A scanning electron microscopic study of denervated neuromuscular junctions

The morphological changes of the neuromuscular junctions (NMJs) caused by nerve transection have been examined by scanning electron microscopy in the peroneus longus muscle of the Chinese hamster. The synaptic grooves in the normal NMJs are deep labyrinthine depressions partitioned by ridges and contain numerous slit-like subsynaptic folds. After denervation, the grooves become shallower with lower sarcoplasmic ridges, and the subsynaptic area of the muscle fibers gradually flattens as a whole. The subsynaptic area shows a plate-like sarcoplasmic elevation by 4 weeks and persists as a fusiform focal bulge on the atrophied muscle fiber after 4 weeks. Concurrently the subsynaptic folds decrease in number and transform into shallow pit-like invaginations. Any subsynaptic specialization has not been discernible after 16 weeks. Changes in acetylcholinesterase stainability at the NMJs have also been observed by light microscopy. No remarkable changes were noted until 4 weeks after denervation, thereafter, however, acetylcholine positive area became smaller showing more diffuse staining pattern. Atrophied muscle fibers often exhibit longitudinal splitting and the satellite cells tend to detach from the muscle surface. These evidences suggest a regenerative process which may take place during muscle degeneration.     

Histochemical study of external urethral sphincter in the rabbit. Analysis with construction of histograms

Male rabbit's external urethral sphincter was examined by histochemical muscle fiber typing (myosin ATP-ase staining), and the analysis with construction of histograms regarding to muscle fiber types were performed. Rabbit's external urethral sphincter was predominantly composed of fast twitch (type 2) fibers (87.3%) as a whole. But the proportion of constituent fiber types varied according to the layers, i.e., the slow twitch (type 1) fibers constituted a relatively high percentage (33.4%) in the inner third layer, while few of the type 1 fibers were found in the outer third layer. The all histograms regarding to fiber type in different layers were normal bell-shaped distribution curves. The mena diameter of type 2 fibers (14.7 microns) was evidently larger than that of type 1 fibers (20.5 microns). All three kinds of muscle fibers equally tended to increase in size toward the outer direction, and in every three layers, the diameter of type 2 was larger than that of type 1 also. The definite differences in the proportion of fiber types and fiber sizes between layers may implicate that the inner and outer layers play different roles, i.e., continuous tonic constriction in the former and sporadic strong constriction of short duration in the latter, under different neural regulations. As far as rabbit's external urethral sphincter is concerned, sporadic strong constriction should be mainly dependent on the muscle fibers of large size composing the outer layer, especially the fast twitch fibers. It is possible that the rabbit is so adapted that it could interrupt urination promptly.     

C Histomorphology of neuromuscular junction in Duchenne muscular dystrophy

BACKGROUND: Our objective was to better understand neuromuscular junction (NMJ) morphology in children with Duchenne muscular dystrophy (DMD). METHODS: We examined the NMJs of four children, age 6-13 years, who were diagnosed with DMD. Using our previously established staining technique, we examined the gross appearance of the NMJs in patients with DMD and evaluated the spread of acetylcholine receptors (AChRs) in relationship to the NMJs. We used a computerized algorithm to measure the area of staining corresponding to AChRs and NMJ. RESULTS: Abnormal shape and morphological appearance of some of the NMJs was clearly evident. The spread of AChRs in DMD patients is comparable with the spread of AChRs in nonDMD patients. CONCLUSIONS: The distribution of AChRs in relationship to the boundaries of NMJs in DMD children is similar to the distribution of NMJs in the erector spinae muscles of idiopathic scoliosis patients.     

Coexistence of fast and slow myosin isozymes in human external urethral sphincter. A preliminary report

Human external urethral sphincter muscle was isolated for biochemical research. Myosin samples were prepared and pyrophosphate gel electrophoresis and two-dimensional electrophoresis were performed. Assignment in pyrophosphate gel of myosin isozymes of human external urethral sphincter was carried out by making comparisons with those from human rectus abdominis muscle. Myosin light chains in human external urethral sphincter were identified using two-dimensional electrophoresis. Human external urethral sphincter, similar to other voluntary muscles, is composed of fast and slow twitch muscle fibers, since fast and slow myosin components were shown in both pyrophosphate gel and two-dimensional electrophoretograms.     

