Identification Of Fiber Types Research Articles

Need for speed: Human fast-twitch mitochondria favor power over efficiency

ObjectiveHuman skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation. While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic. MethodsHere, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity. ResultsMaximal complex I+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per mitochondrial volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for complex I+II, which was primarily mediated through elevated complex II respiration. Furthermore, the abundance of complex II protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid–or complex I respiration. ConclusionMitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.

Fiber Type Identification of Human Skeletal Muscle.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy. The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples. These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

THRIFTY-A High-throughput Single Muscle Fiber Typing Method Based on Immunofluorescence Detection.

Skeletal muscle consists of a mixture of fiber types with different functional and metabolic characteristics. The relative composition of these muscle fiber types has implications for muscle performance, whole-body metabolism, and health. However, analyses of muscle samples in a fiber type-dependent manner are very time consuming. Therefore, these are often neglected in favor of more time-efficient analyses on mixed muscle samples. Methods such as western blot and myosin heavy chain separation by SDS-PAGE have previously been utilized to fiber type-isolated muscle fibers. More recently, the introduction of the dot blot method significantly increased the speed of fiber typing. However, despite recent advancements, none of the current methodologies are feasible for large-scale investigations because of their time requirements. Here, we present the protocol for a new method, which we have named THRIFTY (high-THRoughput Immunofluorescence Fiber TYping), that enables rapid fiber type identification using antibodies towards the different myosin heavy chain (MyHC) isoforms of fast and slow twitch muscle fibers. First, a short segment (<1 mm) is cut off from isolated muscle fibers and mounted on a customized gridded microscope slide holding up to 200 fiber segments. Second, the fiber segments attached to the microscope slide are stained with MyHC-specific antibodies and then visualized using a fluorescence microscope. Lastly, the remaining pieces of the fibers can either be collected individually or pooled together with fibers of the same type for subsequent analyses. The THRIFTY protocol is approximately three times as fast as the dot blot method, which enables not only time-sensitive assays to be performed but also increases the feasibility to conduct large-scale investigations into fiber type specific physiology. Graphical Overview Graphical overview of the THRIFTY workflow. Cut off a small segment (0.5 mm) of an individually dissected muscle fiber and mount it onto the customized microscope slide containing a printed grid system. Using a Hamilton syringe, fixate the fiber segment by applying a small droplet of distilled water on the segment and let it fully dry (1A). The remaining large segment of the fiber should be placed in the corresponding square on a black A4 paper (1B). Once the microscope slide has been fully mounted with fiber segments, submerge the slide in a polypropylene slide mailer (illustrated as a Coplin jar in the figure) containing acetone to permeabilize the fiber segments. Thereafter, incubate the slide with primary antibodies targeting MyHC-I and MyHC-II. Following washes in PBS solution, incubate the slides with fluorescently labeled secondary antibodies, wash again, and mount with a cover glass and antifade reagent (2). Identification of fiber type can be performed using a digital fluorescence microscope (3), whereafter the remaining pieces of the fiber segments (large) are pooled together according to their fiber type or individually collected for experiments on single fibers (4). Image modified from Horwath et al. (2022).

Multimodal high-resolution nano-DESI MSI and immunofluorescence imaging reveal molecular signatures of skeletal muscle fiber types.

The skeletal muscle is a highly heterogeneous tissue comprised of different fiber types with varying contractile and metabolic properties. The complexity in the analysis of skeletal muscle fibers associated with their small size (30-50 μm) and mosaic-like distribution across the tissue tnecessitates the use of high-resolution imaging to differentiate between fiber types. Herein, we use a multimodal approach to characterize the chemical composition of skeletal fibers in a limb muscle, the gastrocnemius. Specifically, we combine high-resolution nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) with immunofluorescence (IF)-based fiber type identification. Computational image registration and segmentation approaches are used to integrate the information obtained with both techniques. Our results indicate that the transition between oxidative and glycolytic fibers is associated with shallow chemical gradients (<2.5 fold change in signals). Interestingly, we did not find any fiber type-specific molecule. We hypothesize that these findings might be linked to muscle plasticity thereby facilitating a switch in the metabolic properties of fibers in response to different conditions such as exercise and diet, among others. Despite the shallow chemical gradients, cardiolipins (CLs), acylcarnitines (CAR), monoglycerides (MGs), fatty acids, highly polyunsaturated phospholipids, and oxidized phospholipids, were identified as molecular signatures of oxidative metabolism. In contrast, histidine-related compounds were found as molecular signatures of glycolytic fibers. Additionally, the presence of highly polyunsaturated acyl chains in phospholipids was found in oxidative fibers whereas more saturated acyl chains in phospholipids were found in glycolytic fibers which suggests an effect of the membrane fluidity on the metabolic properties of skeletal myofibers.

