苏木精-ATP酶新组化法在骨骼肌细胞核与肌纤维型检查中的应用

常规的苏木精和曙红组织化学染色是用于检测一般的肌细胞核表达方法，而用于确定肌纤维型表达的还有两种普遍公认的方法，即肌动球蛋白ATP酶酸、碱组织化学染色法。对骨骼肌异常肥大和肌萎缩症区分的一个重要因素是观察肌细胞核的形态变化。一种适宜的技术方法就是同时检查肌纤维型与肌细胞核两者比值，能应用于肌肉生长发育，肌萎缩症或某种程度的肌营养不良病。一般肌细胞核形态的检查和肌纤维构成的确定，通常需要使用以上描述的方法制作一系列肌纤维切片并做切片染色。我们目前设计的新方法用于联合检查冰冻肌组织切片内的肌纤维型与肌细胞核的同步显示，不失为是一种简便易行的好方法。     

Simplified technique for histochemical determination of three fiber types in equine skeletal muscle

For determination of 3 muscle fiber types in equine skeletal muscle, a comparison of 2 preincubation buffers, each followed by myosin adenosine triphosphatase staining, was made. Serial sections of the muscle samples (n = 75) were preincubated in an acid buffer (pH 4.6) or a formaldehyde-glycine buffer (pH 7.25) and then were stained for myosin adenosine triphosphatase. Differentiation of muscle fibers into type I, IIA, and IIB was identical with both techniques; however, in the samples prepared at pH 4.6, type I fibers were black; type IIA, light gray; and type IIB, dark gray. In the samples prepared at pH 7.25, types I, IIA, and IIB fibers were white, light gray, and dark gray respectively. The formaldehyde-glycine preincubation buffer (at pH 7.25) gave more consistent results, was easier to prepare, and retained cytoarchitecture better, compared with the samples prepared at pH 4.6.     

Simultaneous histochemical determination of three fiber types in single sections of ovine, bovine and porcine skeletal muscle

Classification of muscle fiber types involves tedious comparisons of serial muscle tissue sections. Procedures that would incorporate pertinent histochemical properties into one simplified assay for the successful differentiation and determination of fiber types were evaluated using muscle samples from three different species (ovine, bovine and porcine). For ovine and porcine muscle tissue, the best staining procedure involved initial preincubation of the tissue section at 4.35 (for ovine) and 4.30 (for porcine) followed by incubating for succinate dehydrogenase (SDH) activity, then staining for myofibrillar adenosine triphosphatase (ATPase) activity after an acid preincubation and, finally, counterstaining with hematoxylin. For bovine muscle tissue, the procedure involves first incubating for SDH activity, after which the section is incubated at pH 4.15 and then stained for myofibrillar ATPase activity after an acid preincubation, with a concluding counterstaining using hematoxylin. These procedures permit successful differentiation and identification of three fiber types (beta R, alpha R and alpha W) from a single section of muscle tissue with approximately a 65% saving in time and materials. The results of histochemically typing muscle fibers in a single tissue section stained with these techniques were similar to those obtained with the traditional methods comparing serial sections.     

Metabolic response to racing and fiber properties of skeletal muscle in standardbred and thoroughbred horses

Muscle biopsies were obtained from 17 Standardbred trotters (S) and 22 Thoroughbred racehorses (T) after races which varied in length from 1200 to 2840 m. Biopsies were taken between 2 and 14 minutes from the conclusion of the race. Histochemical and biochemical analyses were performed on all muscle samples. Standardbred horses possessed fewer Type II fibers, especially Type IIB fibers, and demonstrated a higher oxidative and lower glycolytic capacity in muscle than Thoroughbred horses. Intramuscular glycogen and lac-tate concentrations were lower in Standardbred horses after racing (mean 420 and 82 mmol/kg d.w. respectively) com-pared to Thoroughbred horses (mean 545 and 148 mmol/kg d.w. respectively). Low levels of intramuscular adenosine triphosphate (ATP) (mean S 17, T 15 mmol/kg d.w.) and creatine phosphate (mean S 28, T 42 mmol/kg d.w.) were recorded in both breeds after racing. Differences in muscle enzyme activities and metabolic response to exercise between breeds were related to the different muscle fiber compositions of Standardbred and Thoroughbred horses. Within both breeds, a high percentage of Type IIB fibers was associated with a low oxidative and high glycolytic capacity in muscle and a greater lactate accumulation after racing. In Standardbred horses low intramuscular ATP concentrations post-race were associated with a high percentage of Type IIB fibers and high lactate levels. No relationship was found between placing or speed of racing and intramuscular lactate and ATP concentrations post-race. This may have been due to many other factors which affected the outcome of a race and also due to the fact that it was not known when during a race lactate accumulation and ATP depletion occurred. Since the properties of fiber types appeared to be very different, the metabolic response within individual fiber types may be more closely related to fatigue than the metabolic response measured in whole muscle.     

