The effects of hypercarbia on intracellular pH, cardiac function and metabolism in Danio rerio embryos, using microfluidic chip technology

Abstract

O2 consumption (MO2), heart rate and changes in intracellular pH (pHi) were measured in developing embryos of the zebrafish (Danio rerio) at different stages of development following the onset of tissue differentiation. Despite some extensive research in adult fish species, the capacity for pH regulation and its effects on physiology have scarcely been explored in embryonic fish. The rapid development and ease of in vivo examination of critical tissue in the zebrafish embryo make this species an ideal model for use in addressing this topic. Embryos were exposed to a severe hypercarbia challenge of 5% CO2 (~35 mmHg) to induce a severe respiratory acidosis. Microfluidic lab-on-chips (LOCs) were used to enable the hydrodynamic positioning of embryos in a single addressed location which allowed traceable, repeated measures on individual embryos as well as a high level of control of perfused solutions. The single excitation/dual emission intracellular pH indicator dye SNARF-1 was loaded into D. rerio embryos aged 24 and 48 hours post fertilisation (hpf) via diffusion assisted by the addition of pluronic. Images of the heart and white muscle tissue were acquired, during normocarbia and following 30 minutes exposure to hypercarbia, using laser scanning confocal microscopy. These images were analysed for changes in pHi as indicated by a change in the fluorescence ratio, calculated from the selected dual emission spectra. The heart rate of D. rerio embryos 24 and 48 hpf was video recorded in vivo during normocarbia, after 30 minutes exposure to hypercarbia and after a recovery of 30 minutes return to normocarbia. MO2 was measured using an OXY-4 mini 4 channel oxygen meter with PSt3 fiber optic oxygen mini sensors (PreSens, Germany) during normocarbia and following 30 minutes exposure to hypercarbia. Zebrafish embryos experienced a significant acidosis in heart and white muscle tissue at both 24 and 48 hpf. This was found to be associated with a significant chronotropic impairment of the cardiac tissue, with heart rate decreasing from 79± 1.7 beats per minute (bpm) to 64± 2.4 bpm at 24 hpf and from 135± 2.5 bpm to 121± 2.3 bpm at 48 hpf. In addition to this response, upon return to normocarbia for 30 minutes, heart rate was found to increase significantly to frequencies actually above those exhibited pre-exposure, indicating no persistent damage to cardiac tissue. Recorded MO2 responses were novel, with MO2 increasing significantly following hypercarbic exposure in embryos aged 24 hpf but decreasing significantly in embryos aged 48 hpf suggesting that the strategy employed to respond to a respiratory acidosis is highly age dependent. This is the first study to provide empirical evidence that tissue performance impairment is associated with pHi perturbation, but not persistent damage, in vivo, in an aquatic, vertebrate model.