THRIFTY; A Novel Method For Rapid Fiber Type Identification Of Isolated Skeletal Muscle Fibers

Human skeletal muscle is composed of a mixture of fiber types with distinct contractile and metabolic properties, and adaptions to exercise, aging and disease have been reported to occur in a fiber type specific manner. Fiber type specific analysis of skeletal muscle is therefore crucial to fully understand its heterogenous response to external stimuli. However, such analyses are rarely performed due to the labor-intensive process of fiber typing. Purpose: To develop a valid and fast method that enables high-throughput fiber typing of individually isolated muscle fibers. Methods: Employing THRIFTY (High THRoughput Immunofluorescence Fibre Typing), a ≤ 0.5 mm end was cut off individual fibers. The fiber ends were fixed onto a microscope slide with a pre-printed coordinated grid system. Slides were then submerged in acetone to permeabilize the fiber ends. Subsequently, the slides were stained with primary antibodies targeting MHC I & II (BA-F8 & SC-71, respectively), washed three times in PBS, and stained with fluorescent secondary antibodies. After three additional washes, a cover glass was mounted on the slide using anti-fade reagent. Fiber ends were then typed using a fluorescent microscope under 4X magnification. The THRIFTY protocol was then compared to a previously validated protocol for fiber typing using dot blot 1. A time requirement comparison between the two protocols was also carried out by a technician novice to both methods. Results: Both THRIFTY and dot blot produced pools of type I & type II muscle fibers free from contamination validated with SDS-PAGE and silver stain. Out of 400 fibers stained with both THRIFTY and dot blot, 9 fibers were considered contaminated in one of the methods, while being identified as free from contamination in the other. No fibers were identified as BA-F8 positive in one method, and SC-71 positive in the other, or vice versa. The agreement between the two methods was considered strong, κ = 0,955 (95% CI, 0,928 to 0,982), p < 0.001. Time requirements for THRIFTY was less than half that of the dot-blot protocol. Conclusion: THRIFTY is a novel and valid method for fiber typing individual muscle fibers yielding fiber pools free from contamination while drastically reducing the labor requirement, thus enabling a high throughput analyses for large scale investigations. 1) PMID: 31019216

A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus,τ2was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle,τ2was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). Fromτ2distributions, it follows that an FDB fiber withτ2> 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.

Lifetime of autofluorescence: A novel method to determine murine skeletal muscle fiber types

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes derived from a two-exponential fit (τ1 and τ2) in freshly dissected mouse FDB and soleus muscles. While τ1 values did not change between muscles, the distribution of τ2 shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes in cryosections of both muscles and paired them with immunofluorescence images of myosin heavy chain isoforms (MHC), which allow identification of fiber types. In soleus, τ2 was 3.1 ns for type I (SEM = 0.009, n = 49), 3.4 ns for type IIA (SEM = 0.01, n = 30), and 3.3 ns for type IIX (SEM = 0.01, n = 21). In FDB muscle, τ2 was 3.17 ns for type I (SEM = 0.04, n = 18), 3.5 ns for type IIA (SEM = 0.03, n = 27), and 3.62 ns for type IIX (SEM = 0.03, n = 22). From the distribution of measures, it follows that an FDB fiber with τ2 >3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first- and second-class errors estimated at 0.06 and 0.27, respectively. Studies in progress aim at further elucidating the reasons for the different lifetimes, not just among fiber types but between fibers of the same type in the two muscles. Preliminary results point at differences in both the oxidation-reduction and protein-bound versus free states of flavins as causes for the observed divergence of fluorescence lifetimes. Lifetime maps of autofluorescence therefore constitute a tool to identify fiber type that, being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.

Muscle Fiber Type Transitions with Exercise Training: Shifting Perspectives.

