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Currently, there are plenty of histochemical methods to classify pig muscle fibers, which confused the naming and classification of muscle fibers. This study aims to analyze the difference and correlation of 6 different histochemical methods and select the most suitable method for muscle fiber classification at the molecular and histomological levels by in-situ RT-PCR and enzyme histochemical methods. Muscle fiber samples, including psoas (PM), semitendinosus (SM) and trapezius muscle (TM), were collected from Large Spotted (LS), Lantang (LT) and Landrace (LR) pigs at their market-ages (LS at 150 d, LT at 210 d, and LR at 150 d). 6 kinds of histochemical methods combining actomyosin adenosine triphosphatase (AM-ATPase) with succinate dehydrogenase (SDH) enzyme were conducted to differentiate fiber types. 2 types of fibers (I and II) were differentiated by acid 2-fibre (2-AC) or alkaline 2-fibre classification(2-AL), 3 types of fibers (βR, αR and αW) by 3-AC or 3-AL, and 4 types of fibers (I, IIa, IIx and IIb) by 4-AC, or 4-AL. Results showed that AC and AL muscle-fiber classification were consistent in reflecting the characteristics of muscle fibers(P > 0.05), but the color of each muscle fiber type was just opposite. AC methods may be superior to AL methods because of their clear staining background, the sensitivity to staining condition. But there were breed differences and tissue specificity in the optimal preincubation condition. The optimal acid preincubation condition for classifying muscle fibers was pH4.30 for LT, while pH 4.35 for the LS and LR pigs. Meanwhile the optimal acid preincubation condition was pH4.35 for PM, while pH4.40 for TM or SM. For further selection from 2, 3, 4-AC, in-situ RT-PCR was applied to detect the mRNA distribution of myosin heavy chain I (MyHC-I). By combining in-situ PCR with enzyme histochemistry methods, MyHC-I gene and its product – Type I fibrocytes were directly located in cells at both molecular level and morphological level. Compared with the cross-sectional area (CSA) of different muscle fibers (i.e. I, II, βR, αR, αW, IIa, IIx and IIb) identified by enzyme histochemistry, it was found that the CSAs with stronger mRNA expression signal of MyHC-Ⅰ were closer to those of the Type I muscle fiber measured by 4-AC enzyme histochemistry (P > 0.05). Therefore, 4-AC may be considered as the most proper muscle typing method to study muscle fiber typing as well as meat quality. And the combination of in-situ RT-PCR and histochemistry may help better understand porcine muscle fiber characteristics and meat quality in pigs.

The tub gurnard Chelidonichthys lucerna (Linnaeus, 1758), Triglidae, is an opportunistic, demersal carnivorous fish. Data on the digestive enzymes of tub gurnard have not been reported in the literature. Therefore, the aim of this research was to investigate the distribution and intensity of alkaline phosphatase, acid phosphatase, non-specific esterase, and aminopeptidase in the digestive tract of tub gurnard. To investigate data about those enzymes tissue samples of the esophagus, anterior and posterior part of the stomach, pyloric caeca, anterior, middle and posterior part of the intestine proper, and rectum were taken. Azo-coupling methods were used to detect the enzymatic reactions. The intensities of the reactions were measured using ImageJ software. Alkaline phosphatase, acid phosphatase, and non-specific esterase activities were found in all parts of the digestive tract. The brush border of the pyloric caeca and intestine proper were the main sites of alkaline phosphatase reaction, with intensity decreasing toward the posterior parts of the digestive tract. The high intensities of acid phosphatase were found in the epithelium of the anterior part of the stomach, pyloric caeca, anterior part of the intestine proper, and in the rectum. The intensity of non-specific esterase was mainly increased from the anterior to the posterior parts of the digestive tract. Aminopeptidase activity was found in the esophagus, pyloric caeca, and intestine proper. Our results suggest that the entire digestive tract of the tub gurnard is involved in the digestion and absorption of dietary components. [ABSTRACT FROM AUTHOR]

