Oxygen reperfusion damage in an insect

Abstract

The deleterious effects of anoxia followed by reperfusion with oxygen in higher animals including mammals are well known. A convenient and genetically well characterized small-animal model that exhibits reproducible, quantifiable oxygen reperfusion damage is currently lacking. Here we describe the dynamics of whole-organism metabolic recovery from anoxia in an insect, Drosophila melanogaster, and report that damage caused by oxygen reperfusion can be quantified in a novel but straightforward way. We monitored CO(2) emission (an index of mitochondrial activity) and water vapor output (an index of neuromuscular control of the spiracles, which are valves between the outside air and the insect's tracheal system) during entry into, and recovery from, rapid-onset anoxia exposure with durations ranging from 7.5 to 120 minutes. Anoxia caused a brief peak of CO(2) output followed by knock-out. Mitochondrial respiration ceased and the spiracle constrictor muscles relaxed, but then re-contracted, presumably powered by anaerobic processes. Reperfusion to sustained normoxia caused a bimodal re-activation of mitochondrial respiration, and in the case of the spiracle constrictor muscles, slow inactivation followed by re-activation. After long anoxia durations, both the bimodality of mitochondrial reactivation and the recovery of spiracular control were impaired. Repeated reperfusion followed by episodes of anoxia depressed mitochondrial respiratory flux rates and damaged the integrity of the spiracular control system in a dose-dependent fashion. This is the first time that physiological evidence of oxygen reperfusion damage has been described in an insect or any invertebrate. We suggest that some of the traditional approaches of insect respiratory biology, such as quantifying respiratory water loss, may facilitate using D. melanogaster as a convenient, well-characterized experimental model for studying the underlying biology and mechanisms of ischemia and reperfusion damage and its possible mitigation.

Figure 1. Entry into, and recovery from, a single bout of anoxia.

Typical effects of 60 minutes of anoxia on the CO₂ emission rate (VCO₂; black), water loss rate (WLR; red) and activity (green; no units shown) of a male Drosophila melanogaster, mass 0.916 mg, at 25°C. B = baselines. A = initiation of anoxia. N = return to normoxia, S = secondary CO₂ peak after reperfusion to normoxia. Note the increase in VCO₂ after recovery from anoxia (recovery is evident in the activity trace).

Figure 2. Effects of anoxia duration.

The relation between anoxia duration and A) the volume of the secondary peak of CO₂ emission after reperfusion to normoxia (measured by integration of the secondary peak against the background level of the primary peak, using sloping baselines). The secondary peak attains a maximum value after 30 minutes of anoxia exposure. Anoxia duration significantly affected the secondary peak areas (F_{4, 32} = 12.73, P<10⁻⁶); B) the minimum level of water loss rate (WLR) attained after reperfusion to normoxia, which is inversely related to the integrity of spiracular function. The longer the exposure to anoxia, the less adequately the spiracular control system recovered (F_{5, 42} = 9.94, P<10⁻⁵); C) the time required to resume voluntary activity (recovery). The longer the exposure to anoxia, the longer the time required for recovery. The dimensionless slope of the line is 0.399 (F_1,36 = 169.0, P<10⁻⁶). Each point shown is the mean value for the number of flies (out of 8) that recovered; these are 8 for all anoxia durations except 90 minutes (5 recovered) and 120 minutes (only one recovered). Error bars are standard errors. The curves enclose the 95% confidence limits of the line fitted to data pooled by anoxia duration.

Figure 3. Effects of an oxygen reperfusion event followed by anoxia.

Typical effects of 60 minutes of anoxia, interrupted by A) 1 minute of reperfusion with normoxia after 30 minutes, or B) multiple, 1 minute durations of reperfusion with normoxia, on the CO₂ emission rate (VCO₂; black), water loss rate (WLR; red) and activity (green; no units shown) of a male Drosophila melanogaster at 25°C. B = baselines. A = initiation of anoxia. R = reperfusion for 1 minute. N = return to normoxia. E = excretion events in the water vapor trace (red) after return to normoxia (approximately 1.75 and 2 hours); note the fast rise-times of these events.

Figure 4. Effects of multiple oxygen reperfusions.

The effect of successive reperfusions on A) water loss rate (WLR which is inversely related to spiracular control integrity). Successive reperfusions rapidly elevated water loss rates, and thus diminished spiracular control integrity (F_{5, 30} = 19.91, P<10⁻⁶). B) the volume of CO₂ released by mitochondrial activity. Successive reperfusions rapidly reduced mitochondrial activity (F_{5, 30} = 19.91, P<10⁻⁶). Considered as a linear regression using the points shown, reperfusion number explained >95% of mitochondrial activity variance (F_{1, 3} = 62.0, P = 0.004). The curves enclose the 95% confidence limits of the fitted line. The five reperfusions lasted for 60 seconds each and were spaced 20 minutes apart. Each point shown is the mean value for 6 flies. Error bars are standard errors.