Muscle-derived neurotrophin-3 reduces injury-induced proprioceptive degeneration in neonatal mice.

During perinatal development, proprioceptive muscle afferents are quite sensitive to nerve injury. Here, we have used transgenic mice that overexpress neurotrophin-3 (NT-3) in skeletal muscle (myo/NT-3 mice) to explore whether NT-3 plays a neuroprotective role for perinatal muscle afferents following nerve injury. Measurements of NT-3 mRNA using RT-PCR revealed that levels of endogenous NT-3 mRNA in wild-type muscles remained constant during the first postnatal week following nerve crush or nerve section on postnatal day (PN) 1. In comparison, myo/NT-3 mice had significantly elevated levels of NT-3 mRNA that were maintained or increased following injury. To assess whether muscle-derived NT-3 could prevent injury-induced neuronal death, neuron survival in the DRG was analyzed in mice 5 days after sciatic nerve crush on PN3. Retrograde prelabeling of muscle afferents and parvalbumin immunocytochemistry both revealed that overexpression of NT-3 in muscle significantly reduced neuronal loss following injury. Similar neuroprotective effects of NT-3 were observed in wild-type mice injected with exogenous NT-3 in the gastrocnemius muscles. To test whether NT-3 could prevent muscle spindle degeneration, spindle number and morphology were assessed 3 weeks after sciatic nerve crush or section on PN1. No spindles were present in either wildtype or myo/NT-3 muscles after nerve section, demonstrating that NT-3 overexpression cannot maintain spindles following complete denervation. Moreover, NT-3 overexpression could not prevent moderate spindle loss in muscle and did not stimulate new spindle formation following nerve crush. Our results demonstrate that in addition to its early actions on sensory neuron generation and naturally occurring cell death, NT-3 has important neuroprotective effects on muscle afferents during postnatal development.     

Overexpression of neurotrophin-3 in skeletal muscle alters normal and injury-induced limb control

Transgenic overexpression of neurotrophin-3 (NT-3) in mice increases the number of surviving proprioceptive sensory components, including primary sensory neurons, gamma motoneurons and muscle spindles. The numbers of surviving alpha motoneurons are not affected by NT-3 overexpression (Wright et al., Neuron 19: 503- 517, 1997). We have assessed the consequences NT-3-stimulated increase in the proprioceptive sensory system by measuring locomotive abilities of mice that overexpress NT-3 in all skeletal muscles ( myo/NT-3 mice). In adulthood, one myo/NT-3 transgenic line continues to express NT-3 at high levels in muscle and maintains a hypertrophied proprioceptive system (high-OE myo/NT-3 mice). Compared to wildtypes, high-OE myo/NT-3 mice have nine times the amount of NT-3 protein in the medial gastrocnemius at six weeks of age. Although appearing normal during ordinary activity, high-OE myo/NT-3 mice display a distinct clasping phenotype when lifted by the tail. High-OE myo/NT-3 mice show severe locomotor deficits when performing beam walking and rotorod testing. These mice also demonstrate aberrant foot positioning during normal walking. However, following sciatic nerve crush, overexpression of NT-3 prevents further abnormalities in paw positioning, suggesting NT-3 may attenuate sensorimotor deficits that occur in response to sciatic nerve injury. Our results suggest that increases in proprioceptive sensory neurons, spindles and gamma motoneurons, along with continued postnatal NT-3 overexpression in muscle significantly disrupt normal locomotor control. Importantly, however, NT-3 may lessen initial deficits and thus improve functional recovery after peripheral nerve injury, suggesting these mice may serve as a good model to study NT-3's role in neuroprotection of proprioceptive afferents.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats

Summary Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag₂) only rather than two fibers (bag₁ and bag₂) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18–E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats.

Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag2) only rather than two fibers (bag1 and bag2) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18-E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

The effect of neonatal deafferentation or defferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat

Muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to neonatal soleus muscles of rats at a time when spindles are formed but when intrafusal muscle fibers are structurally and immunocytochemically immature. Experimental muscles were excised two months after nerve section. Control and experimental spindles were examined using monoclonal antibodies specific for myosin heavy chains of slow-tonic (ALD58) and fast-twitch (MF30) chicken muscles. Only intrafusal fibers bound these antibodies in intact soleus muscles. The deefferented spindles exhibited a pattern of ALD58 and MF30 binding similar to that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. Thus intact sensory innervation is essential for myosin heavy chain expression in intrafusal muscle fibers during postnatal development of rat spindles.     

