Hypoxia protects horizontal cells from low glucose-induced Ca²⁺ dysregulation in the anoxia-tolerant goldfish

Abstract

Purpose : Intracellular Ca²⁺ concentration [Ca²⁺]_i rises in neurons during ischemia or hypoxia, and induces cell death. The retina of anoxia-tolerant fish from the genus Carassius is resistant to hypoxia, but the underlying mechanisms at the cell level have not been investigated. Horizontal cells (HCs) of goldfish (C. auratus) exhibit spontaneous Ca²⁺ action potentials (APs), which may provide insights into HC Ca²⁺ dynamics during ischemia in the anoxia-tolerant retina. We tested the hypotheses that during hypoxia or ischemia Ca²⁺ APs in goldfish are reduced, and baseline [Ca²⁺]_i is maintained, in a novel in vitro model.

Methods : Dissociated HCs were loaded with the Ca²⁺ indicator, Fura-2, and superfused for 40 min with HEPES-buffered extracellular solution (ECS). HCs were treated for 20 min with hypoxic ECS (N=6), glucose-free ECS (N=6), oxygen-glucose-deprivation (OGD) ECS (N=10), or no treatment (N=10). Baseline [Ca²⁺]_i was monitored and spontaneous Ca²⁺ transients were analyzed for amplitude, time-to-peak, area-under-the-curve (AUC), duration, and frequency. The fold-change for each parameter was obtained by normalizing data to pre-application values. The Kruskal-Wallis test and Dunn’s post test were used for statistical analysis. Means ± S.E.M. are presented.

Results : Baseline [Ca²⁺]_i was significantly higher following administration of 0 glucose ECS, and was 3.2±0.6-fold higher than controls (p<0.01). Interestingly, when 0 glucose was combined with hypoxia during OGD application, baseline was unaffected. Hypoxia alone had no effect upon [Ca²⁺]_i baseline. Moreover, AP frequency was significantly affected (p<0.05). 0 glucose ECS abolished APs in nearly all cells, but frequency was preserved in hypoxic (p<0.05) and OGD conditions (p<0.05) over 0 glucose ECS. No significant changes in other parameters were observed.

Conclusions : Our results are consistent with the hypothesis that [Ca²⁺]_i baseline is maintained in OGD and hypoxic conditions, but do not support the hypothesis that Ca²⁺ APs are down-regulated in hypoxic or OGD conditions. The data suggest that glucose deprivation (which occurs during ischemia) causes Ca²⁺ dysregulation in goldfish HCs, but hypoxia preserves low [Ca²⁺]_i in spite of the concurrent low-glucose challenge. Further research will be needed to clarify the mechanisms by which hypoxia maintains Ca²⁺ homeostasis in the anoxia-tolerant goldfish.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.