Transcriptomic and morphological characterization reveals distinct regeneration kinetics in immediate versus delayed nerve repair

Peripheral nerve injury is a significant clinical challenge due to its limited regenerative capacity. While immediate repair is generally preferred, delayed repair is often necessary in clinical scenarios where primary intervention is not feasible. Understanding the molecular and genetic mechanisms underlying both conditions is essential for optimizing functional recovery. Thus, this study aimed to investigate key pathways involved in nerve regeneration by conducting the first transcriptomic analysis of a regenerating nerve within a conduit, while also comparing immediate and delayed nerve repair over time in a rat model. Immediately after injury or following a delay of 3 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 repair 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 the immediate repair and delayed repair groups at 21 days. To correlate morphometric data with transcriptomic profiles, RNA sequencing was conducted. When comparing regenerating nerves at different time points with a healthy nerve, 25,596 genes were differentially expressed in the immediate group and 25,868 genes in the delayed repair group. These genes were mainly involved in pathways related to inflammatory response, phagocytosis, cell signaling, and response to lipoprotein particles. However, only 137 genes were differentially expressed when comparing the delayed repair group versus immediate repair groups. Gene ontology analysis revealed that the most enriched pathways were related to angiogenesis, particularly at 7 days, which aligned with the higher vessel density observed at 14 days in the delayed group. Overall, the comparison between the experimental groups indicated that immediate repair initiated a more rapid regenerative response, while delayed repair followed a slower, yet ultimately convergent trajectory, highlighting that regeneration is postponed and partially impaired. Notably, the nerve caliber was reduced in the delayed repair group compared to the immediate repair group. These findings emphasize the importance of early intervention and address a critical gap in the field by providing the first transcriptomic comparison of delayed versus immediate repair within conduits, thereby contributing to the development of novel molecular therapies to enhance recovery.