Measurement of contractile force of skeletal and extraocular muscles: Effects of blood supply, muscle size and in situ or in vitro preparation

Contractile forces can be measured in situ and in vitro. To maintain metabolic viability with sufficient diffusion of oxygen, established guidelines for in vitro skeletal muscle preparations recommend use of relatively thin muscles (≤1.25 mm thick). Nevertheless, forces of thin extraocular muscles vary substantially between studies. Here, we examined parameters that affect force measurements of in situ and in vitro preparations, including blood supply, nerve stimulation, direct muscle stimulation, muscle size, oxygenated or non-oxygenated buffer solutions and the time after interruption of vascular circulation. We found that the absolute forces of extraocular muscle are substantially lower when examined in vitro. In vitro preparation of 0.58 mm thick extraocular muscle from 3-week-old birds underestimated contractile function, but not of thinner (0.33 mm) muscle from 2-day-old birds. Our study shows that the effective criteria for functional viability, tested in vitro, differ between extraocular and other skeletal muscle. We conclude that contractile force of extraocular muscles will be underestimated by between 10 and 80%, when measurements are made after cessation of blood supply (at 5-40 min). The mechanisms responsible for the declining values for force measurements are discussed, and we make specific recommendations for obtaining valid measurements of contractile force.