Introduction: This is the first study mapping the duration of action of in vivo photobiomodulation (PBM) on cytochrome-c-oxidase (CCO). In cellular bioenergetics, CCO is the terminal rate-limiting enzyme in the mitochondrial electron transport chain, which catalyzes oxygen utilization for aerobic energy production. PBM using transcranial infrared laser stimulation (TILS) is a promising intervention for non-invasively modulating CCO in the brain. TILS of the human prefrontal cortex directly causes CCO photo-oxidation, which is associated with increased cerebral oxygenation and improved cognition. Methods: This experiment aimed to map the duration of action of in vivo PBM on CCO activity in discrete neuroanatomic locations within rat brains up to 4 weeks after a single TILS session (50 s, 1064 nm CW, 250 mW/cm2). Control brains from rats treated with a sham session without TILS (laser off) were compared to brains from TILS-treated rats that were collected 1 day, 2 weeks, or 4 weeks post-TILS. Cryostat sections of the 36 collected brains were processed using quantitative enzyme histochemistry and digitally imaged. Densitometric readings of 28 regions of interest were recorded and converted to CCO activity units of oxygen utilization using calibration standards. Data analysis (ANCOVA) compared each laser-treated group to sham with whole-brain average as a covariate. Results: The prefrontal infralimbic cortex showed the earliest significant increase in CCO activity between 1-day post-TILS and sham groups, which continued elevated for 2–4 weeks post-TILS. Significant differences in CCO activity between 2-weeks and sham groups were also found in the lateral septum, accumbens core, CA3 of the hippocampus, and the molecular layer of the hippocampus. The medial amygdala showed a significant decrease in CCO activity between 4-weeks and sham. Further analyses showed significant inter-regional CCO activity correlations among the brain regions as the result of TILS, with the most pronounced changes at 4-weeks post-stimulation. Discussion: The time course of changes in CCO activity and network connectivity suggested that TILS caused different neuroplasticity types of bioenergetic changes at different time scales, depending on brain region and its depth from the cortex. In conclusion, this controlled CCO histochemical study demonstrated a long-lasting duration of action of PBM in the rat brain. [ABSTRACT FROM AUTHOR]

At the occasion of the 65th anniversary of Histochemistry and Cell Biology, we browse through its first ten years of publication and highlight a selection of papers from the early days of enzyme, protein, and carbohydrate histochemistry. In addition, we narrate recent progress to identify, quantify, and precisely determine the tissue localization of proteins and lipids, and small molecules by the combination of spectroscopic techniques and histology. [ABSTRACT FROM AUTHOR]

This study provides a combined histochemical method for detecting enzyme activity of chloroacetate esterase simultaneously with immunolabeling of the components of a specific tissue microenvironment on formalin-fixed, paraffin-embedded specimens. Chromogenic detection of the molecular targets within and outside the mast cells provides novel options in determining the histoarchitectonics of organ-specific mast cell populations, studying the functional significance of chloroacetate esterase and specifying the immune landscape of the tissue microenvironment. [ABSTRACT FROM AUTHOR]

Background: The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. Methods: The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). Results: The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved. [ABSTRACT FROM AUTHOR]

Background The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. Methods The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). Results The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved.

Selective neuronal vulnerability (SNV) of specific neuroanatomical regions such as frontal cortex (FC) and hippocampus (HC) is characteristic of age-associated neurodegenerative diseases (NDDs), although its pathogenetic basis remains unresolved. We hypothesized that physiological differences in mitochondrial function in neuroanatomical regions could contribute to SNV. To investigate this, we evaluated mitochondrial function in human brains (age range:1–90 y) in FC, striatum (ST), HC, cerebellum (CB) and medulla oblongata (MD), using enzyme assays and quantitative proteomics. Striking differences were noted in resistant regions- MD and CB compared to the vulnerable regions- FC, HC and ST. At younger age (25 ± 5 y), higher activity of electron transport chain enzymes and upregulation of metabolic and antioxidant proteins were noted in MD compared to FC and HC, that was sustained with increasing age (≥65 y). In contrast, the expression of synaptic proteins was higher in FC, HC and ST (vs. MD). In line with this, quantitative phospho-proteomics revealed activation of upstream regulators (ERS, PPARα) of mitochondrial metabolism and inhibition of synaptic pathways in MD. Microtubule Associated Protein Tau (MAPT) showed overexpression in FC, HC and ST both in young and older age (vs. MD). MAPT hyperphosphorylation and the activation of its kinases were noted in FC and HC with age. Our study demonstrates that regional heterogeneity in mitochondrial and other cellular functions contribute to SNV and protect regions such as MD, while rendering FC and HC vulnerable to NDDs. The findings also support the "last in, first out" hypothesis of ageing, wherein regions such as FC , that are the most recent to develop phylogenetically and ontogenetically, are the first to be affected in ageing and NDDs. [Display omitted] • The processes contributing to region-specific selective neuronal vulnerability (SNV) and their accrual during aging is underexplored in human brains. • We noted that regional mitochondrial, antioxidant and synaptic heterogeneity based on expression and phosphorylation dynamics underlies the SNV of frontal cortex (FC) and hippocampus (HC) to neurodegeneration. • Expression and phosphorylation profile of microtubule associated protein tau (MAPT) and associated kinases during ageing in FC and HC, also probably contributes to SNV. • Regions that develop first [medulla oblongata (MD)] are defined by relatively high mitochondrial, metabolic and antioxidant function contributing to neuroprotection, compared to vulnerable areas such as FC, HC, and striatum (ST), that develop later, providing the biological basis for the "last in first out" hypothesis of ageing. [ABSTRACT FROM AUTHOR]

