Localized Delivery of Indoleamine-2,3-dioxygenase (IDO)-Galectin 3 Fused Enzyme Using Decellularized Rat Sciatic Nerve for Immune-modulation and 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 the United States alone. However, to date, no treatment is available to promote locomotor recovery in SCI patients. The presence of neuroinflammation and prominent glial scar formation mediated by reactive astrocytes and macrophages/microglial cells after SCI hampers the axonal regeneration process, limiting functional recovery. 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. The potential of IDO for SCI treatment is unexplored. 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 the site of injury after SCI. IDO-Gal3 was incorporated within pro-regenerative injectable hydrogel fabricated using a decellularized rat sciatic nerve (Schematic Figure 1). The potential of localized delivery of IDO-Gal3 using decellularized rat peripheral nerve-based injectable hydrogel to decrease neuroinflammation and promote axonal regeneration and locomotor recovery was assessed in the rat spinal cord injury model. Overall, the combinatorial approach of IDO-Gal3 and the injectable hydrogel is a novel approach to ameliorating neuroinflammation and promoting axonal regeneration at the SCI site.

Materials and

Methods: IDO was fused with Gal3 using our previously published protocol 1. The rat sciatic nerve was isolated and decellularized according to our previously published protocol 2. The decellularized nerves were digested using pepsin enzyme to form an injectable hydrogel, and a pre-gel solution was made. Sprague Dawley rats (female, 250-300 g) underwent moderately contused SCI at thoracic T9-T11 region (n=8 animals/group), using infinite impactor (150 kDynes force) as per UF IACUC approved protocol (Schematic Figure 1). Injury alone, Sham (laminectomy only) group, IDO-Gal3 only, Hydrogel only, and IDO-Gal3 within hydrogel (IDO-Gal3 + Hydrogel) served as experimental groups for this study. Two different doses of IDO-Gal3 (~2.5 and ~6 µg) were incorporated into the pregel solution of neutralized digested solution of rat sciatic nerve and then administered using a Hamilton syringe at the site of injury after 1 week of the injury (within sub-acute phase). MRI and diffusion tension imaging (n=3/group) were conducted on ex vivo spinal cord tissue isolated after 4 weeks of treatment. Immunohistochemistry (IHC) (n=5/group) was performed on paraformaldehyde-fixed cryosectioned tissue slices for various neuroinflammation markers; astrocytes (GFAP), and microglial/macrophages cells (CD206, CD86, Arginase-1, and iNOS). The axonal regeneration was measured by staining for 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 of injury. Results were analyzed using One Way ANOVA, Tukey t test.

Results, Conclusions, and Discussions: The major lacunae for IDO enzyme delivery are rapid enzyme dissipation from the site of injury. Initially, using immunostaining, we saw that IDO-Gal3 delivered using developed injectable hydrogel was able to retain at the SCI site. The presence of Gal3 enabled the enzyme to bind to local proteoglycan and retain the enzyme at the SCI site. This localized IDO-Gal3 was then able to modulate neuroinflammation at the SCI site. As shown in Figure 1, IDO-Gal3+Hydrogel group animals have significantly lower GFAP expression as compared to the injury group, indicating a decrease in astrocyte reactivity. Additionally, IDO-Gal3+Hydrogel treated animals had lower CD86 expression as compared to injured animals, showing a decrease in microglial-mediated inflammation. The decrease in astrocyte reactivity mediated by IDO-Gal3 and the presence of pro-regenerative cues afforded by injectable hydrogel also allowed axonal development as evident in Figure 2. IDO-Gal3+Hydrogel has a significant increase in neuronal development (increase in β-tubulin III, NF-200, and GAP43 expression) as compared to the injury group. Additionally, as shown in Figure 3, MRI and DTI imaging shows that as compared to the injury group, IDO-Gal3 + Hydrogel has better neuronal and axonal development as compared to the injury alone, hydrogel alone, and IDO-Gal3 alone group. The decrease in neuroinflammation and axonal development at the SCI site leads to functional locomotor recovery. As shown in Figure 4, IDO-Gal3+Hydrogel has shown better locomotor recovery (higher BBB score and horizontal ladder score) as compared to the injury group. Overall, our results show that localized IDO-Gal3 delivered using our injectable hydrogel has been shown to decrease neuroinflammation and promote axonal regeneration in moderately contused SCI model. This study provides a novel biomaterial, that has a clinical translation potential to treat SCI in humans.

Acknowledgements (Optional): The authors would like to thanks National Institute of Health, NIH R21NS123, Pruitt Chair Funding

References:
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