Striated muscle fiber compositions of human male urethral rhabdosphincter and levator ani

PURPOSE: We clarified the contractile properties of human male periurethral striated muscle fibers to better understand how the rhabdosphincter and the levator ani maintain urinary continence. MATERIALS AND METHODS: Muscle specimens were obtained from 52 male patients who underwent radical prostatectomy or radical cystectomy. The specimens were frozen in liquid nitrogen. Frozen sections (10 microm thick) were stained with myofibrillar ATPase at different pH values (pH 4.2, 4.6 and 10.6), and evaluated for quantitative parameters and fiber type distribution. Myosin heavy chain analysis was performed using SDS-PAGE. RESULTS: Of all 52 cases 37 provided specimens that could be divided into the 2 major fiber types, type 1 (slow twitch) and type 2 (fast twitch). Although type 1 muscle fibers were predominant in RS and LA muscle groups (RS 69.6 +/- 2.7%, LA 67.0 +/- 2.0%), mean muscle fiber size was significantly smaller in RS (mean area 906 +/- 86 microm(2)) than in LA (mean area 2,967 +/- 170 microm(2)) (p <0.0001). In 11 specimens type 2 muscle fibers could be subdivided into types 2A (fast fatigue resistant) and 2B (fast fatigable). Type 2A fibers were significantly more prevalent than type 2B fibers (p <0.05). Likewise, MHC analysis of these 11 specimens found a significantly higher percentage of fiber type 2A expression products (MHC 2A) than of fiber type 2B expression products (MHC 2X) (p <0.05). CONCLUSIONS: RS and LA contribute to urinary continence mechanism by slow contraction. Moreover, the smaller mean size of muscle fibers in RS suggests more fatigue resistance compared with muscle fibers in LA because small fibers have a shorter diffusion distance for metabolic substrates. These results should help contribute to a more detailed understanding of the function of periurethral striated muscles in the human male.     

Differential sensitivity of fast and slow fibers in mammalian nerve. III. Effect of etidocaine and bupivacaine on fast/slow fibers

Etidocaine and bupivacaine are long acting local anesthetics with contrasting effects on motor and sensory function. The effect of these drugs on fast-conducting (large, motor) and slow-conducting nerve fibers (small, pain) in the isolated rabbit vagus nerve was examined. Both drugs had an equivalent effect on slow fibers. Etidocaine had a short latency and bupivacaine a prolonged latency of effect on fast fibers. During this long latency of effect by bupivacaine on fast fibers, only the slow fibers were blocked. This period of differential effect on fast and slow fibers is believed to be the explanation for the early effect of bupivacaine on pain fibers followed by a later block of motor function. This difference is believed to be due to the lower lipid solubility solubility and greater ionization of bupivacaine, which impedes diffusion across the permeability barriers present in fast-conducting A fiber.     

Slow or Fast lsokinetic Training after - Knee Ligament Surgery*

In order to evaluate the importance of training speed and training dose and its effect on muscle morphology, 16 patients operated on for an anterior cruciate ligament injury were randomly divided into two groups for isokinetic training. One group trained with slow speed (60 O/sec), 10 x 10 repetitions and one group with fast speed (180 O/sec), 10 x 15 repetitions. The effect of 8 weeks training three times per week was evaluated with Cybex I/@st rength tests, muscle biopsies, and computed tomography of the thigh muscles. No significant differences between the two groups were noted although the fast speed training group showed tendencies to increase in strength more at higher speeds than did the slow speed training group. Within the fast speed training group the increases at higher speeds were significantly larger than at slower speeds. There was also a tendency toward a larger increase of the cross-sectional area of the fast twitch fibers in the fast speed training group. There was no significant difference in the increase of the cross-sectional area of the quadriceps muscles between the two groups. The large dose of training did not yield larger increases in strength compared to one of our earlier studies that used 3 x 10 repetitions. J Orthop Sports Phys Ther 1987;8(10):475-479.     