Histopathological analysis of muscle biopsy is a prerequisite in the evaluation of neuromuscular disorders, particularly inflammatory myopathies, metabolic myopathies, congenital myopathies, muscular dystrophies and differentiating myopathies and neurogenic disorders with overlapping clinically features. It not only provides useful information that helps in the diagnosis but also treatment and management. Fundamental skills and basic knowledge regarding handling, processing and analyzing a muscle biopsy are required in any specialized or a general pathology lab supporting neuromuscular clinical services. Care during transport of the muscle biopsy, sample receipt in the laboratory and grossing is very important. Standard operating procedure should be followed for the preanalytical steps (freezing and cryomicrotomy), routine and special staining (enzyme and non enzymatic) and immunohistochemistry. A well organized neuromuscular laboratory with good quality management system is necessary for the practice of myopathology. This article gives an overview of establishing such a laboratory.

Within the history of neuromuscular diseases (NMD), congenital myopathies (CM) represent a relatively new category introduced in the mid-nineteen hundreds upon advent and subsequent application of enzyme histochemistry and electron microscopy by establishing the three major CM, central core disease, nemaline myopathy, and centronuclear myopathy which later pluralized each when the molecular era began at the end of last century. Quickly, during the following 5 decades, many new CM entities were described, based on muscle biopsies and their CM-characteristic myopathology, the former a prerequisite to recognizing an individual CM, the latter of the nosological hallmark of the individual CM. When the molecular era ushered in immunohistochemistry the spectrum and nosography of CM altered in that some CM became allelic to other cohorts of NMD, e.g., congenital muscular dystrophies, other muscular dystrophies, distal myopathies based on different or identical mutations in the same gene. The nosological spectrum of a defective gene also enlarged by recognizing several entities with mutations in the same gene, and same or similar nosological conditions originated from mutations in different genes. Lately, however, CM were reported which lacked any individual myopathological hallmarks, but were clearly based on molecular defects, a fair number of them being newly identified ones. Few CM still remain without any molecular clarification. This nosographic development rendered the original definition of such new CM questionable and brought uncertainty to their classification and nomenclature.

The colonial ascidian Botryllus schlosseri possesses an innate immunity, which plays fundamental roles in its survival, adaptability, worldwide spread and ecological success. Three lines of differentiation pathways of circulating haemocytes are known to be present in the haemolymph, starting from undifferentiated haemoblasts: (i) the phagocytic line (hyaline amoebocytes and macrophage-like cells), (ii) the cytotoxic line (granular amoebocytes and morula cells) and (iii) the storage cell line (pigment cells and nephrocytes). Many questions remain about their origin, and thus, observations during various stages of development were undertaken in this study. Haemocytes were detected beginning from the early tailbud embryo stage. Haemoblasts were always present and morula cells were the first differentiated haemocytes detected. In both the next stage, just before hatching, and the swimming tadpole larva stage, hyaline amoebocytes and pigment cells were also recognisable. Some morula cells containing active phenoloxidase migrated from the haemolymph into the tunic after having crossed the epidermis, and this behaviour could be related to the preparation of a defensive function for spatial competition. During larval metamorphosis, macrophage-like cells appeared with their phagosomes positive to acid phosphatase activity and containing apoptotic cells from tail tissue degeneration. After metamorphosis, in the filter-feeding oozoid stage, nephrocytes involved in nitrogen catabolism finally appeared. In both the subendostylar sinus and the peripheral blind-sac vessels (ampullae), clusters of haemoblasts were recognisable, some of which showed incipient specialisations, considering the hypothesis of the presence of putative niches of haemolymph stem cells. [ABSTRACT FROM AUTHOR]

