Background & Aims
Significant age-dependent alterations in the organization of neuronal circuits in the spinal dorsal horn occur during the early postnatal period. Neonatal tissue damage evokes both short-term and long-term changes in synaptic transmission within the dorsal horn which increase the excitability of the spinal nociceptive network. While astrocytes clearly govern synaptic function across the CNS and contribute to the sensitization of the adult dorsal horn under pathological conditions, the degree to which the structural and functional properties of dorsal horn astrocytes change with age, or as the result of neonatal injury, has never been investigated. This gap in knowledge impedes our understanding of the mechanisms underlying the developmental plasticity of spinal nociceptive circuits. Therefore, we sought to elucidate the effects of neonatal surgical injury on the structure and transcriptional profile of astrocytes in the developing dorsal horn.
Methods
At postnatal day 3 (P3), mice expressing tdTomato in astrocytes (Aldh1l1-CreERT2 x Ai9) received either a unilateral incision of the hindpaw skin and underlying muscle or anesthesia only as a control. We then imaged individual astrocytes in the ipsilateral dorsal horn at P4, P10, and P24 using confocal microscopy. To capture entire astrocytes, we generated 100 ?m thick sections and treated tissue with a clearing medium to allow for accurate 3D reconstruction. Using Imaris software we quantified cell volume, domain volume, and astrocyte filament properties in 3D space. For RNA sequencing experiments, we used mice with green fluorescent protein-expressing astrocyte nuclei (Aldh1l1-CreERT2 x CAG-Sun1/sfGFP) and used INTACT (Isolation of Nuclei Tagged in Specific Cell Types) to isolate astrocyte nuclei at P4, P10 or P24. We then extracted RNA from these nuclei and performed bulk RNA sequencing to identify differentially expressed genes between P3-incised animals and naïve controls.
Results
Morphological analysis demonstrates that spinal astrocytes progressively increase in cell volume, domain volume, and 3D Sholl intersections from P4 to P24. We found that astrocytes in the P3 incision group have reduced cell volume at P24 compared to naïve controls, but not at P4 or P10. For metrics of complexity, astrocytes in the incised group have greater 3D Sholl intersections at P4, but fewer branch points, terminal points, and Sholl intersections at P10 and P24. RNA sequencing of astrocyte nuclei revealed 76 differentially expressed genes (DEG) between naïve and incision groups at P4, 2 DEGs at P10, and 8 DEGs at P24. Among these DEGs, we found that genes coding for extracellular matrix proteins such as Thbs1 and Efemp1 and cytoskeletal genes such as Acta1, Acta2, and Tpm2 are upregulated at P4. Using gene ontology term analysis, we found that genes involved in cell migration, cell motility, and cytoskeletal fiber organization are upregulated in incised animals at P4.
Conclusions
Our data suggest that there are significant changes in astrocyte size, complexity, and transcriptional profile during the first three weeks of postnatal development, and that these changes are impacted by neonatal incision. We found a transient increase in astrocyte complexity 1-day post-incision, followed by a significant decrease in complexity 1 week and 3 weeks post-incision. In conjunction with our morphological findings, RNA sequencing of spinal astrocytes revealed upregulation of genes that relate to cytoskeletal organization and cell migration processes shortly after incision. This study harnesses the power of modern imaging and bioinformatics techniques to identify nuanced changes to astrocyte structure and function after early life trauma which may influence nociceptive processing in the developing spinal cord.
References
1. Kim Y, Ganduglia-Cazaban C, Chan W, Lee M, Goodman DC. Trends in neonatal intensive care unit admissions by race/ethnicity in the United States, 2008–2018. Sci Rep. 2021;11:23795. doi:10.1038/s41598-021-03183-1
2. Walker SM. Long-term effects of neonatal pain. Semin Fetal Neonatal Med. 2019;24(4):101005. doi:10.1016/j.siny.2019.04.005
3. Li J, Baccei ML. Excitatory synapses in the rat superficial dorsal horn are strengthened following peripheral inflammation during early postnatal development. Pain. 2009;143(1-2):56-64. doi:10.1016/j.pain.2009.01.023
4. Li J, Kritzer E, Craig PE, Baccei ML. Aberrant synaptic integration in adult lamina I projection neurons following neonatal tissue damage. J Neurosci Off J Soc Neurosci. 2015;35(6):2438-2451. doi:10.1523/JNEUROSCI.3585-14.2015
5. Allen NJ, Eroglu C. Cell biology of astrocyte-synapse interactions. Neuron. 2017;96(3):697-708. doi:10.1016/j.neuron.2017.09.056
6. Chih-Wei Hsu, Juan Cerda III, Jason M Kirk, Williamson D Turner, Tara L Rasmussen, Carlos P Flores Suarez, Mary E Dickinson, Joshua D Wythe (2022) EZ Clear for simple, rapid, and robust mouse whole organ clearing https://doi.org/eLife 11:e77419
7. Dourson AJ, Willits A, Raut NGR, Kader L, Young E, Jankowski MP, Chidambaran V. Genetic and epigenetic mechanisms influencing acute to chronic postsurgical pain transitions in pediatrics: Preclinical to clinical evidence. Can J Pain. 2022 May 10;6(2):85-107. doi: 10.1080/24740527.2021.2021799. PMID: 35572362; PMCID: PMC9103644.
Presenting Author
Judy J. Yoo
Poster Authors
Topics
- Mechanisms: Biological-Molecular and Cell Biology