Characterization of the train-of-four response in fast and slow muscles: effect of d-tubocurarine, pancuronium, and vecuronium

The in vivo cat soleus and gastrocnemius muscles were used to compare isometric contraction strength and the train-of-four (T4) response (2 Hz for 2 s) of two muscle types (fast and slow) during onset of competitive neuromuscular blockade in order to determine the extent of the correlation between twitch depression and T4 fade. Prior to drug administration the muscles that were studied differed significantly in that the T4 ratio was 1.0 in the gastrocnemius and only 0.87 in the soleus. Three competitive neuromuscular-blocking agents were compared: d-tubocurarine, pancuronium, and vecuronium. d-Tubocurarine was found to produce a close correlation between the degrees of twitch strength depression and T4 for both muscles. However, these muscles demonstrated significantly different ED50 values (105 micrograms/kg for gastrocnemius, 150 micrograms/kg for soleus). Pancuronium also produced a similar relationship between twitch strength depression and T4 decrement for each muscle. In this case, however, there was little difference in their ED50 values for twitch depression (11.5 micrograms/kg for gastrocnemius, 13 micrograms/kg for soleus). The effects of vecuronium were quite different from the other two muscle relaxants. Although vecuronium produced a comparable correlation between twitch tension and T4 fade in fast muscle, no such relationship was found to exist in slow muscle. Even when the twitch strength was blocked to 18% of control, the soleus T4 response was depressed to only 75% of control. These results highlight major differences among competitive neuromuscular-blocking agents and suggest multiple sites of action.     

Changes in skeletal muscle contractile properties in streptozocin-induced diabetic rats and role of polyol pathway and hypoinsulinemia

Functional changes in slow-twitch soleus and fast-twitch extensor digitorum longus muscles were assessed after 2 mo of streptozocin-induced diabetes in rats. For soleus, there was a slowing of twitch times both for contraction and relaxation and a reduction of maximum tetanic relaxation rate. There was little effect on strength performance assessed by maximal tetanic tension production. Treatment with the aldose reductase inhibitor ponalrestat largely prevented relaxation defects but had little effect on contraction. For the fast muscle, twitch times were relatively unaffected, but maximum tetanic relaxation rate was reduced. In addition, tetanic tension output decreased. These changes were largely prevented by ponalrestat treatment. The effects of partial insulin therapy were also investigated. This regimen reduced hypoinsulinemia, but sufficient hyperglycemia remained to stimulate the polyol pathway. It prevented the slowing of soleus twitch contraction but had no effect on relaxation. For extensor digitorum longus, insulin produced further deleterious effects on tetanic tension and maximum relaxation rate, which were antagonized by ponalrestat. A 1% dietary myo-inositol supplement had little effect on contractile function in slow or fast muscles. It was concluded that polyol-pathway activity is an important factor underlying skeletal muscle functional changes in diabetes, probably acting through disruption of Ca2+ handling. Hypoinsulinemia was considered a secondary factor causing atrophy, particularly of fast muscles. There was no evidence of effects dependent on neuropathy.     

Power spectrum analyses of electromyographic activity. Discriminators in the differential assessment of patients with chronic low-back pain.

Some parameters of the electromyographic power spectrum of the paraspinal muscles were recorded and analyzed with regard to their ability to discriminate between normal controls and patients with back pain classified as "avoiders" or "confronters" by their responses to the Pain Behavior Checklist. In terms of the activity of the multifidus muscle, the avoider group displayed spectral changes towards lower frequencies, reduced variability in the density spectrum, and higher values of estimated initial frequencies. Measures of the activity of the iliocostalis lumborum did not contribute in any significant way to these group differences. Based on these findings, and on the available literature concerning the histology and physiology of paraspinal muscles, it is proposed that the observed characteristics of the avoider group may be due to a reduced ratio of slow twitch to fast twitch muscle fibers in the multifidus.     