Human muscle fibers are generally classified by myosin heavy chain (MHC) isoforms characterized by slow to fast contractile speeds. Type I, or slow-twitch fibers, are seen in high abundance in elite endurance athletes, such as long-distance runners and cyclists. Alternatively, fast-twitch IIa and IIx fibers are abundant in elite power athletes, such as weightlifters and sprinters. While cross-sectional comparisons have shown marked differences between athletes, longitudinal data have not clearly converged on patterns in fiber type shifts over time, particularly between slow and fast fibers. However, not all fiber type identification techniques are created equal and, thus, may limit interpretation. Hybrid fibers, which express more than one MHC type (I/IIa, IIa/IIx, I/IIa/IIx), may make up a significant proportion of fibers. The measurement of the distribution of fibers would necessitate the ability to identify hybrid fibers, which is best done through single fiber analysis. Current evidence using the most appropriate techniques suggests a clear ability of fibers to shift between hybrid and pure fibers as well as between slow and fast fiber types. The context and extent to which this occurs, along with the limitations of current evidence, are discussed herein.

MyoSight\u2014semi-automated image analysis of skeletal muscle cross sections

BackgroundManual analysis of cross-sectional area, fiber-type distribution, and total and centralized nuclei in skeletal muscle cross sections is tedious and time consuming, necessitating an accurate, automated method of analysis. While several excellent programs are available, our analyses of skeletal muscle disease models suggest the need for additional features and flexibility to adequately describe disease pathology. We introduce a new semi-automated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses.ResultsWe describe staining and imaging methods that generate high-quality images of immunofluorescent-labelled cross sections from mouse skeletal muscle. Using these methods, we can analyze up to 5 different fluorophores in a single image, allowing simultaneous analyses of perinuclei, central nuclei, fiber size, and fiber-type distribution. MyoSight displays high reproducibility among users, and the data generated are in close agreement with data obtained from manual analyses of cross-sectional area (CSA), fiber number, fiber-type distribution, and number and localization of myonuclei. Furthermore, MyoSight clearly delineates changes in these parameters in muscle sections from a mouse model of Duchenne muscular dystrophy (mdx).ConclusionsMyoSight is a new program based on an algorithm that can be optimized by the user to obtain highly accurate fiber size, fiber-type identification, and perinuclei and central nuclei per fiber measurements. MyoSight combines features available separately in other programs, is user friendly, and provides visual outputs that allow the user to confirm the accuracy of the analyses and correct any inaccuracies. We present MyoSight as a new program to facilitate the analyses of fiber type and CSA changes arising from injury, disease, exercise, and therapeutic interventions.

Moving human muscle physiology research forward: an evaluation of fiber type-specific protein research methodologies.

Human skeletal muscle is a heterogeneous tissue composed of multiple fiber types that express unique contractile and metabolic properties. While analysis of mixed fiber samples predominates and holds value, increasing attention has been directed toward studying proteins segregated by fiber type, a methodological distinction termed "fiber type-specific." Fiber type-specific protein studies have the advantage of uncovering key molecular effects that are often missed in mixed fiber homogenate studies but also require greater time and resource-intensive methods, particularly when applied to human muscle. This review summarizes and compares current methods used for fiber type-specific protein analysis, highlighting their advantages and disadvantages for human muscle studies, in addition to recent advances in these techniques. These methods can be grouped into three categories based on the initial processing of the tissue: 1) muscle-specific fiber homogenates, 2) cross sections of fiber bundles, and 3) isolated single fibers, with various subtechniques for performing fiber type identification and protein quantification. The relative implementation for each unique methodological approach is analyzed from 83 fiber type-specific studies of proteins in live human muscle found in the literature to date. These studies have investigated several proteins involved in a wide range of cellular functions that are important to muscle tissue. The second half of this review summarizes key findings from this ensemble of fiber type-specific human protein studies. We highlight examples of where this analytical approach has helped to improve understanding of important physiological topics such as insulin sensitivity, muscle hypertrophy, muscle fatigue, and adaptation to different exercise programs.

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle.