Comparative study of muscle fiber type composition in the middle gluteal muscle of andalusian, thoroughbred and arabian horses

Samples were taken from the right middle gluteal muscle of 25 Andalusian horses, 20 Thoroughbreds and 10 Arabian horses, all aged between 5-and 12-years-old and not involved in training. Histochemical analysis was carried out using myosin ATPase after acid preincubation (pH 4.4) and NADH-tr in order to determine the percentage of different fiber types. Statistical analysis showed highly significant differences (P<0.001) in the proportion of type 1 and type 2 fibers between breeds. The Thoroughbred was found to have a significantly higher proportion of type 2A fibers (P<0.001) than the other 2 breeds, although differences in the proportion of type 2B fibers in different breeds were not found to be statistically significant. The Andalusian horse was found to have a higher proportion of fast-twitch fibers of low oxidative capacity (type FT) than the other 2 breeds (P<0.001). These results not only confirm those obtained in previous studies of different breeds of horses, but highlight the histochemical differences in the skeletal muscle of breeds of great commercial and sporting interest in Spain.     

Histochemical differentiation of fiber types in neonatal canine skeletal muscle

Differentiation of fiber types in developing canine skeletal muscle was studied, using morphologic, morphometric, and histochemical techniques. Sample collections were made from 6 muscles from the pectoral and pelvic limbs of 16 healthy pups between 1 day and 12 weeks of age. In newborn pups, 90% to 95% of the fibers in the 6 muscles were classified as undifferentiated or type IIC; the remaining fibers were classified either normal or large-size type I. Large-size type I fibers usually accounted for 2% to 4% of the total population and were considered analogous with the B fiber of Wohlfart. These fibers were larger than all other fiber types and disappeared after pups reached 4 to 5 weeks of age. After 2 to 4 weeks, the number of undifferentiated fibers decreased with the appearance of, and the concomitant numerical increases of, normal size type I and type IIA fibers. The percentages of type I and IIA fibers approached proportions of the adult dog by 12 weeks, at which time a type IIA fiber predominance was present in biceps femoris, lateral head of the gastrocnemius, cranial tibial, and long head of the triceps. Type I fibers predominated in medial head of the triceps and superficial digital flexor after 4 to 5 weeks. The mean fiber diameters of type I and IIA fibers were similar to any given muscle throughout the postnatal development. All fiber types stained uniformly with the oxidative stain nicotinamide adeninedinucleotide-tetrazolium reductase during the first 12 weeks of life, whereas a distinction between type I and II fibers was evident after 3 to 4 weeks with the periodic acid-Schiff stain reaction.     

Histochemical identification of fiber types in canine skeletal muscle.

Proximal and distal skeletal muscles from pectoral and pelvic limbs were histochemically examined in 18 neuromuscular disease-free dogs. On the basis of the human system of classification and nomenclature and results of standard adenosine triphosphatase (ATPase) and glycine-formaldehyde preincubation procedures, the fiber types identified in immature and mature canine skeletal muscles were I, IIA, and IIC.     

Oxidative capacity of skeletal muscle fibres in racehorses: histochemical versus biochemical analysis