The effect of neonatal deafferentation or deefferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat

Summary Muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to neonatal soleus muscles of rats at a time when spindles are formed but when intrafusal muscle fibers are structurally and immunocytochemically immature. Experimental muscles wereexcised two months after nerve section. Control and experimental spindles were examined using monoclonal antibodies specific for myosin heavy chains of slow-tonic (ALD58) and fast-twitch (MF30) chicken muscles. Only intrafusal fibers bound these antibodies in intact soleus muscles. The deefferented spindles exhibited a pattern of ALD58 and MF30 binding similar to that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. Thus intact sensory innervation is essential for myosin heavy chain expression in intrafusal muscle fibers during postnatal development of rat spindles.     

Innervation of regenerated spindles in muscle grafts of the rat

Features of the nerve supply and the encapsulated fibers of muscle spindles were assessed in grafted and normal extensor digitorum longus (EDL) muscles of rats by analysis of serial 10-microns frozen transverse sections stained for enzymes which delineated motor and sensory endings, oxidative capacity and muscle fiber type. The number of fibers was significantly more variable, and branched fibers were more frequently observed in regenerated spindles than in control spindles. Forty-eight percent of regenerated spindles received sensory innervation. Spindles reinnervated by afferents had a larger periaxial space than did spindles which were not reinnervated by afferents. Regenerated fibers innervated by afferents had small cross-sectional areas, equatorial regions with myofibrils restricted to the periphery of fibers, unpredictable patterns of nonuniform and nonreversible staining along the length of the fiber for 'myofibrillar' adenosine triphosphatase (mATPase) after acid and alkaline preincubation. In contrast, regenerated fibers devoid of sensory innervation resembled extrafusal fibers in that they usually exhibited myofibrils throughout the length of the fiber, no central aggregations of myonuclei, uniform staining for mATPase and a reversal of staining for mATPase after preincubation in an acid or alkaline medium. Approximately thirty percent of encapsulated fibers devoid of sensory innervation stained analogous to a type I extrafusal fiber, a pattern of staining never observed in intrafusal fibers of normal spindles. Groups of encapsulated fibers all exhibiting this pattern of staining reflect that either these fibers may have been innervated by collaterals of skeletomotor axons that originally innervated type I extrafusal fibers or that fibers innervated by only fusimotor neurons express patterns of staining for mATPase similar to extrafusal fibers in the absence of sensory innervation. Sensory innervation may also influence the reestablishment of multiple sites of motor endings on regenerated intrafusal fibers. Those regenerated fibers innervated by afferents had more motor endings than did regenerated fibers devoid of sensory innervation. Differences in size, morphology, and patterns of staining for mATPase and numbers of motor endings between fibers innervated by afferents and fibers devoid of sensory innervation reflect that afferents can influence the differentiation of muscle cells and the reestablishment of motor innervation other than during the late prenatal/early postnatal period when muscle spindles form and differentiate in rats.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

Formation of muscle spindles in regenerated avian muscle grafts

Summary Pigeon muscles lacking muscle spindles were grafted into sites which normally have a muscle containing spindles. The reciprocal transplantations were also made. After two to eight months, the graft of the donor muscle without spindles had regenerated into a muscle containing muscle spindles. The reciprocal grafts, muscles containing spindles transplanted to a site lacking spindle innervation, had neither muscle spindles nor remnants of the spindles. These experiments demonstrate that 1) the innervation is required for formation of the spindle; 2) the original spindles do not survive transplantation; and 3) parts of the original spindle are not required for spindle regeneration.This work was supported in part by NSF grants PCM 77-15960 and PCM 79-16540     

Laminin α4 and Integrin α6 Are Upregulated in Regenerating dy/dy Skeletal Muscle: Comparative Expression of Laminin and Integrin Isoforms in Muscles Regenerating after Crush Injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin α2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and “preactivated” myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin α1 was not detectable in skeletal muscle. Laminin α2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin α2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin α chains in early myogenic differentiation, such as laminin α4 and α5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin β1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin α3 was not expressed in uninjured or regenerating muscle, while integrin α6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin α6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin α6 occurred on some newly formed myotubes. Integrin α7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin α7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin α7 negative. No marked difference was observed in integrin α7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin α4 and integrin α6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin α4 and integrin α6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin α2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.     