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin's concordance correlation coefficients and Bland and Altman's graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

Spectral reflectance characterization and fiber type discrimination for common natural textile materials using a portable spectroradiometer

Component identification of textile materials is an important issue for studies of cultural heritage collections and science conservation. Portable spectroradiometer technology as a potential technique can be utilized to discriminate mass natural fibers rapidly and nondestructively. The goal of this study was to examine the feasibility of portable spectroradiometer to natural fiber type discrimination in the spectral range covering visible and near infrared (VNIR) (350–1000 nm), shortwave infrared 1 (SWIR1) (1000–1850 nm) and SWIR2 (1850–2500 nm). For this purpose, reflection spectra of four types of natural fibers commonly used in archaeological textiles were measured using a portable spectroradiometer under standardized conditions. The spectral features of four categories of natural fibers in different wavelength ranges were extracted and compared. To further explore the potential of discriminating the fiber types using portable spectroradiometer, principal component analysis in combination with statistical tests was applied. The results indicated that fiber type had a strong influence on the spectral features, which was much more remarkable than the influences of the intra-class variation and physical properties. Moreover, with large inter-class variation and small intra-class variation, SWIR2 was more suitable for fiber type identification than VNIR and SWIR1, which was also validated in the statistical analysis. Besides, the approach of selecting effective principal components in different wavelength ranges could provide a more optimized combination of differential information for the correct assignment of the investigated natural fiber types of textile materials. In general, portable spectroradiometer could be a powerful technique for on-site fiber type discrimination.

Automated Fiber Type Identification in SDN-Enabled Optical Networks

Network design margins are introduced by quality of transmission estimator inaccuracies. Some of those inaccuracies are due to uncertainty on the fiber type deployed in optical networks, and on the value of the chromatic dispersion of deployed fibers. We propose, in this paper, a method to identify all unknown fiber types (and estimate chromatic dispersion) in an optical network to reduce said uncertainties. We monitor, collect, centralize, and correlate chromatic dispersion values accumulated over each established light path, already measured by coherent receivers to autonomously identify fiber types without traffic interruption. Thanks to this method, we can identify the fiber type of all the 41-unknown links for a 28-node network built with four different fiber types and only 100 light paths. Our method is also capable of distinguishing two fiber types that only differ by their dispersion slope. We have also deployed and demonstrated this fiber type identification method at the ECOC 2018 demo session with real-time communication with our Lab from the demo floor. The SDN-enabled optical network is based on ONOS controller that periodically provides real-time chromatic dispersion values of active light paths needed for the autonomous fiber type identification.

Lower-Limb Dominance, Performance, and Fiber Type in Resistance-trained Men.

Large imbalances between limbs are common and potentially dangerous, yet few studies have simultaneously examined performance and physiological asymmetries. The current study examined the associations between lower-limb dominance, drop-jumping kinematics, maximal strength, and myosin heavy-chain (MHC) fiber type in the vastus lateralis. Thirteen resistance-trained men (age, 24.3 ± 2.7 yr; height, 181.4 ± 6.6 cm; mass, 87.7 ± 11.3 kg) identified their dominant (DOM) and nondominant (ND) limb, performed drop jumps (30 cm) and maximal knee extensions (1-repetition maximum, or 1RM), and provided biopsies from both vastus lateralis muscles for single-fiber (109 ± 36 per limb per person) MHC fiber-type identification (FT%). All participants selected "right" as the "preferred kicking limb" (DOM). DOM displayed a trend for a greater eccentric knee angular velocity (EKV; P = 0.083) and a significantly greater concentric knee angular velocity (CKVl P = 0.002) during drop jump. DOM also tended to be stronger than ND (64.3 ± 11.3 vs 61.0 ± 8.8 kg, P = 0.063). Slow-twitch (MHC I) fibers were more prevalent in DOM (P < 0.025), whereas ND contained more fast-twitch (MHC IIa; P < 0.025). No correlations existed between categories (jumping, 1RM, and FT%). Asymmetries of >5% were present in 6 of 12 participants for EKV, 2 of 12 for CKV, 6 of 13 for 1RM, 12 of 13 for MHC I, and 11 of 13 for MHC IIa. However, only a single participant expressed asymmetries of >5% in all dependent variables (EKV, CKV, 1RM, MHC I, and MHC IIa). Several statistically and clinically relevant asymmetries were identified. The FT% differences between lower limbs were large and common. The findings also seem to conclude that DOM was stronger, moved faster, and contained more MHC I. However, only 23% of participants actually displayed that result. This highlights the need to analyze and report both group and individual data, particularly when interpreting findings across multiple related, but not necessarily causal, measurements.

