Bruce R. Pachter

Neuromuscular plasticity following limb immobilization.

SummaryThe effects of immobilization on the ultrastructure of the rat neuromuscular junction of type I and type II muscle fibres were studied both qualitatively and quantitatively. Muscle fibre areas were measured as well The plantaris muscle was immobilized in a shortened position by applying a plaster cast for three weeks. Immobilized muscles were then compared to normal litter mates. Both type I and type II immobilized muscle fibres atrophied. Endplates from type II muscle fibres exhibited greater amounts of degeneration than type I endplates. Degeneration consisted of nerve terminal disruption, exposed junctional folds, postsynaptic areas which contained little or no postjunctional folds, and subjunctional sarcoplasmic masses. In addition to degeneration, the type II endplates also exhibited regeneration in the same endplate consisting of small terminals associated with large expanses of junctional folds, several small terminals occurring within the same primary synaptic cleft, and several axons wrapped by the same Schwann cell. These observations suggest terminal axonal regeneration. Our results demonstrate that limb immobilization produces muscle atrophy as well as denervation-like changes at the neuromuscular junctions which leads to terminal axonal sprouting and an ultrastructural remodelling.

The effect of electrical stimulation on reinnervation of rat muscle: contractile properties and endplate morphometry

Denervated extensor digitorum longus muscles of Wistar rats were electrically stimulated in vivo for 4 days (2h per day) after peroneal nerve crush 1 cm from the muscle. Isometric contractile properties and endplate ultrastructure were measured on days 11 and 18. On day 11, the time to peak (116% of control) and 1/2-relaxation time (136% of control) for the twitch tensions of stimulated muscles measured in vivo were significantly less than those (127% and 157% of controls, respectively) of non-stimulated muscles. Peak twitch and tetanic tensions were not significantly different. The postsynaptic area of endplates for stimulated muscles were closer in size to controls than those for the non-stimulated ones. On day 18, no difference was found in the contractile responses between stimulated and non-stimulated groups. Similarly, the postsynaptic areas were the same for both groups. These results demonstrate that denervated muscle stimulated electrically for 4 days prior to reinnervation can preserve the structure of the endplate as well as accelerate recovery of normal function in reinnervated muscle fibers after 11 days of denervation.

Light and electron microscopic serial analysis of mouse extraocular muscle: Morphology, innervation and topographical organization of component fiber populations

Mouse superior rectus extraocular muscle was examined in serial section by light and electron microscopy. By such analysis, it was possible to discriminate single versus multiple innervation, characteristics of internal cell morphology, and topographical distribution of the respective fiber populations within the muscle. Singly innervated (SIF) and multiply innervated fibers (MIF) were observed, both in an orbital surface layer and in the underlying global region of the muscle. Five morphologically distinct fiber types (three SIF and two MIF) were discriminable in terms of fiber diameter, mitochondrial richness, development of the sarcoplasmic reticulum, and myofibrillar size. Many fibers both SIF and MIF, terminated variously along the length of the muscle. The diameter of orbital MIF typically varied from one end of the fiber to the other by a factor of about three; the global MIF were of essentially constant diameter. The junctional complexity varied among the respective types of SIF. The MIF of both the global and orbital regions exhibited comparable ranges of complexity in their neuromuscular junctions.

Passive Exercise and Reinnervation of the Rat Denervated Extensor Digitorum Longus Muscle after Nerve Crush

Denervated extensor digitorum longus muscles of Wistar rats were passively exercised for 4 days (2 h/day) after peroneal nerve crush 1 cm from the muscle. Isometric contractile properties and end plate ultrastructure were measured 11 days post crush. No significant differences were observed in tension characteristics between the exercised and non exercised muscles on day 11. However, the postsynaptic area of end plates for exercised muscles were closer in size to controls than those for the non exercised ones. In addition, the end plates from the exercised muscles exhibited increased numbers of axonal sprouts and terminals than non exercised muscles. These results demonstrate that denervated muscles exercised 4 days before reinnervation can preserve the structure of the end plate as well as enhance reinnervation and sprouting at these end plates after 11 days of denervation.

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.

Myotonic muscle in mouse. A light and electron microscopic study in serial sections.

