Background: Reducing the migration of leukocytes after secondary damage in the lesion site helps to preserve the spinal cord tissue and improves movement ability. Although their initial penetration rate is increased within 48 to 72 hours and may continue for up to 7 days, their presence and activity should be reduced over time. Due to the ability of nano-sized MSCs-EVs for passing through blood-brain barrier and their immunomodulatory properties, they may be effective to modulate the leukocyte infiltration following spinal cord injury (SCI). Methods: In the present study, hTERT-immortalized human adipose tissue-derived mesenchymal stem cells were used, in which the ectopic expression of human IDO1 gene is stably increased. As a control group, hTERT-MSCs-GFP were applied. To isolate extracellular vesicles (EVs), 72-hour conditioned media were collected under EV-depleted culture conditions. Following the pre-clearing steps, the extraction of small EVs (sEVs) performed based on the ultrafiltration method. Freshly prepared sEVs were mixed with modified chitosan hydrogel under sterile conditions and were kept on ice. Female rats (N=60, ~230 g) were used and divided into five groups including Sham, SCI, Hydrogel, hTERT-MSCs-GFP-EVs, and hTERT-MSCs-IDO1-EVs. Laminectomy of the T10 was performed based on the previously described method. The drake aneurysm clip is placed on the spinal cord for 1 minute. 60 µl of hydrogel or hydrogels containing 100 µg sEVs from control GFP and IDO1 groups were locally injected immediately after SCI. After 72 h and 8 weeks, blood samples were collected and Giemsa staining was done to determine the differential leukocyte counts. Results: No significant differences observed between the groups 72 hours after SCI (P>0.05). In the first 3 days, the highest monocyte counts were reported for IDO1 (4.5±1.52) and GFP –EVs (4.33±1.63) groups. 8 weeks later, monocytes were remarkably decreased in IDO1 (1.78± 1.48) and GFP –EVs (1.83± 1.60) groups. These changes were statistically significant in comparison to the hydrogel (4.67± 1.37) and sham (4.17± 1.94) groups. In addition, MSCs-IDO1-EVs reduced the lymphocyte infiltration in comparison to other groups (8 weeks). Conclusion: The administration of EVs, enriched from the conditioned media of genetically modified MSCs, in the benefit of IDO1 may be an effective approach to manage the inflammatory status and reducing tissue damages following the SCI.

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