Multi-color Immunofluorescence Procedure for Rapid Identification of MHC Expression in Human Skeletal Muscle

We have previously outlined an immunofluorescence staining protocol for evaluating the fiber type composition of rat and mouse skeletal muscles. The main advantage of this protocol compared to typical myosin ATPase and immunoperoxidase procedures is that multiple myosin heavy chain (MHC) isoforms can be identified on a single cross-section, eliminating the need for multiple incubations. Additionally, detection of MHC expression directly allows for more accurate fiber type identification as differential pH-dependant inactivation of myosin ATPase has been noted across species. However, antibody cross-reactivity with human muscle must also be considered. PURPOSE: To examine the effectiveness of a rapid immunofluorescence fiber-typing procedure on human skeletal muscle. METHODS: Cross-sections of vastus lateralis muscle from eight subjects were incubated with antibodies against MHCI (BA-F8), IIa (SC-71 or 2F7), and IIx (6H1). An additional antibody, specific for all MHC isoforms except IIx (BF-35) was also used. Fiber-type specific oxidative and glycolytic capacity was measured by metabolic enzyme activity staining for succinate dehydrogenase (SDH) and glycerophosphate dehydrogenase (GPD), respectively. RESULTS: Fibers demonstrating strong reactivity with BA-F8 were classified as type I (48.9%). A considerable number of fibers stained intensely for SC-71 and 2F7 and were classified as type IIA (42.8%). A very small subset of fibers stained intermediate for both BA-F8 and SC-71 and were classified as type I/IIA (0.1%). A population of fibers displayed reactivity against 6H1; however, this population also showed intermediate staining for both MHCIIa antibodies. These fibers were identified either as pure type IIX based on their non-reactivity with BF-35 (5.6%), or as type IIAX hybrid fibers based on intermediate staining for both 6H1 and BF-35 (2.6%). Type I fibers were observed as being most oxidative and type IIX fibers as being most glycolytic, with hybrid fibers displaying enzyme activity between their respective pure counterparts. CONCLUSIONS: These results suggest that a multi-color approach using three commercially available antibodies simultaneously is effective for identifying both pure and hybrid fibers in human muscle.

Asbestos Fiber Content of Lungs With Diffuse Interstitial Fibrosis: An Analytical Scanning Electron Microscopic Analysis of 249 Cases

Asbestosis is one of many forms of diffuse interstitial pulmonary fibrosis. Its histologic diagnosis rests on the pattern of fibrosis and the presence of asbestos bodies by light microscopy in lung biopsies. To determine the asbestos fiber burden in patients with diffuse pulmonary fibrosis (DPF) who had a history of asbestos exposure, but whose biopsies did not meet established criteria for asbestosis, and compare it with the fiber burden in confirmed asbestosis cases. Fiber burden analysis was performed using scanning electron microscopy and energy-dispersive x-ray analysis of lung parenchyma from 86 patients with DPF and 163 patients with asbestosis. The correlation of the number of asbestos fibers found for a quantitative degree of fibrosis was analyzed. The fibrosis scores of the asbestosis cases correlated best with the number of uncoated commercial amphibole fibers. Seven DPF cases fell within the 95% interval of asbestos body count by light microscopy and 3 cases within that of the total commercial amphibole fiber count. Strict histologic criteria are useful for positive identification of asbestosis among cases of advanced pulmonary fibrosis. Few DPF patients with history of asbestos exposure whose biopsies did not meet the criteria for asbestosis may have asbestos fiber counts in the range seen in asbestosis, and fiber type identification by scanning electron microscopy with energy-dispersive x-ray analysis should be considered in these rare instances to avoid false-positive and false-negative diagnoses of asbestosis.

