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-?B 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. Overall design: 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.

在成年哺乳动物中，脊髓损伤（spinal cord injury, SCI）后失调的程序性细胞死亡（programmed cell death, PCD）会通过引发神经元丢失、加重炎症反应及破坏组织修复过程，损害再生能力。然而，PCD通路间存在复杂的相互调控作用，这要求建立针对SCI的特异性全面细胞死亡（pan-PCD）分析框架，以量化PCD动态变化并筛选治疗靶点。本研究发现，与成年个体不同，新生哺乳动物因PCD反应处于平衡状态而具备更优异的再生能力，因此可作为模拟pan-PCD水平以促进再生的理想模型。

为此，我们构建了“Thanatoset”——一款pan-PCD基因组合，可全面解析SCI后的细胞死亡动态变化。在多种细胞类型中，小胶质细胞被鉴定为pan-PCD的关键介导因子，且在成年小鼠中表现出更高的易感性。通过计算机药物筛选，我们筛选得到化合物Withaferin A（WFA），其可将小胶质细胞的PCD重编程为新生样再生状态。体内外实验均证实，WFA能够调控NF-κB信号通路，减轻炎症反应、改善运动功能、减少瘢痕形成并促进神经元恢复。上述研究结果凸显了调控pan-PCD以恢复成年个体脊髓损伤后再生能力、改善SCI预后的治疗潜力。

整体实验设计：
新生C57BL/6小鼠（出生后第2天，P2）采用异氟烷进行麻醉，成年雌性C57BL/6小鼠（体重18~20 g）则通过腹腔注射3%戊巴比妥钠溶液（40 mg/kg）实现麻醉。脊髓压迫损伤模型的构建参照既往报道的方法：于胸段（T9-10）位置进行椎板切除术，充分侧方暴露脊髓；随后使用5号Dumont镊子（Fine Science Tools）在镊子尖端宽度为0.1 mm的末端5 mm区段压迫脊髓。假手术组仅实施椎板切除术，不进行脊髓损伤操作。术后护理包括涂抹双氯芬酸钠软膏以镇痛，并使用加热垫维持动物体温过夜；新生小鼠需用母鼠粪便覆盖后放回母鼠饲养笼，每日手动排空膀胱直至小鼠被处死。

新生C57BL/6小鼠（出生后第2天，P2）采用异氟烷进行麻醉，成年雌性C57BL/6小鼠（体重18~20 g）则通过腹腔注射3%戊巴比妥钠溶液（40 mg/kg）实现麻醉。脊髓压迫损伤模型的构建参照既往报道的方法：于胸段（T9-10）位置进行椎板切除术，充分侧方暴露脊髓；随后使用5号Dumont镊子（Fine Science Tools）在镊子尖端宽度为0.1 mm的末端5 mm区段压迫脊髓。假手术组仅实施椎板切除术，不进行脊髓损伤操作。术后护理包括涂抹双氯芬酸钠软膏以镇痛，并使用加热垫维持动物体温过夜；新生小鼠需用母鼠粪便覆盖后放回母鼠饲养笼，每日手动排空膀胱直至小鼠被处死。

损伤后1、3、7天，取损伤区域1 cm组织进行转录组测序。