An Anesthesia-Free Pharmacological Assay for Zebrafish Heart Failure Models Using a Microfluidic Platform

Background: The zebrafish model is extensively utilized for phenotypic screening, particularly in heart failure research. However, traditional photography-based data collection requires tricaine methanesulfonate (MS-222) anesthesia, which can suppress cardiac function and introduce artifacts. This study presents a microfluidic platform designed to facilitate anesthesia-free, noninvasive in vivo photography of zebrafish heart failure models. Methods: Isoproterenol (ISO) induced heart failure in 1 day post-fertilization (dpf) zebrafish embryos. Larvae were assigned to three groups: Control (E3 embryo medium only), Model (1 mM ISO for 48 h from 1 dpf), and Positive (1 mM ISO + 10 μM propranolol for 48 h from 1 dpf). Subsequently, a microfluidic chip featuring tapered immobilization channels was adopted. The chip facilitated the photography and collection of cardiac function parameters from lateral and supine positions, including cardiac output (CO), blood flow velocity (BFV), ejection fraction (EF), stroke volume (SV), and fractional shortening (FS). All imaging was performed in completely independent parallel experiments, with no individual larva shared between the two imaging methods. Data obtained under hydrodynamic confinement were compared with those acquired under MS-222 anesthesia. Results: Cardiac parameters measured using the microfluidic platform were significantly higher than those under MS-222 anesthesia: CO (0.339 ± 0.032 vs. 0.279 ± 0.023 nL/s, p < 0.001), BFV (681 ± 53 vs. 604 ± 38 μm/s, p < 0.001), EF (10.3 ± 0.7% vs. 6.6 ± 0.7%, p < 0.001), SV (184,519 ± 12,822 vs. 97,979 ± 5306 μm³, p < 0.001), and FS (7.3 ± 1.8% vs. 3.7 ± 0.9%, p < 0.001). Strong correlations (R² > 0.85, p < 0.0001) revealed that anesthesia underestimated parameters by 11.3-49.3%. Conclusions: Compared with the traditional anesthesia-based method, the microfluidic platform enables more precise cardiac parameter collection in a zebrafish heart failure model than when anesthesia is used, offering a reliable tool for phenotypic screening of zebrafish embryos.