Avian Muscular Dystrophy: Muscle Ultrastructure and Histochemistry Following Isaxonine Treatment

Abstract

Due to many similar properties, including high serum creatine phosphokinase (CPK) levels and histological signs of muscle degeneration, inherited muscular dystrophy of the chicken has been used for over 20 years as a model for hereditary muscular dystrophy in humans. Line 413 dystrophic chickens were selected for this drug study because they are homozygous for muscular dystrophy (disease appears with 100% incidence), they have a characteristic elevation in CPK activity, they have extensive signs of muscle autolysis, and they demonstrate a reproducible and measurable onset of functional disability in a flip-test procedure. Since the earliest detectable clinical sign of the disease is the inability of the dystrophic bird to right itself from the supine position, any therapeutic agent which can delay the onset of this condition is usually considered beneficial therapy. In this study we investigated the drug, Isaxonine (N-isopropyl amino pyrimidine phosphate) which has been shown to have a nerve growth-promoting action. Dystrophic chickens (413) were injected daily with Isaxonine (50mg/kg weight) from day 3 exc ovo to day 40 and compared to normal controls (412) and non-injected dystrophics. In addition to the flip test, we measured the effect of this drug on serum CPK levels and muscle histology and ultrastructure. The results of these experiments indicate that 14., 20 and 40 day Isaxonine injected dystrophic animals are greatly improved compared to non-injected dystrophic animals. For example, Figure 1 is an electron micrograph of pectoralis skeletal muscle from a 20-day-old normal chicken injected daily with Isaxonine (CPK 560mU/ml plasma, Flip 25) showing normal ultrastructure. Figure 2 is an electron micrograph of pectoralis skeletal muscle from a 20-day-old uninjected dystrophic chicken (CPK 1370mU/ml plasma, Flip 2) illustrating extensive ultrastructural deterioration. Figure 3 is a representative micrograph of pectoralis skeletal muscle from a 20-day-old dystrophic chicken injected daily with Isaxonine (CPK 840mU/ml plasma, Flip 13). Note that the ultra-structural morphology appears more similar to the normal than to the dystrophic animals. Figure 4 is an electron micrograph of pectoralis skeletal muscle from a 20-day-old dystrophic chicken injected every other day with Isaxonine (CPK 1218mU/ml plasma, Flip 3). The ultrastructure of this treatment group more closely resembles the dystrophic rather than the normal control muscle. As is apparent in these figures and confirmed with light microscopic histologic sections of similar tissue, the daily injected animals appear to have greatly improved muscle ultrastructure compared to the dystrophic or every-other-day injected animals. Even though the mode of action of Isaxonine is unknown at present, it does appear that it must be injected daily to have therapeutic value and that cessation of drug administration results in the dystrophic phenotype reappearing. Finally, in another dystrophic strain of chickens (304), we have found Isaxonine to be beneficial, as measured by a 50% reduction in serum CPK levels after 20 days of drug administration.