injury Facts and Figures at a Glance. Birmingham, AL: University of Alabama at Birmingham, 2024.
[2] Farhadi et al., Locally anchoring enzymes to tissues via extracellular glycan recognition, Nature Communications, 2018.
[3] Bousalis et al., Decellularized peripheral nerve as an injectable delivery vehicle for neural applications, J Biomed Mater. Res., 2021.