Objective: Congenital myopathies (CMs) are rare neuromuscular disorders. Through this article, authors want to present a clinicopathological study of 10 cases of CM. Materials and Methods: The study included patients with histopathologically confirmed CM attending the neurology services at the Institute of Human Behavior and Allied Sciences for 2 years. After collecting the demographic data, all patients were subjected to comprehensive workup including a detailed neurological examination and investigations, including muscle biopsy from representative involved muscle. Results: Ten patients diagnosed with CM. The most common CM type was congenital fiber-type disproportion (CFTD) seen in four cases followed by centronuclear myopathy in two cases and one each in desmin-related myopathy, central core disease, nemaline myopathy, CM with type II fiber hypoplasia. Clinically, they have variable features. Conclusion: This study from India highlights the importance of specific clinical features to look for when suspecting a CM coupled with specific features in histopathology. However, studies with longer duration are needed to find out the true prevalence and various spectra of CMs. [ABSTRACT FROM AUTHOR]

The colonial ascidian Botryllus schlosseri possesses an innate immunity, which plays fundamental roles in its survival, adaptability, worldwide spread and ecological success. Three lines of differentiation pathways of circulating haemocytes are known to be present in the haemolymph, starting from undifferentiated haemoblasts: (i) the phagocytic line (hyaline amoebocytes and macrophage-like cells), (ii) the cytotoxic line (granular amoebocytes and morula cells) and (iii) the storage cell line (pigment cells and nephrocytes). Many questions remain about their origin, and thus, observations during various stages of development were undertaken in this study. Haemocytes were detected beginning from the early tailbud embryo stage. Haemoblasts were always present and morula cells were the first differentiated haemocytes detected. In both the next stage, just before hatching, and the swimming tadpole larva stage, hyaline amoebocytes and pigment cells were also recognisable. Some morula cells containing active phenoloxidase migrated from the haemolymph into the tunic after having crossed the epidermis, and this behaviour could be related to the preparation of a defensive function for spatial competition. During larval metamorphosis, macrophage-like cells appeared with their phagosomes positive to acid phosphatase activity and containing apoptotic cells from tail tissue degeneration. After metamorphosis, in the filter-feeding oozoid stage, nephrocytes involved in nitrogen catabolism finally appeared. In both the subendostylar sinus and the peripheral blind-sac vessels (ampullae), clusters of haemoblasts were recognisable, some of which showed incipient specialisations, considering the hypothesis of the presence of putative niches of haemolymph stem cells.

Objective: To assess the distribution pattern of Langerhans cells in oral lichen planus using adenosine triphosphatase (ATPase) histochemistry and to correlate this with the duration of symptoms. Method and Materials: Included were fresh unfixed tissues from previously untreated cases that were clinically and histologically diagnosed as oral lichen planus (n = 18). Healthy oral mucosal tissues were used as controls (n = 5). ATPase activity in the Langerhans cells from the tissue samples was assessed by enzyme histochemistry. Results: The difference in the distribution pattern of Langerhans cells in the superficial and basal half of the epithelium in both the lesional and control tissue was significant (P < .001, Mann-Whitney U test). With an increase in duration of symptoms, the median migration of cells from superficial position to the basal half was significant (P = .001, Kruskal-Wallis test). A negative correlation (Pearson correlation coefficient) was seen between duration of symptoms and migration of cells. Conclusion: The initial increase in the number of Langerhans cells in the lesional tissues compared to controls suggests that these cells are critically required for both the initiation and progression of the mucosal immune response in oral lichen planus. Variation in their number and distribution bear clinical importance as objective assessment of long-standing lesions could be made with reference to treatment planning. [ABSTRACT FROM AUTHOR]

