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. Overall design: 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.