An improved method for studying mouse diaphragm function.

Dysfunction in the contractile properties of the diaphragm muscle contributes to the morbidity and mortality in many neuromuscular and respiratory diseases. Methods that can accurately quantify diaphragm function in mouse models are essential for preclinical studies. Diaphragm function is usually measured using the diaphragm strip. Two methods have been used to attach the diaphragm strip to the force transducer. The suture method is easy to adopt but it cannot maintain the physiological orientation of the muscle fibers. Hence, results may not accurately reflect diaphragm contractility. The clamp method can better maintain diaphragm muscle fiber orientation but is used less often because detailed information on clamp fabrication and application has never been published. Importantly, a side-by-side comparison of the two methods is lacking. To address these questions, we engineered diaphragm clamps using mechanically highly durable material. Here, we present a detailed and ready-to-use protocol on the design and manufacture of diaphragm clamps. Also, we present a step by step protocol on how to mount the diaphragm strip to the clamp and then to the muscle force measurement system. We compared the diaphragm force from the same mouse with both suture and clamp methods. We found the clamp method yielded a significantly higher muscle force. Finally, we validated the utility of the clamp method in the mdx model of Duchenne muscular dystrophy. In summary, the clamp method described in this paper yields reliable and consistent diaphragm force data. This method will be useful to any laboratory interested in performing mouse diaphragm function assay.