Currently, there are plenty of histochemical methods to classify pig muscle fibers, which confused the naming and classification of muscle fibers. This study aims to analyze the difference and correlation of 6 different histochemical methods and select the most suitable method for muscle fiber classification at the molecular and histomological levels by in-situ RT-PCR and enzyme histochemical methods. Muscle fiber samples, including psoas (PM), semitendinosus (SM) and trapezius muscle (TM), were collected from Large Spotted (LS), Lantang (LT) and Landrace (LR) pigs at their market-ages (LS at 150 d, LT at 210 d, and LR at 150 d). 6 kinds of histochemical methods combining actomyosin adenosine triphosphatase (AM-ATPase) with succinate dehydrogenase (SDH) enzyme were conducted to differentiate fiber types. 2 types of fibers (I and II) were differentiated by acid 2-fibre (2-AC) or alkaline 2-fibre classification(2-AL), 3 types of fibers (βR, αR and αW) by 3-AC or 3-AL, and 4 types of fibers (I, IIa, IIx and IIb) by 4-AC, or 4-AL. Results showed that AC and AL muscle-fiber classification were consistent in reflecting the characteristics of muscle fibers( P > 0.05), but the color of each muscle fiber type was just opposite. AC methods may be superior to AL methods because of their clear staining background, the sensitivity to staining condition. But there were breed differences and tissue specificity in the optimal preincubation condition. The optimal acid preincubation condition for classifying muscle fibers was pH4.30 for LT, while pH 4.35 for the LS and LR pigs. Meanwhile the optimal acid preincubation condition was pH4.35 for PM, while pH4.40 for TM or SM. For further selection from 2, 3, 4-AC, in-situ RT-PCR was applied to detect the mRNA distribution of myosin heavy chain I (MyHC-I). By combining in-situ PCR with enzyme histochemistry methods, MyHC-I gene and its product - Type I fibrocytes were directly located in cells at both molecular level and morphological level. Compared with the cross-sectional area (CSA) of different muscle fibers (i.e. I, II, βR, αR, αW, IIa, IIx and IIb) identified by enzyme histochemistry, it was found that the CSAs with stronger mRNA expression signal of MyHC-Ⅰ were closer to those of the Type I muscle fiber measured by 4-AC enzyme histochemistry ( P > 0.05). Therefore, 4-AC may be considered as the most proper muscle typing method to study muscle fiber typing as well as meat quality. And the combination of in-situ RT-PCR and histochemistry may help better understand porcine muscle fiber characteristics and meat quality in pigs., Competing Interests: We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript., (© 2024 The Author(s).)

Acrylamide is an important industrial chemical; it also is formed in starch-rich foodstuffs during baking, frying and roasting. Most acrylamide exposure occurs by ingestion of processed foods. We investigated possible immunotoxic effects of extended administration of low doses of acrylamide in rats. To do this, we measured alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) activities in peripheral blood lymphocytes. Male and female weanling Wistar rats were administered 2 or 5 mg acrylamide/kg/day in drinking water for 90 days. Peripheral blood was sampled at the end of the administration period. We found ANAE staining in eosinophils and T-lymphocytes, but not in monocytes, platelets, B-lymphocytes and neutrophils. ACP-ase was found in B-lymphocytes. We found a significant reduction of the ratio of ANAE:ACP-ase in lymphocytes of the experimental animals compared to controls. We found no statistically significant differences between the doses or sexes. We found that acrylamide ingested in processed foods might affect the immune system adversely by decreasing the population of mature T- and B-lymphocytes. [ABSTRACT FROM AUTHOR]

Abstract: Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age‐related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh‐frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase‐deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels – this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX‐deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell‐by‐cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Pompe disease (PD) is a lysosomal storage disorder characterized by glycogen accumulation in muscle, with infantile-onset (IOPD) and late-onset (LOPD) types. Nineteen cases of PD were diagnosed over a 14-year period on muscle biopsy by ultrastructural examination. Pools of glycogen (intralysosomal and cytoplasmic) and excessive phagocytosis were seen in myofibers on electron microscopy. Glycogen was noted in endothelial cells in IOPD. Although PD accounts for a small fraction of muscle diseases, timely accurate diagnosis is imperative as it is treatable. Ultrastructural examination is necessary to confirm the diagnosis in cases with non-diagnostic light microscopic features, especially in adult LOPD patients. [ABSTRACT FROM AUTHOR]

