4. DESPLIEGUE DEL PROGRAMA DE MEDIDAS
4.4. Clasificación de las medidas según el subtipo IPH
2.4.1 Introduction
Cardiac function in 10 dpf zebrafish larvae was hypothesized to respond to pharmacological challenges with acetylcholine (ACh) and norepinephrine (NE). All previous toxicological studies in larval zebrafish have examined cardiac function only under unstimulated conditions (Henry et al., 1997; Andreasen et al., 2002a; Antkiewicz et al., 2005; Carney et al., 2006; Yamauchi et al., 2006). However, at least once basic biological study in developing zebrafish has utilized pharmacological tools to assess larval cardiovascular function (Park et al., 2000). Therefore, the goal of these preliminary experiments was to first assess how the conditions used for immobilizing larval zebrafish affected heart function under unstimulated conditions after developmental exposure to AhR ligands such as BaP and TCDD. Second, the ability of heart rate in normal larvae to respond to exogenously applied norepinephrine and acetylcholine was also examined.
2.4.2 In vivo cardiac function measurements
The larvae were prepared in agarose according to methods described by Fritsche et al., (2000). Briefly, zebrafish at 10 dpf were anesthetized in 50 mg/L ethyl 3-aminobenzoate methanesulfonate (MS-222), a common fish and amphibian anesthetic which functions by blocking sodium channels (Wang et al., 1994). The anesthetized larvae were embedded in a lateral position using low melt agarose dissolved in dechlorinated water with 50 mg/L MS-222. The agarose was covered with 1 ml aerated dechlorinated water and fish imaged with an Olympus transilluminated upright microscope equipped
connected to a Canon HV20 high definition digital camcorder. Larvae were imaged using brightfield illumination at 200x magnification. The heart rate was imaged by recording near the trunk of the larvae. Heart rate in beats per minute (bpm) was calculated by counting the number of heart beats in 10 seconds and multiplying by 6.
The effects of the conditions used for immobilization on heart rate were assessed over a time period of 10 minutes. The fish were embedded in agarose as described above and the beating heart was recorded for 10 minutes. Heart rates between treatment groups were assessed at each time point using a repeated measures ANOVA.
Pharmacological challenges were carried out by adding 100 μl of either water (for baseline measurements), acetylcholine (1 and 10 mM), or norepinephrine (1 and 10 mM) to the water on top of the agarose. Before addition of drug, a baseline recording was taken after addition of water and heart rate monitored for 1.5 minutes. Then, acetylcholine was added to produce a final concentration of 1 mM and effects monitored for 1.5 minutes.
Finally, acetylcholine was added again to produce 10 mM and heart rate monitored for an additional 1.5 minutes. Larvae were euthanized at this point and new larvae used to examine effects of norepinephrine in a similar manner.
2.4.3 Effects seen on in vivo cardiac function
The time course experiment indicated that there were no significant changes in unstimulated heart rate in the zebrafish larvae from each treatment group at 10 dpf over the 10 minute observation period (Figure 2.4; p>0.05 for time and treatment in repeated measures ANOVA). There was also no difference observed between groups of larvae exposed developmentally to AhR ligands such as BaP and TCDD (Figure 2.4). In normal larvae, no significant change occurred after the addition of either pharmacological agent ACh or NE at 10 dpf (Figure 2.5).
Figure 2.4: Time course of heart rates in larval zebrafish at 10 days post-fertilization (dpf) embedded in agarose from 0 to 10 minutes. Results are mean ± SEM. Larvae were exposed from fertilization until 4 dpf to aryl hydrocarbon receptor agonists benzo(a)pyrene (BaP; 5000 ng/L) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 ng/L) alone and in combination with antagonists resveratrol (Res; 300 μg/L) and α-naphthoflavone (ANF; 100 μg/L). Data were analyzed using repeated measures ANOVAs, but no significant differences were detected with either time or treatment. n=8-9 fish per treatment group.
Figure 2.5: Heart rates (beats per minute) of normal larval zebrafish at 10 days post-fertilization after treatment with 1 mM and 10 mM norepinephrine or acetylcholine.
Results are mean values. Equivalent volumes of dechlorinated system water were added to the untreated fish instead of drug solution. Arrows indicate addition of pharmacological agents at specified concentrations. n=2 fish per treatment group.
2.4.4 Methodological challenges with in vivo analysis of cardiac function
It was apparent that the unstimulated heart rate did not significantly change over time and the method of immobilization was not lethal. There also appeared to be no need for a stabilization period since the heart rate was fairly constant, therefore heart rate was measured immediately from the time of embedding for at least 2 minutes for all experiments in Chapter 4.
In contrast, the attempt to establish methods to challenge the larval zebrafish heart with drugs was a failure. There are a number of reasons that may have led to the inability to see drug-induced changes in heart rate using this method of measuring in vivo larval cardiac function. The first reason is that the agarose may have been too thick and the drug did not permeate through to the fish. The second reason is that the drugs permeated the agarose but were not absorbed in sufficient amounts by the larvae to produce an effect. The third reason is that the drugs may actually have been oxidized or metabolized before they were able to cause an effect in the fish. Therefore, all subsequent experiments measured only in vivo larval cardiac function under unstimulated conditions. Previous successful experiments using pharmacological tools to manipulate cardiovascular function in agarose-embedded larval zebrafish have used microinjection techniques, which appear to be necessary for success with this technique (Fritsche et al., 2000).