颈总动脉周交感神经网剥脱切除术后痉挛肌肉内酶与肌纤维结构的研究

目的　探讨交感神经切断后,痉挛肌肉内酶与肌纤维结构的改变。方法　将20只wistar大白鼠作成痉挛性模型,分成两组,随机选择1组行颈总动脉周交感神经网剥脱切除术,另1组作为对照,于术后第20天切取两组大鼠部分肱三头肌组织,采用(Ellman)爱尔蒙法及AU1000检测仪测定肌组织内乙酰胆碱酯酶活性和肌酸激酶含量,在IBAS图像分析仪上观察两组肌组织内快收缩肌纤维与慢收缩肌纤维的改变情况。结果　交感神经切断后,乙酰胆碱酯酶活性和肌酸激酶含量明显下降,分别从（3.37±1.01）U/g降至（2.15±1.42）U/g（P＜0.01）;（3582.90±1561.7）IU/L降至（420.10±73.55）IU/L（P＜0.01）,快肌纤维明显减少,从（275727.31±98240.23）μm     

Electrical conditioning of in situ skeletal muscle for replacement of myocardium

Eight canine right hemidiaphragms were electrically stimulated at 2Hz (N = 3) and 10 Hz (N = 5) for up to 135 days. ATPase histochemical studies showed nearly total transformation of type II fast twitch fatigable muscle fibers to type I slow twitch fatigue resistant muscle fibers. The thickness and vascular perfusion of the stimulated muscle increased significantly. Electrical thresholds remained unchanged. The muscle contracted vigorously and without fatigue for up to 135 days at 10 Hz stimulation. Electrical conditioning of canine diaphragm skeletal muscle at 10 Hz frequency for 5 weeks results in a 95% and greater population of slow twitch fatigue resistant muscle fibers. This change in diaphragmatic skeletal muscle should make it a more suitable autograft for replacement or augmentation of diseased cardiac muscle.     

The Effects of d-Tubocurarine, Pancuronium and Atracurium on the Responses of Gastrocnemius and Soleus Muscles in the Cat

In vivo, the effects of d-tubocurarine (0.20 mg kg-1), pancuronium (0.015 mg kg-1) and atracurium (0.15 mg kg-1) on the responses of the indirectly stimulated cat gastrocnemius (fast) and soleus (slow) muscles to a twitch, train-of-four and tetanic stimuli were studied. The soleus muscle demonstrated a greater degree of fade than the gastrocnemius in response to tetanic stimuli (50 Hz). There was no difference between the responses of the two muscles to twitch or train-of-four stimuli with any of the drugs. Recovery of train-of-four ratio occurred more rapidly than did the tetanic fade ratio. At a time when train-of-four ratio exceeded 0.7, tetanic fade was still evident, especially in the soleus muscle.     

Comparison of the histochemical and contractile properties of human gastrocnemius muscle

Contractile and histochemical properties of the human gastrocnemius muscle were compared in 21 male athletes ranging from 20 to 29 years of age. Surface electrical stimulation was used to determine muscle twitch parameters. The contractile variables of the muscle twitch were: latency (L), time to peak force (TPF1 and TPF2), peak force (Pf1 and Pf2), half-contraction time (1/2 CT), and half-relaxation time (1/2 RT). Muscle samples from the belly of the medial gastrocnemius muscle were obtained using the needle biopsy technique. The samples were treated histochemically for myosin ATPase in order to classify the fibers as slow twitch (ST) or fast twitch (FT) and to determine fiber areas. Multiple correlations were performed between the contractile and histochemical variables. The results demonstrated significant (p < 0.05) positive relationships between the calculated twitch recordings and percentage of FT fibers (R = 0.85), percentage of ST fibers (R = 0.85), relative percent area of FT fibers (R = 0.84), and relative percent area of ST fibers (R = 0.84). The results suggest that fiber type percentage and relative percent area determined using myosin ATPase are related to electrically stimulated isometric contractile properties. J Orthop Sports Phys Ther 1991;13(6):322-328.     