Aim: The present study was aimed at elucidating the distribution of various enzymes in the submandibular salivary gland of buffalo during prenatal development and correlation of these enzyme systems with the secretory activity of the gland. Materials and Methods: The study was carried out on submandibular salivary gland of 15 buffalo fetuses ranging from 11 cm curved crown-rump length (CVRL) (78 days) to 100 cm CVRL (full term). The fetuses were categorized into three groups based on their CVRL. Results: A weak activity of phosphatases and oxidoreductases was observed in the acinar cells and ductular epithelium at 11-19 cm CVRL (78-114 days). From 28 to 37 cm CVRL (136-157 days), a weak to moderate diffused granular alkaline phosphatase (AKPase) activity was observed in the seromucous acini whereas oxidoreductases showed moderate activity. The enzyme activity showed progressively increased with the advancement of the gestation period. The AKPase activity was more in the lumen of acini and along the intercellular canaliculi at 42-100 cm of CVRL (168 days - full term). Large ducts exhibited strong activity for oxidoreductases indicating increased metabolic activity of the cells. Conclusion: The fetuses of Group I showed a uniform weak activity in the acinar cells and ductular epithelium of the gland. In Group II, the enzymes showed a weak to moderate activity which progressively increased with the advancement of gestation period. The enzymes related to Group III showed a strong positive activity for enzymes which reflected higher secretory activity of the gland.

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised. [ABSTRACT FROM AUTHOR]

The presence of lesions on the pig carcass is an indicator of poor animal welfare and has economic impact as it downgrades the carcass value. The assessment of the age of lesions on the carcass may help identify risk factors and ultimately prevent their occurrence. The aim of this study was to assess the age of lesions on pig carcasses through spectrophotometric color evaluation and to relate the results with gene expression and histological and histochemical parameters. A total of 96 barrows were mixed 4 times over 3 d before slaughter and 80 lesions were selected after skin lesion observations to define 4 age categories: < 7 h (T1), 7-25 h (T2), 25-30 h (T3), and 49-54 h (T4). A nonlesioned skin area was used as a control. At slaughter, 3 biopsies per lesion and control skin were taken immediately after bleeding for analyses of gene expression (CCL2, COX2, IL6, IL8, IL10, ITGA3, MMP1, TNFα, TIMP1, SERPINE1), skin histological characteristics (inflammation, erosion or ulceration, and necrosis), and enzyme activity (alkaline phosphatase and adenosine triphosphatase). The number of lesions was counted on each carcass, and the color was assessed visually by a pictorial chart and instrumentally through a spectrophotometer. Delta values (Δ) were calculated as the difference between the value of the lesion and the value of the control for all measures, except for the histological analysis. Results indicated that visual color observation was not sufficiently accurate to discriminate lesions by time of infliction (P > 0.10), while the spectrophotometer ΔL* and Δa* values variation allowed the identification of < 7 h or > 25 h old lesions (P < 0.05). Similarly, the expression of CCL2, IL6, ITGA3, MMP1, and SERPINE1 genes was higher (P < 0.05) in < 7 h old lesions, while TIMP1 gene expression was higher (P < 0.05) in < 25 h old lesions. As for the histological analysis, the severity of inflammation was correlated with the age of the lesion (lower score in < 7 h old lesions and higher score in > 25 h old lesions; P < 0.05). To conclude, the spectrophotometric color assessment of the carcass lesions at slaughter appears to be a reliable method to discriminate between fresh and older lesions on the carcass at the abattoir. [ABSTRACT FROM AUTHOR]

Objective To investigate the influence of increasing the occlusal vertical dimension (iOVD) on the fibre-type distribution and ultrastructure of deep masseter of rat at different ages. Design A total of forty-eight male Wistar rats were divided into two groups according to age: ‘teenage’ group (n = 24, 1.5 months) and ‘young adult’ group (n = 24, 8 months). Both the teenage and the young adult rats were then randomly divided into the control group (n = 12) and the experimental group (n = 12). The occlusal vertical dimensions of the rats in the experimental groups were increased by placing composite resin on all maxillary molars. The fibre-type distribution and ultrastructure of the deep masseter were subsequently observed on day 7 and day 14 after iOVD. Results In the teenage experimental group, the proportion of type IIa fibres increased, while the proportion of type IIb and type IIx fibres decreased by day 7 after iOVD ( P < 0.05). However, no significant fibre phenotype transformation was observed in the young adult experimental group until day 14 after iOVD. In addition, the proportion of type IIa in the teenage experimental group was higher than that of the young adult experimental group on day 7 and 14 ( P < 0.05). Under the transmission electron microscope, muscle fibre reconstruction and the compensatory increase in the number and volume of mitochondria appeared earlier in the teenage experimental group. The cellular traumatic reaction was less than that in the young adult experimental group. Conclusion The teenage rat alters masseter muscle structure to a slower phenotype earlier and to a greater degree than that of the young adult rat when increasing the occlusal vertical dimension. [ABSTRACT FROM AUTHOR]

