Pharmacological Modulation of Hemodynamics in Adult Zebrafish In Vivo

Abstract

Introduction: Hemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo.

Materials and methods: Adult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations.

Results: Under control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 μmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001). Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01) and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03).

Discussion: In this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.

Fig 1. Workflow of routine investigations to measure hemodynamic and cardiac parameters in vivo.

After an anesthesia with 0.04% tricaine, the fish were injected i.p. with 12 μl (1 nmol/L) of the paralyzing agent μ-conotoxin GIIIB (μ-CTX). After paralysis was observed, the fish was adapted to the oral perfusion for 10 min. Thereafter, baseline values have been acquired between t₀ and t₈. Drug solutions were orally administered with the perfusate at 9.5 min.

Fig 2. Electrocardiographic (ECG) measurements to validate heart rate (HR) stability during paralysis.

(A) Placement of the electrodes for the ECG. The reference electrode was coupled to the perfusion needle. (B) Optical measurements of HR of non-treated zebrafish (n = 5). The HR remained constant with deviations of less than ±5%. (C) Example of an ECG recorded during stopped oral perfusion. (D) Representative image of plotted PP-intervals in a non-perfused window.

Fig 3. Optical measurement of heart rates in response to isoprenaline, tricaine and sodium nitroprusside (SNP) in vivo.

The heart rates of the same fish were followed over a period of 30 min at intervals of 2 min. Values obtained between t₁₀ and t₃₀ are expressed as percentage of the baseline (t₀-t₈). Drug solutions were administered at 9.5 min (indicated by the dashed line).

Fig 4. Blood velocity and shear stress measurements in the submental vein.

(A) Ventral view of the head of an adult zebrafish. White arrowheads indicate the submental vein. (B) Still image of a 3 second-movie of the blood flow in the submental vein. (C+D) Blood velocity at the inlet and shear stress plotted in percent as deviation from baseline (t₀-t₈). (C’+D’) Simulations of blood velocity and shear stress performed at the time points designed in C+D.

Fig 5. Hemodynamic response in the submental vein to application of 50 μmol/L Iso and 1 mmol/L SNP.

Blood flow velocity, shear stress and an example of a numerical simulation of (A) control animals, (B) isoprenaline-treated, and (C) SNP-treated animals. Control fish were observed after 10 min of oral perfusion with system water (“before”) and 10 min later (“after”).