The characteristic phenotype of the masseter muscle fibers is established after birth]

Muscle biopsies were taken from the superficial portion of the M. masseter in 10 foetuses (aged between 12 and 38 weeks), in a child of 18 months and in an adult without any neuromuscular disease. Serial frozen sections were processed for immunocytochemistry using antibodies specific for the embryonic, foetal, slow and fast myosin heavy chains (MHCs). Diameter of the different types of fibers were measured with a Leitz ASM 68 K; the results have been expressed as average diameters and histograms. During foetal development, the masseter is formed from two successive generations of muscle fibers in a manner very similar to that which has been previously described for other skeletal muscles. After birth, a particular phenotype appears. This phenotype is characterised by the persistence of embryonic and foetal MHCs and by the presence of two distinct populations of fibers: small diameter fibers which coexpress embryonic, foetal and fast myosin isoforms but never express the slow MHC; large diameter fibers which express slow myosin either exclusively or in variable associations with the other isoforms.     

Skeletal muscle fiber composition, nutritional status and subjective fatigue during surgical convalescence

Samples from the lateral vastus femoris muscle were obtained by needle biopsy from 12 patients before and 20 days after elective abdominal surgery. The content of fast and slow twitch muscle fibers and their lesser diameter were determined and related to subjective degree of fatigue and changes in nutritional parameters. Fatigue increased from a mean preoperative level of 2.2 +/- 0.4 arbitrary units to 4.3 +/- 0.5 (p less than 0.001) on postoperative day 20. Body weight, triceps skin fold and slow twitch fiber diameter decreased postoperatively (p less than 0.05). There was no significant postoperative change in the distribution of muscle fiber types. Fatigue increase correlated to weight loss and reduction of triceps skin fold (r = 0.50 and 0.54, p = 0.08 and 0.06, respectively). Postoperative changes in slow and fast twitch fiber diameter correlated to loss of body weight (r = 0.60 and 0.74, p less than 0.05 and less than 0.01, respectively), but correlation was not found between postoperative fatigue and changes in fiber-type distribution (r = 0.34 and 0.29).     

Isoform-dependent Effects of Halothane in Human Skinned Striated Fibers

Background: Reports of the effects of halothane on isoform contractile proteins of striated muscles are conflicting. To determine whether halothane affects cardiac and skeletal contractile proteins differently, the authors examined the effects of two doses of halothane (0.44 and 1.26 mM, equivalent to 0.75 and 2.25 vol%, respectively) on the Calcium sup ++ sensitivity and maximal force in human skinned cardiac, type I (slow twitch), and type II (fast twitch) skeletal muscle fibers.

Methods: Left ventricular muscle strips and skeletal muscle biopsy specimens were obtained from eight and ten patients undergoing cardiac and orthopedic surgery, respectively. Sarcolemma and sarcoplasmic reticulum were destroyed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus Brij 58. Calcium sup ++ sensitivity was studied by observing the isometric tension developed by skinned fibers challenged with increasing concentrations of Calcium sup ++. Muscle fiber type was determined in each skeletal fiber by the difference in strontium-induced tension measurements.

Results: Halothane shifted the Calcium sup ++ tension curves toward higher Calcium sup ++ concentrations and increased the Calcium sup ++ concentration for half-maximal activation in both cardiac and type I skeletal muscle fibers (from 1.96 micro Meter and 1.06 micro Meter under control conditions to 2.92 micro Meter and 1.71 micro Meter in presence of 0.75 vol% halothane, respectively) without changing the slope of this relationship (Hill coefficient). In contrast, no significant effect was observed in type II fibers. Halothane also decreased the maximal activated tension in the three groups of fibers with a lesser effect in type II fibers.     

