Prolonged diaphragm activation using closed loop optogenetic stimulation.

Diaphragm dysfunction in individuals with late stage neuromuscular disease and cervical spinal cord injury is a substantial problem, often resulting in respiratory insufficiencies and ultimately respiratory failure. Therefore, there is a need to develop new methods that can improve diaphragm function in these clinical populations. Here we used an optogenetic method of activating diaphragm myofibers to assess the feasibility of prolonged photostimulation of the diaphragm. Our hypothesis was that closed loop photostimulation can pace the diaphragm over a prolonged period (1 hour) without a decrease in evoked response magnitude. Wild type mice (C57/bl6, n= 5) received an injection of AAV9-CAG-ChR2-mVenus (34 µl, titer: 1.8x1013 vg/ml) to the intrapleural space between the lung and chest wall to target the diaphragm. After an 8-18 week incubation period, diaphragm EMG and respiratory airflow were measured under isoflurane anesthesia. A light stimulus was delivered to the inferior diaphragm surface using a high-power, 472 nm, micro-LED. Photostimulation was delivered in a closed loop fashion using the onset of the inspiratory EMG burst recorded in intact hemi-diaphragm to trigger the stimulus. The photostimulation paradigm consisted of a 250 ms, ramping/decrementing train of light pulses (pulse duration = 0.1 ms, interpulse interval = 0.1 ms). After a 10-minute baseline recording, a unilateral right phrenicotomy was performed to paralyze the right diaphragm. The photostimulation paradigm immediately restored EMG activity to the paralyzed hemi-diaphragm. Further, the magnitude of the evoked diaphragm EMG bursts stayed consistent over the sixty-minute period of closed loop photostimulation, and there was no evidence of a decrement in EMG activation. Post-mortem histology confirmed mVenus expression in diaphragm myofibers. We conclude that stimulation of the diaphragm using optogenetics is effective for up to one hour. This is a first demonstration of prolonged photostimulation of the diaphragm and provides evidence to support the feasibility of this method for the treatment of chronic diaphragm paralysis.