The origins, spatiotemporal distribution and dynamic changes of A1 and A2 astrocytes after spinal cord injury

The dual functions of astrocytes are an indispensable part of nerve damage and repair after spinal cord injury (SCI). A recent definition of neurotoxic A1 and neuroprotective A2 astrocytes may be important for these roles. Increasing evidence has also shown that A1 astrocytes can transform into A2 astrocytes. However, the origins, spatiotemporal distribution and dynamic changes of A1 and A2 astrocytes after SCI remain poorly understood. Through transgenic mice and lineage tracing, we investigated the origins of A1 and A2 astrocytes after SCI. Juvenile and adult mouse SCI models, primary astrocyte culture, real-time PCR and immunofluorescence staining were used to observe the spatiotemporal distribution and dynamic changes in A1 and A2 astrocytes. RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq) and bioinformatic analysis were used to reveal the dynamic changes in A1 and A2 astrocyte transcripts and the signaling pathways regulating their transformation. We found that resident astrocytes produced both A1 and A2 astrocytes, whereas ependymal cells regenerated only A2 astrocytes in the lesion area. Furthermore, A1 astrocytes were predominantly activated in adult mice with SCI, and A2 astrocytes were hyperactivated in juvenile mice with SCI, and this relationship was closely associated with the prognosis of SCI. Importantly, we observed that A1 and A2 astrocytes had a dynamic transformation process at different times, and a majority of intermediate states of A1 and A2 astrocytes were found during transformation in vitro and vivo. RNA-seq and scRNA-seq results further confirmed that the transcripts of A1 astrocytes and their lipid toxicity were gradually increased with time and age. In contrast, A2 astrocyte transcripts increased as early as the first day to the third day after injury and then gradually decreased. Bioinformatics analysis further revealed the dynamic changes in several signaling pathways regulating A1 and A2 astrocyte transformation. Our results provide insight into the origins, spatiotemporal distribution and dynamic changes of A1 and A2 astrocytes after SCI, which will be valuable resources to further target A1 and A2 astrocytes and for therapeutic research after SCI.