Florianne E. Schoot Uiterkamp

Purpose: Optic nerve crush (ONC) is a model for studying optic nerve trauma. Unilateral ONC induces massive retinal ganglion cell (RGC) degeneration in the affected eye, leading to vision loss within a month. A common assumption has been that the non-injured contralateral eye is unaffected due to the minimal anatomical decussation of the RGC projections at the chiasm. Yet, recently, microglia, the brain-resident macrophages, have shown a responsive phenotype in the contralateral eye after ONC. Whether RGC loss accompanies this phenotype is still controversial. Methods: Using the available RGCode algorithm and developing our own RGC-Quant deep-learning-based tool, we quantify RGC’s total number and density across the entire retina after ONC. Results: We confirm a short-term microglia response in the contralateral eye after ONC, but this did not affect microglia number. Furthermore, we cannot confirm the previously reported RGC loss between naïve and contralateral retinas five weeks after ONC induction across the commonly used Cx3cr1 ^creERT2 and C57BL6/J mouse models. Neither sex nor the direct comparison of the RGC markers Brn3a and RBPMS, with Brn3a co-labeling, on average, 89% of the RBPMS ⁺ -cells, explained this discrepancy, suggesting that the early microglia-responsive phenotype does not have immediate consequences on the RGC number. Conclusions: Our results corroborate that unilateral optic nerve injury elicits a microglial response in the uninjured contralateral eye but without RGC loss. Therefore, the contralateral eye should be treated separately and not as an ONC control.

Balanced and dynamic mitochondrial networks are essential for cell survival and function. Mitochondrial networks remodel their connectivity, content and subcellular localization to support optimized energy production under conditions of increased stress. In vivo , stressors can arise from the environment, such as in neuronal injury, or from mutation-induced cellular dysfunction. Cells programmed to identify and respond to these stress signals, like microglia, rely on optimized mitochondrial function, however we know very little about mitochondrial networks of microglia in vivo or their adaptation to environmental or cellular stressors. Here, we define the mitochondrial networks of retinal microglia in physiological conditions in vivo and evaluate network alterations by taking advantage of a microglia-selective mitochondria-labeled mouse model. First, we demonstrate significant differences in the mitochondrial networks of microglia in vivo and in vitro. Then, we induced neuronal injury in the in vivo environment using optic nerve crush, where responsive microglia exhibit more fragmented mitochondrial networks with increased content and perinuclear localization, supporting a state of increased cellular stress. Surprisingly, when we selectively increase cellular stress by knocking out the mitochondria-associated gene uncoupling protein 2 (UCP2), only male UCP2 ^KO microglia establish a hyperfused mitochondrial network after injury, indicating sex differences in microglial stress mitigation. Ovariectomy in UCP2 ^KO females elicits a shift toward the male hyperfused mitochondrial phenotype suggesting that circulating estrogens are a contributing factor to the differences in microglial stress mitigation.