Simulating Neonatal Microglial Pan-Programmed Cell Death to Unlock Regenerative Potential in Adult Mice Spinal Cord Injury [RNA-seq]

In adult mammals, dysregulated programmed cell death (PCD) following spinal cord injury (SCI) impairs regeneration by causing neuronal loss, exacerbating inflammation, and disrupting tissue repair. However, the complex interplay of PCD pathways necessitates the establishment of an SCI-specific diverse cell-death (pan-PCD) framework to quantify PCD dynamics and identify therapeutic targets. Here, we found that, unlike adults, neonatal mammals exhibit superior regenerative capacity due to a balanced PCD response, making them a valuable model for mimicking pan-PCD levels to promote regeneration. To address this, we introduce "Thanatoset," a pan-PCD gene panel, to comprehensively profile cell death dynamics in SCI. Among various cell types, microglia were identified as key mediators of pan-PCD, exhibiting heightened susceptibility in adult mice. Through computational drug screening, we identified Withaferin A (WFA), a compound capable of reprogramming microglial PCD to a neonatal-like regenerative state. In vitro and in vivo experiments demonstrated that WFA modulates NF-κB signaling, reducing inflammation, improving motor function, decreasing scar formation, and facilitating neuronal recovery. These findings underscore the therapeutic potential of modulating pan-PCD to restore regeneration and improve SCI outcomes in adults. Neonatal C57BL/6 mice (P2) were anesthetized using isoflurane, while adult female C57BL/6 mice (weighing 18–20 g) were anesthetized via intraperitoneal injection of 3% sodium pentobarbital solution (40 mg/kg). Spinal cord crush injury was performed as previously described. Briefly, a laminectomy was performed at the thoracic level (T9-10) to fully expose the spinal cord laterally. The spinal cord was then crushed using No. 5 Dumont forceps (Fine Science Tools) at the final 5 mm portion of the forceps, with a tip width of 0.1 mm. In the sham-operated group, only the laminectomy was performed without subsequent spinal cord injury. Postoperative care included the application of diclofenac sodium ointment for analgesia and the use of a heating pad to maintain the animals' body temperature overnight. Neonatal mice were returned to the maternal cage after being covered with maternal mouse feces. Manual bladder expression was performed daily until the mice were euthanized. Neonatal C57BL/6 mice (P2) were anesthetized using isoflurane, while adult female C57BL/6 mice (weighing 18–20 g) were anesthetized via intraperitoneal injection of 3% sodium pentobarbital solution (40 mg/kg). Spinal cord crush injury was performed as previously described. Briefly, a laminectomy was performed at the thoracic level (T9-10) to fully expose the spinal cord laterally. The spinal cord was then crushed using No. 5 Dumont forceps (Fine Science Tools) at the final 5 mm portion of the forceps, with a tip width of 0.1 mm. In the sham-operated group, only the laminectomy was performed without subsequent spinal cord injury. Postoperative care included the application of diclofenac sodium ointment for analgesia and the use of a heating pad to maintain the animals' body temperature overnight. Neonatal mice were returned to the maternal cage after being covered with maternal mouse feces. Manual bladder expression was performed daily until the mice were euthanized.After injury, take 1cm of the injury area for transcriptome sequencing on 1, 3, and 7 days.