Early life injury alters spinal astrocyte development

Neonatal injury alters synaptic transmission and plasticity in the spinal superficial dorsal horn (SDH), resulting in aberrant amplification of ascending nociceptive transmission. Astrocytes orchestrate synapse development and function across the CNS and have been shown to play a critical role in the emergence and maintenance of persistent pain. However, very little is currently known about the postnatal development of spinal astrocytes, nor about how the maturation of SDH astrocytes is impacted by early life injury. Here, we used a hindpaw incision model of postsurgical pain in postnatal day (P) 3 mice to elucidate the effects of neonatal injury on the maturation of SDH astrocytes. Three-dimensional morphological analysis of individual astrocytes revealed that incision elicits age-dependent changes to astrocyte structure. At P4, spinal astrocytes in incised mice show increased size and complexity compared to naïve controls. This is reversed at P10 and P24, as astrocytes from incised mice are smaller and less ramified compared to their naïve counterparts. Transcriptomic analysis of spinal astrocytes demonstrated that injury-evoked changes to astrocyte gene expression occur acutely. We found 76 differentially expressed genes (DEGs) at P4, many of which are related to cell motility and cytoskeletal organization (Thbs1, Efemp1, Acta1, Acta2, Tpm2, Fgf14) but very few DEGs at P10 and P24. Lastly, we identified that microglial engulfment of astrocytes occurs in the developing dorsal horn, and that this process is altered by neonatal injury in a sex-dependent manner. These data illustrate, for the first time, that neonatal injury alters the postnatal development of spinal astrocytes. Neonatal mouse pups underwent unilateral hindpaw incision at postnatal day (P)3, or were exposed to isoflurane only as a control. Pups are Aldh1l1-creERT2xSun1-GFP, in which astrocytes are tagged with the nuclear envelope protein Sun1 fused to eGFP (Sun1-GFP). Ipsilateral dorsal horn L3-L5 was harvested 1, 7, or 21 days after incision, corresponding to ages P4, P10, and P24. Homogenate of intact spinal cord nuclei was prepared from each harvested sample and sorted on the basis of GFP+ fluorescence to selectively isolate spinal dorsal astrocyte nuclei from each sample. Nuclear RNA was isolated from the sorted astrocytes for each mouse, and differential gene analysis was performed to identify incision-induced transcriptional changes as well as normal developmental changes across the three developmental ages.

新生儿期损伤可改变脊髓浅层背角（spinal superficial dorsal horn, SDH）内的突触传递与可塑性，导致上行伤害性传递出现异常放大。星形胶质细胞（astrocytes）调控中枢神经系统（central nervous system, CNS）内的突触发育与功能，且已被证实对持续性疼痛的发生与维持发挥关键作用。然而，目前学界对脊髓星形胶质细胞的出生后发育过程，以及早期生命损伤如何影响SDH星形胶质细胞的成熟，仍知之甚少。
本研究采用出生后第3天（postnatal day, P）小鼠的后足切口术后疼痛模型，旨在阐明新生儿期损伤对SDH星形胶质细胞成熟的影响。对单个星形胶质细胞的三维形态学分析显示，切口手术可引发星形胶质细胞结构出现年龄依赖性改变。在出生后第4天（P4），接受切口手术的小鼠脊髓星形胶质细胞的体积与复杂程度均高于空白对照组小鼠；而在P10与P24时，该变化趋势出现反转：手术组小鼠的星形胶质细胞体积更小、分支更少，相较于同年龄段的空白对照组小鼠。
脊髓星形胶质细胞的转录组学分析显示，损伤诱导的星形胶质细胞基因表达变化呈急性发生特征。我们在P4时检测到76个差异表达基因（differentially expressed genes, DEGs），其中多数与细胞运动及细胞骨架组织相关（Thbs1、Efemp1、Acta1、Acta2、Tpm2、Fgf14）；而在P10与P24时，差异表达基因的数量极少。
最后，本研究发现发育中的背角内存在小胶质细胞对星形胶质细胞的吞噬过程，且该过程可因新生儿期损伤出现性别依赖性改变。上述数据首次证实，新生儿期损伤可改变脊髓星形胶质细胞的出生后发育进程。
本研究中，新生小鼠于出生后第3天（P3）接受单侧后足切口手术，对照组小鼠仅暴露于异氟烷环境。实验所用小鼠为Aldh1l1-creERT2xSun1-GFP品系，其星形胶质细胞可通过融合于增强绿色荧光蛋白（enhanced green fluorescent protein, eGFP）的核膜蛋白Sun1（Sun1-GFP）进行标记。分别于切口术后1、7、21天（对应小鼠年龄P4、P10、P24）采集同侧L3-L5段脊髓背角组织。从每份采集的样本中制备完整脊髓细胞核匀浆，基于GFP阳性荧光分选，以选择性分离脊髓背角星形胶质细胞核。从分选得到的星形胶质细胞中提取核RNA，并进行差异基因分析，以识别切口手术诱导的转录组变化，以及三个发育年龄段内的正常发育性转录变化。