MORPHOLOGICAL AND TRANSCRIPTOMIC ANALYSES HIGHLIGHT DIFFERENTIAL REGENERATION KINETICS IN IMMEDIATE <i>VERSUS</i> DELAYED NERVE REPAIR

Peripheral nerve injuries represent a significant clinical challenge due to their limited regenerative capacity. Although immediate repair is ideal, delayed intervention is often unavoidable. Understanding the mechanisms underlying both conditions is essential for optimizing functional recovery. Thus, this study aimed to uncover key pathways in nerve regeneration through the first transcriptomic analysis of regenerating nerves within a conduit, also comparing immediate and delayed nerve repair over time in a rat model. Immediately after injury, or following a delay of three months, microsurgical intervention with a chitosan tube was performed to repair an 8-mm median nerve gap, and regenerated nerves inside the conduit were collected at 14- and 21-days for morphometric analysis, and at 7-, 14-, and 21-days post-repair for RNA sequencing. Morphometric analysis based on absolute values showed a significant reduction in Schwann cell and axonal areas at 14-days, along with a decreased number of blood vessels and an overall smaller section area at 21-days in the delayed group. However, when normalized to the total section area to assess the relative proportions occupied by Schwann cells, axons, and vessels, no significant differences were observed between immediate and delayed groups at 21-days. To correlate morphometric data with transcriptomic profiles, RNA sequencing was conducted. Approximately 25,000 genes were differentially expressed in regenerating nerves compared to healthy controls, mainly related to inflammatory response, phagocytosis, cell signalling, and response to lipoprotein particles. Only 137 genes differed between delayed and immediate repair. Gene ontology analysis showed that the most enriched pathways were involved in angiogenesis, especially at 7-days, in accordance with the higher density of vessel area observed at 14-days. Overall, the comparison between the experimental groups indicated that immediate repair initiated a more rapid regenerative response, while delayed repair followed a slower, but ultimately convergent, trajectory highlighting that regeneration is postponed and partially impaired. Indeed, it is noteworthy that the nerve calibre was hindered in the delayed compared to the immediate repair. These findings underscore the importance of early intervention, but also suggest that, over time, delayed repair might achieve similar regenerative outcomes, especially if novel therapies will be developed to further enhance recovery.