Gail Schwartz

Treadmill training promotes axon regeneration in injured peripheral nerves.

Physical activity after spinal cord injury promotes improvements in motor function, but its effects following peripheral nerve injury are less clear. Although axons in peripheral nerves are known to regenerate better than those in the CNS, methods of accelerating regeneration are needed due to the slow overall rate of growth. Therefore we studied the effect of two weeks of treadmill locomotion on the growth of regenerating axons in peripheral nerves following injury. The common fibular nerves of thy-1-YFP-H mice, in which a subset of axons in peripheral nerves express yellow fluorescent protein (YFP), were cut and repaired with allografts from non-fluorescent littermates, and then harvested two weeks later. Mice were divided into groups of low-intensity continuous training (CT, 60 min), low-intensity interval training (IT; one group, 10 reps, 20 min total), and high-intensity IT (three groups, 2, 4, and 10 reps). One repetition consisted of 2 min of running and 5 min of rest. Sixty minutes of CT resulted in the highest exercise volume, whereas 2 reps of IT resulted in the lowest volume of exercise. The lengths of regenerating YFP(+) axons were measured in images of longitudinal optical sections of nerves. Axon profiles were significantly longer than control in all exercise groups except the low-intensity IT group. In the CT group and the high-intensity IT groups that trained with 4 or 10 repetitions axons were more than twice as long as unexercised controls. The number of intervals did not impact axon elongation. Axon sprouting was enhanced in IT groups but not the CT group. Thus exercise, even in very small quantities, increases axon elongation in injured peripheral nerves whereas continuous exercise resulting in higher volume (total steps) may have no net impact on axon sprouting.

Sexual Dimorphism in the Rabbit Masseter Muscle: Myosin Heavy Chain Composition of Neuromuscular Compartments

The myosin heavy chain (MyHC) isoform composition of six adult (>7 months old) male and female rabbit masseter muscles was studied using seven monoclonal antibodies. In matched serial tissue sections, muscle fibers in 10 different neuromuscular compartments were analyzed. Nearly all fibers were found to express one of five phenotypes. They either contained one of four different slow/beta MyHC phenotypes (I1–I4), nearly all of which co-express cardiac alpha MyHC, or they contained type IIa MyHC. Very few fibers contained slow/beta or cardiac alpha MyHC only or both the alpha/slow/beta and IIa isoforms. Most, but not all, of the compartments studied contained similar proportions of fibers of the five major phenotypes, at least within sex. For 7 of the 10 compartments studied, significant sex differences in the proportion of I1 and IIa fibers were found. Males contained more IIa fibers and fewer I1 fibers than females. Fibers of the IIa phenotype were significantly larger than fibers of all of the other phenotypes and larger in males than females.

Investigation of sexual dimorphism in the rabbit masseter muscle showing different effects of androgen deprivation in adult and young adult animals.

To evaluate the role played by androgens in the development and maintenance of sex differences in the proportion of muscle fibres of different phenotypes, the effects of castration in adult (>6 months old) and in young adult (2-3 months old) male rabbits was examined. Immunohistochemical methods were used to evaluate the proportion of muscle fibres containing different myosin heavy-chain isoforms in 10 different neuromuscular compartments of the masseter. In young adult animals of both sexes, the proportion of fibres of different phenotypes in different compartments was not significantly different from that of normal adult females. In animals castrated as young adults, the development of adult male phenotype proportions was completely blocked in most compartments. In animals castrated as adults, proportions were not significantly different from those of the intact males. For most masseter compartments, androgens produced permanent changes in muscle fibre phenotype during a critical period of postnatal development. However, in the posterior deep compartment, androgen deprivation in young adults had no effect on phenotype proportions, but castration of adults resulted in a striking increase in the proportion of fibres containing the IIa myosin heavy-chain isoform.

Different phenotypes among slow/beta myosin heavy chain-containing fibres of rabbit masseter muscle : a novel type of diversity in adult muscle

Differences in the phenotype of different mammalian muscle fibres are usually attributed to differences in the expression of the product of different myosin heavy chain (MyHC) genes, which are known as isoforms. We studied differences in phenotype among fibres containing a single MyHC isoform (slow/beta) of the masseter muscle of adult rabbits. Four different monoclonal antibodies to slow/beta MyHC were used to stain serial sections from muscles in males and females. All antibodies recognize a single band on immunoblots and stain the same set of fibres in rabbit postcranial muscles. However, differential staining was observed in the masseter muscles. Antibody BA-D5 reacts with the most fibres, antibody A4.951 reacts with a subset of these fibres, and antibody A4.840 reacts with a subset of the A4.951-positive fibres. Antibody S58 reacts only with an even smaller subset of fibres. Even though differential staining using four antibodies might allow for the expression of as many as 15 different staining patterns, or phenotypes, only four were observed in>99% of over 30000 fibres studied. In females, nearly 40% of the fibres stain exclusively with antibody BA-D5, while in males, fewer than 8% of the fibres express this phenotype. The proportions of fibres of the other phenotypes do not differ so strikingly with gender. We conclude that an epitope diversity exists among muscle fibres in the adult rabbit masseter and that it is not necessarily a consequence of differences in gene expression. We feel that it is a regulated process and that, at least for some phenotypes, this regulation may be hormonally influenced.

Brief Exposure to Testosterone Is Sufficient to Induce Sex Differences in the Rabbit Masseter Muscle

In order to evaluate whether testosterone is responsible for inducing long-lasting sex differences in myosin heavy chain gene expression in the rabbit masseter muscle, we castrated young adult animals and administered supra-physiologic doses of testosterone for 3 or 6 weeks duration. Biopsies were taken from the masseter muscles of these animals at the time of castration and 3, 6 and 9 weeks later. Both immunoblot and immunohistochemical analyses of the myosin heavy chain (MyHC) isoform content of these muscle samples were performed. Exposure to testosterone for either duration resulted in a dramatic decrease in the content of the cardiac alpha MyHC isoform and a comparable increase in the content of the IIa MyHC isoform. The decrease in the cardiac alpha MyHC isoform content persisted for as long as 6 weeks after the end of treatment, but the increase in the content of the IIa MyHC isoform declined to normal during this time. Significant numbers of fibers were found containing both the cardiac alpha (and slow) and the IIa isoforms. Several fibers were encountered that contained both IIa and, presumably, the IIx isoform. Thus, a brief exposure to testosterone during postnatal maturation is able to produce a long lasting myosin heavy chain isoform switch that is similar in magnitude to that found during normal development.