Age-related changes in muscle fiber types in the human thyroarytenoid muscle: an immunohistochemical and stereological study using confocal laser scanning microscopy.

A decline in motor performance contributes to laryngeal dysfunction in the elderly, but the pathogenetic mechanisms are unknown. Quantitative 3-dimensional, age-related changes in the muscle fiber content of the human thyroarytenoid muscle were estimated from geometric probability (stereology) by use of a technique that provided a statistically unbiased sample of all possible section orientations and locations in the entire muscle volume. There was a preferential 27% age-related loss in the length density (L(V type, muscle)) of type 1 (slow) fibers in contrast to the selective type 2 (fast) fiber loss typical of aging limb muscles. In type 2 fibers there was no significant loss in the L(V), but there was an age-related decrease (P < 0.05) in the surface density (S(V type, muscle)) and an increase (P < 0.05) in the atrophy factor, an index of the content of very small, atrophic fibers. There was also an age-related increase in the length fraction (L(L type, all fibers)) of muscle fibers that coexpress both fast and slow myosin heavy-chain isoforms (P < 0.05). These findings demonstrate a type-specific fiber loss and atrophy that differs from that in aging limb muscles and an age-related increase in motor unit remodeling.     

Treatment with a coinducer of the heat shock response delays muscle denervation in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

We undertook a longitudinal study of the histological and biochemical changes at the neuromuscular junction (NMJ) in muscles of SOD1-G93A mice. We also assessed these functions in mice treated with a known heat shock protein inducer, arimoclomol. Tissue samples of treated and untreated mSOD mice were analysed for AChE and ChAT enzyme activities as markers of neuromuscular function. Sections of hindlimb muscles (TA, EDL and soleus) were also stained for succinate dehydrogenase and silver cholinesterase activities as well as for immunohistochemistry. Hsp70 levels were also measured from muscle samples using ELISA. Results showed that denervation and nerve sprouting were present at symptom onset in fast muscles, although slow muscles remained fully innervated. Cholinergic enzyme activities were reduced prior to denervation and declined further with disease progression. Reduction of endplate size, a slow to fast shift in muscle phenotype was also observed. Treatment with arimoclomol delayed the appearance of these changes, increased innervation, cholinergic enzyme activities and endplate size and reversed muscle fibre transformation. These beneficial effects of arimoclomol in muscles were accompanied by an increase in Hsp70 expression. In conclusion, our results indicate that pharmacological targeting of muscles at early stages of disease may be a successful strategy to ameliorate disease progression in ALS.     

Fast and slow myosin as markers of muscle regeneration in mangled extremities: a pilot study

Mangled extremities were classically managed by amputation. But over the past few decades, with the advancement in surgical techniques, an increased number of limb salvages have been possible. As muscles usually get damaged in such grievous injuries, a thorough understanding of muscle regeneration may give a better insight into muscle healing in these injuries. Muscles are composed of slow and fast fibers which can be represented by slow and fast myosin, respectively. There are some animal studies which reported differential regeneration of slow and fast muscle fibers during muscle healing. We conducted this pilot study to find out whether the same holds true for muscle healing in mangled extremities also. This pilot study is designed in 15 patients with lower limb mangled extremities presenting to trauma center of PGIMER, Chandigarh, who were operated within 24 h of injury to see whether muscle healing in mangled extremities follows the same pattern. Biopsies were taken during initial surgery conducted within 24 h of injury and on the 7th day of injury when patient was posted again for secondary wound closure procedure or revision amputation. The biopsy samples were subjected to histopathological and immunohistochemistry examination using antibodies against fast and slow myosin. We found that the regenerating muscle fibers in the biopsy sample taken on the 7th day of injury showed only slow muscle fibers with the absence of fast muscle fibers when compared with the initial biopsy results showing differential regeneration of slow muscle fibers.