Adaptive changes of the ground squirrel retina during hibernation

National Eye Institute

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Abstract

Hibernation confers extraordinary protection against various forms of stress and insults that would be life-threatening to non-hibernators. However, the mechanisms of such promising protection remain elusive, hindering potential therapeutic applications. One of the hallmarks of hibernation is metabolic regulation, such as modifications of in mitochondrial respiration throughout the different stages of hibernation. Nonetheless, the possible link between metabolic regulation and cellular protection is unclear. In the last report, we documented that hibernating ground squirrels are remarkably protected against axonal injury-induced neuronal death. Here we explore the possible mechanism for such neuroprotection. To understand why retinal ganglion cells (RGCs) in active and torpid ground squirrels (GSs) responded differently to the same axonal injury, we performed RNA sequencing analyses on samples of injured optic nerves (from nerve-head to chiasm). We then categorized genes enriched in major cell types in the optic nerve (neurons, astrocytes, microglia/macrophages, oligodendrocytes, and oligodendrocyte precursor cells). Among differentially expressed genes (DEGs), we found, in active animals, most upregulated genes (58.8%; 80/136) were enriched in microglia/macrophages, including many associated with microglia/macrophage activation such as CD68, CD74, C1QB, and TREM2. This suggests a differential innate immune response to ONC in active vs. torpid GS. We then directly examined microglia/macrophages at the ONC injury site. In active GS samples, we found massive aggregation of IBA1+ cells, or myeloid cells (since IBA1 does not differentiate between resident microglia or infiltrating monocytes). Moreover, these cells were positive for CD68 labeling and adopted a round, ameboid shape, indicating that they are activated. In stark contrast, such IBA1+ cell aggregation was absent in samples from torpid GSs. Instead, a cell-sparse region at the crush site was apparent. These findings corroborate the transcriptomic analysis indicating differential macrophage/microglial responses at the ONC site between active and torpid GS. However, RGC cell bodies are located in the retina, some distance (up to several mm) away from the crush site where inflammatory responses may differ. Therefore, we further examined the reaction of IBA1+ cells to ONC within the retina. There, we refer them as microglia for simplicity, as it has been documented that ONC does not appear to trigger peripheral monocyte infiltration in the retina. In retinal samples from active GS following pONC, along with the progressive RGC loss, we observed a progressive accumulation of microglial cells and a gradual increase in their CD68 expression. In contrast, in samples from torpid animals, there was little RGC loss, and neither microglia aggregation nor CD68 upregulation were observed. Intriguingly, we observed that post-ONC microglia extend elaborate processes across the RGC layer to run parallel with astrocytic processes and form numerous close contacts, and many microglia even migrate into the nerve fiber layer. Although the nature of microglia-astrocyte interaction remains unclear, this observation lends strong anatomical support for an interplay between these two cell types after axonal injury. We will further investigate why microglial responses to ONC are muted in torpid GS.