Four neuromuscular electrical stimulation (NMES) paradigms do not result in immediate damage to the sarcolemma.
(A) Experimental overview. At 2 days post-fertilization (dpf), WT siblings and dmd mutants were injected with Evans blue dye (EBD). 4 hr later, zebrafish were imaged for birefringence and EBD before and after a single session of NMES. (B) For NMES, zebrafish are placed in a 3D-printed gym with their heads towards the positive electrode and tails towards the negative electrode. (C, D) NMES delivers a series of square wave pulses that vary in frequency and voltage. We named these paradigms after weightlifting regimes. (E–J) Anterior left, dorsal top, side-mounted birefringence, and EBD fluorescent images. Yellow asterisks denote the same position in embryos before and after NMES. (E) WT sibling control exhibits healthy muscle segments (E1, E2) and no dye entry in the muscle (E1’, E2’) during the first and second imaging sessions. (F) dmd mutant control has significant areas of degenerated muscle (F1) and dye entry (F1’) but no new areas of degeneration or dye entry during the second imaging session (F2, F2’). (G–J) Similar to the dmd mutant control, dmd mutants that receive NMES have significant areas of degenerated muscle and dye entry prior to NMES but no new areas of degeneration or dye entry during following NMES. (K) Quantification of EBD during the first and second imaging sessions.
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