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

Abstract

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 (< or =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.

Fig. 1

Examples of isometric twitch tension of the chicken superior oblique muscle (A, B) and lateral gastrocnemius muscle (C, D) examined at post-hatch day 2 (P2) and P21. The superior oblique muscle was stimulated via the trochlear nerve and the gastrocnemius muscle was stimulated via the tibial nerve. Force of twitch tension increased with animal age in both muscles, the superior oblique and gastrocnemius.

Fig. 2

A and B. Influence of age (muscle thickness), method of stimulation, and blood supply on the contractile force of the chicken superior oblique muscle. Time course of twitch tension in superior oblique muscle elicited (A) via the trochlear nerve or (B) by direct muscle stimulation. Animals were examined at post-hatch day 2 (P2) or P21 with blood flow intact (Control) or without blood flow in the presence of non-oxygenated Krebs buffer (No Blood) or oxygenated Krebs buffer (No Blood/O2 Krebs). Note the substantial decrease in force within 10–15 min of ischemia. Error bars = SEM. n = 4–7 animals per data point.

Fig. 3

Influence of blood supply on contractile force of the chicken superior oblique muscle. Time course of tetanic tension in superior oblique muscle elicited via the trochlear nerve. Animals were examined at post-hatch day 21 (P21) with blood flow intact (Control) or without blood flow in the presence of oxygenated Krebs buffer (No Blood/O2 Krebs). Note the substantial decrease in force within 15 min of ischemia. Error bars = SEM. n = 3 animals per data point.

Fig. 4

A and B. Influence of age (muscle thickness), blood supply, oxygenated and non-oxygenated Krebs buffer, and method of stimulation on the contractile force of the chicken lateral gastrocnemius muscle. Time course of twitch tension in lateral gastrocnemius muscle elicited (A) via the tibial nerve or (B) by direct muscle stimulation. Animals were examined at post-hatch day 2 (P2) or P21 with blood flow intact (Control) or without blood flow in the presence of non-oxygenated Krebs buffer (No Blood) or oxygenated Krebs buffer (No Blood/O2 Krebs). Note the substantial decrease in force with nerve stimulation after 15–20 min of ischemia. Error bars = SEM. n = 4–7 animals per data point.