Abstract The EDL muscle of the Bar Harbor Strain C57BL 6Jdy 2j mouse was studied in serial section by light and electron microscopy. Phase contrast microscopy of 15 μm epon sections revealed fiber abnormalities such as central nucleation, atypical fiber diameter and regions of focal fiber necrosis; fiber splitting detected in such sections was a prominent feature of the myotonic muscle. Ultrathin resectioning of selected 15 μm sections showed additional alterations such as dilation of the sarcoplasmic reticulum, mitochondrial conglomerations and disruptions, sarcolemmal infolding and breakdown, and myofibrillar degeneration. The most notable neuropathic changes included distortion in shape and decrease in numbers of synaptic vesicles as well as axonal retraction, reduplication of the basement membrane, and the presence of dense bodies between axon and junctional sarcolemma. These data indicate the involvement of both muscle fiber and motor end plate in mouse myotonic dystrophy.

Long-term effects of partial denervation on sprouting and muscle fiber area in rat plantaris

The long-term effects of partial denervation on the muscle fiber cross-sectional area, degree of axonal sprouting, and end-plate morphology were examined 1, 3, 6, 9, and 12 months postsurgery in rat plantaris muscle. After 1 month of partial denervation, mean cross-sectional areas of type I and type II myofibers were significantly lower than that of sham-operated controls; fiber atrophy and hypertrophy was observed. After 3 months, we found no statistical difference in the mean cross-sectional fiber area between the two experimental groups. After 6 months, the fiber areas were now significantly larger than controls, possibly the result of compensatory work hypertrophy, due to the overuse of remaining hyperexpanded motor units. Preterminal, intranodal, and intraterminal sprouting were found to significantly increase from 1 to 6 months following partial denervation. While sprouting was enhanced, the number of terminal branch points per end plate did not change. Following 9 months of partial denervation and overuse, the mean fiber areas significantly decreased as compared to controls. The former muscles were found to contain angulated fibers, group atrophy, and increased levels of axonal sprouting. The number of terminal branches per end plate was now significantly increased over control values, possibly a compensatory response to reduced synthesis of neurotrophic factor(s) and/or transmitter-related components. At 12 months, fiber areas, axonal sprouting, and the number of terminal branches per end plate have all decreased. Degenerating end plates, denervated myofibers, angulated fibers, and group atrophy were observed. It would appear that aging-like changes are occurring earlier in chronically stressed, partially denervated muscles.

Cisternal distention in membrane-glycogen complexes of rabbit extraocular muscle.

Arrays of glycogen layers alternating with the double membranes of flattened cisternae were observed in apparently normal muscle. The cisternae of such arrays appeared in both nondistended and evenly distended forms. The distended cisternae retained their structural integrity in contrast to a frequently seen disruption of those that were not distended. Cisternal distention was related to the extent of glycogen deposition on membranes that were partially glycogen-bearing. Randomly organized membrane—glycogen complexes were also seen in which the cisternae consistently appeared in a distended form.

Nerve sprouting and endplate growth induced in normal muscle by contralateral partial denervation of rat plantaris

The incidence of motor nerve and terminal sprouting was quantitatively analyzed in normal unoperated muscles, in homologous muscles contralateral to muscles which have been partially denervated, in partially denervated muscles, and in sham-operated muscles. Muscles were studied by light microscopy after staining motor endplates by a combined silver-cholinesterase stain. In addition, the incidence of endplates containing terminal sprouts, the number of terminal branch points per endplate, and endplate, and endplate size were also assessed in the various groups examined. We observed that following section of the L4 spinal nerve, the incidence of sprouting (preterminal and intranodal) in the contralateral muscle exhibited a 2-fold increase over sham-operated controls. We also found a correlation between nerve terminal sprouting, terminal branch point number and endplate size. All of these parameters were significantly increased in the contralateral muscles as compared to the sham-operated control muscles. These findings suggest that normal muscles undergo sprouting which can be enhanced by contralateral partial denervation. The possible underlying mechanism may be the transneuronal induction of sprouting.

Structural alterations of the intramuscular nerves and junctional region in extraocular muscles of C57BL/Ks (db/db) diabetic mice

SummaryThe extraocular muscles of the C57BL/Ks (db/db) diabetic mutant mouse were examined by electron microscopy. The intramuscular myelinated nerves and the junctional apparatus of the singly and multiply innervated muscle fibers were found to exhibit various anomalies. Lamellated inclusion bodies were found in many of the Schwann cells of the myelinated nerve fibers; intra-axonal inclusion bodies resembling polyglucosan bodies were also observed. Junctional abnormalities consisted of various types of inclusion bodies within the junctional sarcoplasm and within sole-plate nuclei; in addition, hypertrophied endplates were observed and often penetrated by networks of axonal terminal branches. At times, pseudopod-like extensions of the junctional sarcoplasm encompassed and made protracted synaptic contacts with the incoming axons.