Novel myosin heavy chain immunohistochemical double staining developed for the routine diagnostic separation of I, IIA and IIX fibers

The different histochemical ATPase properties of myosins separating the muscle fiber types have been utilized in diagnostic muscle biopsy routine for more than four decades. The ATPase staining method is rather laborious and has several disadvantages, such as weakening of staining over time and non-specific staining of capillaries, making the distinction of extremely atrophic muscle fibers difficult. We have developed a reliable and advanced immunohistochemical myosin double staining method for the identification of fiber types, including highly atrophic fibers in routine diagnostics. With this double staining method, we are able to distinguish among type I (ATPase type 1), IIA (ATPase type 2A), IIX (ATPase type 2B) and remodeled ATPase type 2C fibers expressing both fast and slow myosins using a one slide technique. Immunohistochemical double staining of myosin heavy chain isoforms can be used as an alternative for the conventional ATPase staining method in routine histopathology. The method provides even more detailed information of fast fiber subtypes and highly atrophic fibers on one single slide.

Fiber types in canine muscles: myosin isoform expression and functional characterization

This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called "type 2dog" fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension (P(o)) and maximum shortening velocity (V(o)) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed (V(f)) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between V(o) and V(f) indicated that the diversity in V(o) was due to the different myosin isoform composition. V(o) increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers.

Myogenic changes of the levator ani muscle in premenopausal women: the impact of vaginal delivery and age.

The aim of this study was to evaluate the impact of parity and age on histomorphology of the pelvic floor muscles in female cadavers of reproductive age and to find out whether there is evidence of myogenic or neurogenic muscle injury. In a cross-sectional study 45 premenopausal unfixed and fresh female cadavers were studied. Four groups were defined: nulliparous and parous women under the age of 40 and over 40 years of age. The pelvic floor was biopsied at six standardized locations. For evaluation of the quantitative parameters and fiber type identification, actomyosin ATPase at pH 9.4 was used. For histomorphological evaluation, sections were stained with hematoxylin/eosin, van Gieson, and Gomori trichrome. The circumference of type I fibers is significantly larger in nulliparous women younger than 40 years compared to nulliparae older than 40 years. Comparing these groups, the form factor of type II fibers also increases significantly, presenting a more circular cell form. Compared to nulliparae, vaginal delivery led to a significant difference regarding the presence of centrally located nuclei, fibrosis, and variation in fiber diameter. In nulliparous women, these significant changes were also found with increasing age. In women with a history of vaginal delivery, no further increase in these characteristics could be detected with increasing age. Comparing the three different biopsy sites, all three changes were more pronounced in the ventral part. There was no evidence of grouped fiber atrophy, small angulated fibers, or type grouping in any of the biopsy specimens. Aging and vaginal childbirth lead to histomorphological changes of the pelvic floor muscle that are consistent with changes of myogenic origin. Evidence of neurogenic damage could not be demonstrated.

Myogenic changes of the levator ani muscle in premenopausal women: The impact of vaginal delivery and age

The aim of this study was to evaluate the impact of parity and age on histomorphology of the pelvic floor muscles in female cadavers of reproductive age and to find out whether there is evidence of myogenic or neurogenic muscle injury. In a cross-sectional study 45 premenopausal unfixed and fresh female cadavers were studied. Four groups were defined: nulliparous and parous women under the age of 40 and over 40 years of age. The pelvic floor was biopsied at six standardized locations. For evaluation of the quantitative parameters and fiber type identification, actomyosin ATPase at pH 9.4 was used. For histomorphological evaluation, sections were stained with hematoxylin/eosin, van Gieson, and Gomori trichrome. The circumference of type I fibers is significantly larger in nulliparous women younger than 40 years compared to nulliparae older than 40 years. Comparing these groups, the form factor of type II fibers also increases significantly, presenting a more circular cell form. Compared to nulliparae, vaginal delivery led to a significant difference regarding the presence of centrally located nuclei, fibrosis, and variation in fiber diameter. In nulliparous women, these significant changes were also found with increasing age. In women with a history of vaginal delivery, no further increase in these characteristics could be detected with increasing age. Comparing the three different biopsy sites, all three changes were more pronounced in the ventral part. There was no evidence of grouped fiber atrophy, small angulated fibers, or type grouping in any of the biopsy specimens. Aging and vaginal childbirth lead to histomorphological changes of the pelvic floor muscle that are consistent with changes of myogenic origin. Evidence of neurogenic damage could not be demonstrated. Neurourol. Urodynam. 17:197–205, 1998. © 1998 Wiley-Liss, Inc.