Optical microscope

A 16-channel single-shank intracortical microelectrode array (A1×16-3mm-50-177-Z16, iridium electrode sites, NeuroNexus Technologies, Ann Arbor, MI, USA) was used to record neural action potentials of the motor cortex (M1). Alternatively, a non-functional silicone implant of the same dimension was used to assess neuroinflammation and microbial composition. In a Faraday cage setup, each MEA to be implanted underwent EIS testing with a Gamry Interface 1010E Potentiostat (Gamry Instruments, Warminster, PA, USA) consisting of each electrode site as the working electrode, a platinum wire as a counter electrode, and an Ag|AgCl electrode stored in KCl reference electrode for measurements. EIS was performed in 1x PBS (pH = 7.4) over a range of 1 to 10⁶ Hz (12 points per decade) with an AC voltage of 50 mV. The impedance magnitude at 1 kHz was used to confirm functionality with expected values between 150 – 550 kHz. Following EIS verification, MEAs were cleaned using 70% ethanol and DI water to remove any residual 1x PBS and optically imaged using a Keyence Optical Microscope (Keyence Corporation, Osaka, Japan) at a magnification of 150x for visual inspection. Non-functional dummy implants were cleaned using the same protocol as functional implants. After cleaning, both implant types were sterilized using cold gas ethylene oxide.

Before implantation, the 16-channel (electrode), single-shank intracortical microelectrode array (A1x16-3 mm-100-177-Z16, iridium electrode sites, NeuroNexus Technologies, Ann Arbor, MI, USA) quality was verified via electrochemical impedance spectroscopy (EIS) testing. In a Faraday cage, each MEA to be implanted underwent EIS testing with a Gamry Interface 1010E Potentiostat (Gamry Instruments, Warminster, PA, USA) consisting of each electrode site as the working electrode, a platinum wire as a counter electrode, and an Ag|AgCl reference electrode for measurements. EIS was performed in 1× phosphate-buffered saline (PBS) (pH = 7.4) over 1 to 10⁶ Hz (12 points per decade) with an AC voltage of 50 mV rms. Device verification was determined by measuring the impedance value at 1 kHz. If a device was above 1 MΩ or below 100 kΩ, it was not used for implantation. Following EIS verification, MEAs were cleaned by dipping in 70% ethanol and DI water to remove any residual 1× PBS and optically imaged using a Keyence Optical Microscope (Keyence Corporation, Osaka, Japan) at a magnification of 150× for visual inspection. MEAs were then sterilized under cold-gas ethylene oxide sterilization for surgical use.

After completion of the MRI at 12 weeks of age, the mice were sacrificed, and their hearts were removed and fixed in formalin. Fixed hearts were sliced in the direction of the short axis of the left ventricle. Heart specimens were embedded in paraffin and sectioned at 5 μm; some sections were stained with hematoxylin and eosin (H&E), and some sections were immunostained. The stained tissues were observed using an optical microscope (Keyence Corporation; Osaka, Japan). H&E staining was performed by immersing samples in a hematoxylin solution (5 min) and alcohol–eosin staining solution (3 min). H&E-stained tissues were dehydrated six times with 100% alcohol and then permeabilized using xylene. Immunostaining was performed using the enzyme–antibody method. Dystrophin rabbit polyclonal antibodies (12715-1-P; Proteintech Group, Inc., Rosemont, IL, USA) were used as the primary antibody; the samples were incubated for 1 h. After washing three times for 5 min each, the samples were incubated with the EnVision+ horseradish peroxidase-conjugated anti-rabbit secondary antibodies (K4003; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min.