The oxidative capacity of skeletal muscle fibre types was evaluated histochemically using the nicotinamide dinucleotide diaphorase (NADH-D) staining, and biochemically by measuring the activity of citrate synthase (CS) in both whole muscle samples and in pools of fibres of identified type. Duplicate determinations of the NADH-D staining pattern resulted in standard deviations (sd) between duplicates of 6 and 11 per cent for two observers. The NADH-D pattern was found to differ between observers. Duplicate determinations of CS activity in the same fibre pools resulted in an sd value of 2.9 mumol/g/min. Measurements of whole muscle CS activity did not provide information about the distribution of oxidative capacity among fibre types. The NADH-D stain and CS activity in fibre pools both showed that, in general, type I and IIA fibres had a higher oxidative capacity than type IIB fibres. Biochemical techniques also showed, however, that the CS activity in type I and IIA fibres of different horses could vary as much as twofold, whereas the NADH-D rating showed a high intensity staining for most type I and IIA fibres in all horses. Furthermore, type IIB fibres received a lower NADH-D rating than the other fibre types even when the CS activities were quite similar. For purposes of research, biochemical measurement of oxidative capacity in individual muscle fibre types provides valuable quantitative and comparative information. The ease of histochemical NADH-D staining in comparison to fibre dissections makes this technique more practical for routine estimates of the distribution of oxidative capacity among muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crooked calf disease: a histological and histochemical examination of eight affected calves

Macroscopic, histopathologic, and histochemical investigations were made on a group of eight neonatal Angus X Hereford calves, selected from an ongoing outbreak of crooked calf disease among calving heifers. Arthrogryposis of the forelimbs was seen to varying degrees in all eight animals, and torticollis was present in six calves. Histopathology, using hematoxylin and eosin stain, did not reveal any striking or consistent lesion in the affected animals; the majority of the tissues sampled were normal. Muscle samples were processed for adenosine triphosphatase (ATPase) and NADH-tetrazolium reductase (NADH-tr) histochemistry, and the data suggest that a primary myopathy is not responsible for the congenital anomalies in the affected calves.     

Equine synovial tendon sheaths and bursae: an histological and scanning electron microscopical study

The structure of equine synovial tendon sheaths and bursae has been examined by light and scanning electron microscopy. Tissue samples were obtained from horses of various types and ages with no clinical evidence of sheath or bursal disorders. The interior of both structures was lined by a cellular layer superimposed on a vascular zone supported by a fibrous layer. The pattern of cell distribution of the lining varied from site to site within the same structure depending on the nature of the underlying tissue and on the amount of movement to which the structure was subjected. The cellular layer was predominantly fibrous in nature with scanty, widely separated fibroblasts (eg where it lines the palmar ligament, tendons and paratendons). In the mesotendon and bursal extremities, where the lining is subjected to a positive degree of movement, the cellular layer was areolar in type with well established folds populated by abundant cells oval to round in shape. In foals and yearlings, the supportive layer was mainly around the areolar with patches of adipose tissue; which were gradually replaced by fibrous tissue as the animal grew.     

A comparative histochemical and morphometric study of canine skeletal muscle.

The purpose of this study was to determine whether there were differences in skeletal muscle properties in the hindlimb muscles of different types of dogs. Muscle samples were obtained from the gracilis, sartorius cranial head, sartorius caudal head and tibialis anterior muscles of mixed-breed and hound-type dogs and Beagles. Fiber type, fiber size and capillary morphometry determinations of each muscle from each dog were made from sections stained for myofibrillar ATPase activity. Individual animals were bilaterally symmetric for all measured variables. Fiber type, fiber size and capillary geometry varied between dogs of a given type and muscles within a given dog. There were no differences between dog types for fiber type or fiber size; significant variation in log(muscle)/log(body) mass ratios between dog types was observed for all muscles. The results indicate that for a given muscle, significant variation can occur in skeletal muscle characteristics between different types of dogs and that these differences can be independent of differences in exercise history.     

Morphological,histological and histochemical studies of the gonads of ovine freemartins

Freemartins are XX/XY chimaeras that develop as a result of the fusion of the placental circulation of at least one male and one female fetus. Of 19 freemartin ewes, 13 had testis-like structures, seven of them in an abdominal position and six in an inguinal position. Histologically, their gonads had structures resembling seminiferous tubules and interstitial cells, and grossly, most had structures derived from the mesonephric ducts (vasa deferentia, epididymides and vesicular glands). The other six freemartin ewes had small, undifferentiated gonads that lacked either follicles or seminiferous tubule-like structures. They also lacked any structures derived from the mesonephric ducts. No derivatives of the paramesonephric ducts were detectable in any of the freemartin ewes. The gonads of the male-type freemartins stained immunocytochemically for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and histochemically for alkaline phosphatase (AP) in a similar way to, but more extensively and intensely than, the gonads of normal rams, and the staining was confined to interstitial cell-like structures. The staining in the undifferentiated-type freemartins was weak, but both 3beta-HSD and AP were present in unidentified cell types.