Muscle spindle alterations precede onset of sensorimotor deficits in Charcot–Marie–Tooth type 2E

Charcot–Marie–Tooth (CMT) is the most common inherited peripheral neuropathy, affecting approximately 2.8 million people. The CMT leads to distal neuropathy that is characterized by reduced motor nerve conduction velocity, ataxia, muscle atrophy and sensory loss. We generated a mouse model of CMT type 2E (CMT2E) expressing human neurofilament light E396K (hNF‐L^E396K), which develops decreased motor nerve conduction velocity, ataxia and muscle atrophy by 4 months of age. Symptomatic hNF‐L^E396K mice developed phenotypes that were consistent with proprioceptive sensory defects as well as reduced sensitivity to mechanical stimulation, while thermal sensitivity and auditory brainstem responses were unaltered. Progression from presymptomatic to symptomatic included a 50% loss of large diameter sensory axons within the fifth lumbar dorsal root of hNF‐L^E396K mice. Owing to proprioceptive deficits and loss of large diameter sensory axons, we analyzed muscle spindle morphology in presymptomatic and symptomatic hNF‐L^E396K and hNF‐L control mice. Muscle spindle cross‐sectional area and volume were reduced in all hNF‐L^E396K mice analyzed, suggesting that alterations in muscle spindle morphology occurred prior to the onset of typical CMT pathology. These data suggested that CMT2E pathology initiated in the muscle spindles altering the proprioceptive sensory system. Early sensory pathology in CMT2E could provide a unifying hypothesis for the convergence of pathology observed in CMT.     

Somatosensory unit input to the spinal cord during normal walking

Chronic recording techniques in freely walking cats have been used to sample unitary activity from most large myelinated afferent classes. Cutaneous mechanoreceptors are highly sensitive and generate regular activity patterns predictable from their modalities. Knee joint afferents can fire briskly midrange locomotory movements but appear to be influenced by factors other than joint angle. Golgi tendon organs generate activity consistent with sensitivity to active muscle tension. Muscle spindle afferents do not appear to conform to any single functional pattern for all muscles. It is suggested that degree and rate of stretch are sensed by spindles (possibly under dynamic fusimotor bias) in extensor muscles which normally undergo isometric or lengthening contractions whereas rapidly modulated static fusimotor activity is employed to preserve spindle activity during the rapidly shortening contractions of flexor muscles. Both patterns may be represented in different spindles of bifunctional, biarticular muscles such as rectus femoris and sartorius.     

Bace1 and Neuregulin‐1 cooperate to control formation and maintenance of muscle spindles

The protease β‐secretase 1 (Bace1) was identified through its critical role in production of amyloid‐β peptides (Aβ), the major component of amyloid plaques in Alzheimer's disease. Bace1 is considered a promising target for the treatment of this pathology, but processes additional substrates, among them Neuregulin‐1 (Nrg1). Our biochemical analysis indicates that Bace1 processes the Ig‐containing β1 Nrg1 (IgNrg1β1) isoform. We find that a graded reduction in IgNrg1 signal strength in vivo results in increasingly severe deficits in formation and maturation of muscle spindles, a proprioceptive organ critical for muscle coordination. Further, we show that Bace1 is required for formation and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1‐dependent processing as a molecular mechanism.     

Age‐related differences in synaptic plasticity following muscle unloading

The objective of the present investigation was to determine the effects of muscle unloading—a form of subtotal disuse— on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n = 8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT‐4 expression. Quantitative analyses revealed that aged controls displayed significant (p < 0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls > young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT‐4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 246–256, 2003     

VCAM‐1 upregulation accompanies muscle remodeling following resistance‐type exercise in Snell dwarf (Pit1dw/dw) mice

Snell dwarf mice (Pit1^dw/dw) exhibit deficiencies in growth hormone, prolactin, and thyroid stimulating hormone. Besides being an experimental model of hypopituitarism, these mice are long‐lived (>40% lifespan extension) and utilized as a model of slowed/delayed aging. Whether this longevity is accompanied by a compromised quality of life in terms of muscular performance has not yet been characterized. In this study, we investigated nontrained and trained muscles 1 month following a general validated resistance‐type exercise protocol in 3‐month‐old Snell dwarf mice and control littermates. Nontrained Snell dwarf gastrocnemius muscles exhibited a 1.3‐fold greater muscle mass to body weight ratio than control values although muscle quality, maximum isometric torque normalized to muscle mass, and fatigue recovery were compromised. For control mice, training increased isometric torque (17%) without altering muscle mass. For Snell dwarf mice, isometric torque was unaltered by training despite decreased muscle mass that rendered muscle mass to body weight ratio comparable to control values. Muscle quality and fatigue recovery improved twofold and threefold, respectively, for Snell dwarf mice. This accompanied a fourfold increase in levels of vascular cell adhesion molecule‐1 (VCAM‐1), a mediator of progenitor cell recruitment, and muscle remodeling in the form of increased number of central nuclei, additional muscle fibers per unit area, and altered fiber type distribution. These results reveal a trade‐off between muscle quality and longevity in the context of anterior pituitary hormone deficiency and that resistance‐type training can diminish this trade‐off by improving muscle quality concomitant with VCAM‐1 upregulation and muscle remodeling.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.