Insulin-like growth factor-1 and cardiotrophin 1 increase strength and mass of extraocular muscle in juvenile chicken

Abstract

Purpose: Insulin-like growth factor 1 (IGF1) and cardiotrophin 1 (CT1) are known to increase the strength of extraocular muscles in adult and embryonic animals, but no information is available for the early postnatal period, when strabismus treatment in humans is most urgent. Here the authors sought to determine whether these trophic factors strengthen juvenile maturing extraocular muscles and gain insight into mechanisms of force increase.

Methods: After two injections of IGF1, CT1, or both with different dosages in posthatch chickens, the authors quantified five parameters of the superior oblique extraocular muscle at 2 weeks of age: contractile force, muscle mass, total myofiber area, myofiber diameter, and number of proliferating satellite cells labeled by bromodeoxyuridine.

Results: Treatment with IGF1, CT1, and combination of IGF1 and CT1 significantly increased contractile force by 14% to 22%. CT1 and combination treatment significantly increased muscle mass by 10% to 24%. IGF1/CT1 combination treatment did not have additive effects on strengthening muscles, compared with single-drug treatments. Myofiber area increased significantly with IGF1 and CT1 treatment in proximal, but not distal, parts of the muscle and this was due to increased fiber numbers or length (IGF1) or increased diameters of global layer myofibers (CT1). Trophic factors increased the number of proliferating (bromodeoxyuridine-labeled) satellite cells in proximal and middle segments of muscles.

Conclusions: Exogenous IGF1 and CT1 strengthen extraocular muscles during maturation. They predominantly remodel the proximal segment of juvenile extraocular muscles. This information about muscle plasticity may aid the design of pharmacologic treatment of strabismus in children during the "critical period" of oculomotor maturation.

Figure 1.

Tracing of twitch and tetanic tension of superior oblique muscles from 14-day-old chickens elicited by stimulation of the trochlear nerve. (A) Tracings of isometric twitch tension elicited by a single pulse (15-V, 0.2-ms duration) of a control muscle (left) and an IGF1 (0.5 μg)-treated muscle (right). (B) Tracing of tetanic tension elicited by stimulation with a train of pulses (15-V, 0.2-ms pulse duration, 450-Hz frequency, 200-ms train duration) of a control muscle (left) and an IGF1 (0.5 μg)-treated muscle (right).

Figure 2.

Quantification and comparison of three muscle parameters, twitch tension, tetanic tension, and muscle mass, for eight different conditions of the superior oblique muscle in 14-day-old chickens. Conditions include normal (N), injection of phosphate-buffered saline (PBS), injection of low-dose (0.5 μg) IGF1 (I-l), high-dose (5 μg) IGF1 (I-h), low-dose (0.5 μg) CT1 (C-l), high-dose (5 μg) CT1 (C-h), combination of low-dose IGF1 plus low-dose CT1 (I-l+C-l), and combination of low-dose IGF1 plus high-dose CT1 (I-l+C-h). Error bars, SEM; n = 5–10 animals per data point. *Significant difference (P < 0.05) compared with controls. There was no significant difference between normal and PBS-injected muscles. (A) All trophic factors except for low-dose CT1 increased twitch tension, with low-dose IGF1, high-dose CT1, and low-dose IGF1 plus high-dose CT1 being statistically significant compared with PBS controls. (B) All treatment regimens with trophic factors except for low-dose CT1 increased maximal tetanic tension significantly. (C) High-dose CT1 and IGF1 plus CT1 combination treatment significantly increased muscle mass compared with PBS control.

Figure 3.

Representative examples of the histology of transverse sections through the normal superior oblique muscle of 14-day-old chickens at three levels: (A) proximal segment, (B) middle segment, (C) distal segment (attached to the eyeball). Semithin sections (1 μm) from resin-embedded muscles were stained with 0.7% toluidine blue. (D) Approximate levels of transverse sections in a schematic drawing of the muscle (P, proximal; M, middle; D, distal). In the normal muscle, the middle segment had the largest cross-sectional and myofiber area, whereas the distal segment had the smallest (see Fig. 4A). (E) Example of the orbital-global layer transition zone from a muscle stimulated at tetanic fusion frequencies. (F) Example of the orbital-global layer transition zone from an unstimulated muscle. OL, orbital layer; TZ, transition zone; GL, global layer. Scale bars: 100 μm (A–C); 10 μm (E, F).

Figure 4.

Quantification of three muscle parameters: average myofiber area, average myofiber diameter, and average myofiber number in transverse sections from three segments (proximal, middle, distal) in normal and IGF1- or CT1-treated superior oblique muscle. (A) IGF1 and CT1 treatment significantly increased myofiber area in the proximal segment. (B) CT1 treatment increased myofiber diameter in the proximal segment. (C) IGF1 treatment significantly increased myofiber number in the proximal segment, and CT1 treatment showed a trend toward increased myofiber numbers in the middle and distal segments. Error bars, SEM; n = 3–4. *Statistically significant difference compared with normal (P < 0.05).

Figure 5.

Comparison of the distribution of myofiber diameters in proximal segments of normal and IGF1-treated (A) or normal and CT1-treated (B) or comparison between IGF1- and CT1-treated superior oblique muscles of 14-day-old chickens (C). The first peak (6–15 μm) corresponds largely with orbital layer fibers, and the second peak (17–33 μm) with global layer fibers. (A) IGF1 increases the percentage of large diameter fibers but also shows larger numbers of smaller myofibers (possibly new or elongated fibers; see Fig. 4C). (B) CT1 increases the percentage of the larger myofibers (diameters > 22 μm) more than the percentage of the smaller myofibers (diameters = 8–11 μm), thus resulting in a net increase in the average fiber diameter (Fig. 4B). (C) Direct comparison of IGF1 and CT1 shows a very similar effect, with minimal differences in percentages of fiber diameters. Error bars, SEM; n = 3–4. *Statistically significant difference (P < 0.05).

Figure 6.

(A) Example of bromodeoxyuridine (BrdU) immunolabeling in longitudinally sectioned myofibers of a CT1-treated superior oblique muscle from a 14-day-old chicken. (B) The average number of BrdU-positive nuclei per 100 μm myofiber from three segments (proximal, middle, distal). Both IGF1 (5 μg) and CT1 (5 μg) treatments significantly increased the number of BrdU-positive nuclei in proximal and middle segments. Error bars, SEM; n = 5–7. *Statistically significant difference (P < 0.05).