Calcium elevations after neurotrauma are not only implicated in cell death but may contribute to adaptive plasticity. We now wished to resolve this contradiction by following calcium dynamics after optic nerve crush in vivo. Adult rats received no injury (n = 5), unilateral mild (n = 10) or moderate optic nerve crush (n = 10) (ONC), or axotomy (n = 5). Before surgery, retinal ganglion cells (RGCs) were retrogradely labelled with Oregon Green BAPTA-dextran, a fluorescent calcium marker. Calcium-related fluorescence intensity (FI) was repeatedly measured in individual RGCs in vivo using the in vivo confocal neuroimaging (ICON) method. Four different RGC types were found. Normal RGCs without FI change were found in sham rats and also in both ONC groups. RGCs with mild damage were seen only after mild ONC, showing an initial calcium depression of 26% at day 4 followed by a 169% increase 15 days after ONC. RGCs with moderate damage were found only after moderate ONC and showed calcium hypoactivation followed by a slower return toward baseline and a delayed calcium increase of 72% above baseline. Sixty to sixty-five per cent of the RGCs in both ONC groups and all RGCs in the axotomy group died within 6 days following a fast and massive calcium increase of 316% with a concomitant 156% soma size increase. In conclusion rapid calcium elevation leads to cell death, while an initial calcium depression followed by a delayed and moderate calcium hyperactivation is associated with cell survival. We propose that immediate, massive calcium activation is maladaptive whereas delayed and moderate hyperactivation of surviving cells is adaptive. Implications for pharmacotherapy are discussed.

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With in vivo confocal neuroimaging (ICON), single retinal ganglion cells (RGCs) can be visualized non-invasively, repeatedly, in real-time and under natural conditions. Here we report the use of ICON to visualize dynamic changes in RGC morphology, connectivity and functional activation using calcium markers, and to visualize nanoparticle transport across the blood-retina barrier by fluorescent dyes. To document the versatility of ICON, we studied the cellular response to optic nerve injury, and found evidence of reversible soma swelling, recovery of retrograde axonal transport and a difference in calcium activation dynamics between surviving and dying RGCs. This establishes ICON as a unique tool for studying CNS physiology and pathophysiology in real-time on a cellular level. ICON has potential applications in different research fields, such as neuroprotection/regeneration, degeneration, pharmacology, toxicity and drug delivery.

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After damage many surviving RGCs lose their axon transport, but after approximately 3 weeks, this transport recovers again, a sign of intrinsic axon repair. Contrary to the prediction, axon transport recovery is not associated with better cell survival but rather with a second wave of cell death. Thus, the accelerated cell death associated with recovery of axon transport suggests the existence of a late retrograde cell death signal.