Objective: Congenital myopathies are rare. Through this article, the authors want to present a clinicopathological analysis of 25 new cases. Materials and methods: The clinical data of patients who were diagnosed with congenital myopathy between 2001 and 2006 was retrieved. Muscle biopsies were processed for H&E staining, enzyme histochemistry, and immunohistochemistry. Biopsies were also processed for ultrastructural analysis. Results: During a period of 6 years, 1.12% of the muscle biopsies were diagnosed as congenital myopathies. The most common congenital myopathy was central core disease followed by nemaline rod myopathy and multi-mini core disease. Clinically, they have variable features. The final diagnosis was made with the help of enzyme histochemistry and ultrastructural features. Conclusion: This study emphasizes the importance of enzyme histochemistry and electron microscopic examination in the diagnosis of congenital myopathies especially in the absence of genetic studies.

Peroxidase may play roles in innate host immunity and affect the sperm survivability in hen oviduct. The aim of this study was to localize the peroxidase activity in the oviduct of hens and to examine whether the activity is affected by the developmental stages of the oviduct. The oviducts were collected from immature, laying and non-laying White Leghorn hens. Formalin-fixed oviductal tissues were processed for frozen sections and incubated with reaction mixture containing diaminobenzidine and H2O2. No peroxidase activity products were found in the surface epithelium of the infundibulum, magnum, uterus and uetero-vaginal junction (UVJ), in the tubular glands of magnum, isthmus and uterus, and in the sperm storage tubules in the UVJ. The activity was occasionally observed in the surface epithelial cells in the isthmus. The surface epithelial cells of the vagina contained dense peroxidase reaction products, which were distributed at the bottom region of the secondary folds. The density of reaction products and their distribution were reduced in non-laying hens, and the oviducts of immature hens contained no peroxidase reaction products. These results suggest that the surface epithelium of the vagina contains peroxidase activity in laying hens, which may play roles in innate host immunity and affect the sperm viability passing the vagina. The vaginal peroxidase activity is likely to be changed in association with the developmental stage of the oviduct.

The renal biopsy is a dynamic way of looking at renal disease, and tubular elements are an important part of this analysis. The mitochondria in 20 renal biopsies were examined by immunohistochemical (electron transport chain enzyme: cytochrome C oxidase IV [COX IV]) and enzyme histochemical methods (COX), both by light and electron microscopy. The distal convoluted tubules and thick ascending limbs showed the greatest intensity in the COX immunostains and enzyme activity in controls. The degree of mitochondrial COX protein and enzyme activity diminished as the tubules became atrophic. With proximal hypertrophic changes, there was great variation in both COX activity and protein expression. In contrast, in three cases of systemic lupus erythematosus, biopsied for high-grade proteinuria, the activity was consistently upregulated, whereas protein expression remained normal. These unexpected findings of heterogeneous upregulation in hypertrophy and the dyssynchrony of protein expression and activity may indicate mitochondrial dysregulation. Functional electron microscopy showed COX activity delineated by the intense mitochondrial staining in normal or hypertrophic proximal tubules. With atrophic changes, residual small mitochondria with diminished activity could be seen. With mitochondrial size abnormalities (enlargement and irregularity, adefovir toxicity), activity persisted. In the renal biopsy, mitochondrial analysis is feasible utilizing immunohistochemical and enzyme histochemical techniques. [ABSTRACT FROM AUTHOR]

Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p < 0.05). In addition, 24 h following crush injury, focal areas of highly elevated thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of thrombin in physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Tripeptidyl aminopeptidase I (TPP I) is a lysosomal exopeptidase that is widely distributed throughout the central nervous system (CNS) and internal organs in many mammalian species. The enzyme is involved in the breakdown of collagen and different peptides. The carotid body (CB) is the main peripheral arterial chemoreceptor playing an important role in the control of breathing and the autonomic control of cardiovascular function. In response to hypoxia its neuron-like glomus cells release a variety of peptide transmitters that trigger an action potential through the afferent fibers, thus conveying the chemosensory information to the CNS. In the present study we investigated the histochemical localization of TPP I in the CB of rats. Enzyme histochemistry showed high activity of TPP I in CB glomeruli. In particular, the glomus cells contained many TPP I-positive granules, while the glial-like sustentacular cells displayed a slightly fainter reaction. The interglomerular connective tissue was also weakly stained. The results show that both the parenchymal cells of the rat CB express, albeit with different intensity, TPP I. Taken together with our previous enzyme histochemical investigations on the rat CB, it seems likely that the glomus cells possess enzymatic equipment necessary for the neuropeptide intracellular and collagen extracellular initial degradation. These findings also suggest that TPP I is involved in the general turnover of chemotransmitters between glomus cells and sensory nerve endings which emphasizes its importance for chemoreception under hypoxic conditions. [ABSTRACT FROM AUTHOR]

Proper kidney function depends highly on mitochondria homeostasis. This organelle is the primary source of ATP production in the kidney and regulates other cellular processes such as redox and calcium homeostasis. Although the mitochondria's primary recognized function is cellular energy production, through the function of the Krebs cycle, electron transport system (ETS), as well as oxygen and electrochemical gradient consumption, this function is interconnected with multiple signaling and metabolic pathways, making bioenergetics a central hub in renal metabolism. Furthermore, mitochondrial biogenesis, dynamics, and mass are also strongly related to bioenergetics. This central role is not surprising given that mitochondrial impairment, including functional and structural alterations, has been recently reported in several kidney diseases. Here, we describe assessment of mitochondrial mass, structure, and bioenergetics in kidney tissue and renal-derived cell lines. These methods allow investigation of mitochondrial alterations in kidney tissue and renal cells under different experimental conditions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aim: The present study was aimed at elucidating the distribution of various enzymes in the submandibular salivary gland of buffalo during prenatal development and correlation of these enzyme systems with the secretory activity of the gland. Materials and Methods: The study was carried out on submandibular salivary gland of 15 buffalo fetuses ranging from 11 cm curved crown-rump length (CVRL) (78 days) to 100 cm CVRL (full term). The fetuses were categorized into three groups based on their CVRL. Results: A weak activity of phosphatases and oxidoreductases was observed in the acinar cells and ductular epithelium at 11-19 cm CVRL (78-114 days). From 28 to 37 cm CVRL (136-157 days), a weak to moderate diffused granular alkaline phosphatase (AKPase) activity was observed in the seromucous acini whereas oxidoreductases showed moderate activity. The enzyme activity showed progressively increased with the advancement of the gestation period. The AKPase activity was more in the lumen of acini and along the intercellular canaliculi at 42-100 cm of CVRL (168 days - full term). Large ducts exhibited strong activity for oxidoreductases indicating increased metabolic activity of the cells. Conclusion: The fetuses of Group I showed a uniform weak activity in the acinar cells and ductular epithelium of the gland. In Group II, the enzymes showed a weak to moderate activity which progressively increased with the advancement of gestation period. The enzymes related to Group III showed a strong positive activity for enzymes which reflected higher secretory activity of the gland. [ABSTRACT FROM AUTHOR]

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining. [ABSTRACT FROM AUTHOR]

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar. [ABSTRACT FROM AUTHOR]

We evaluated the histologic and histochemical (succinate dehydrogenase, SDH) changes in cochleas of guinea pigs after non-focused ultrasound (NFU) irradiation. For this purpose, the cochleas of 50 guinea pigs (both ears in Groups 1-10) were irradiated at 2.5-8.0 MHz NFU for 6 h and the histologic/histochemical changes were determined. Our data show that after NFU irradiation for 6 h, no histological changes were observed in the cochleas by silver staining and scanning electron microscopic examination; however, the SDH activity of the basilar membranes and stria vascularises at the corresponding position decreased significantly. The SDH values of basilar membranes and stria vascularis were higher at 8 h than at 30 min after the NFU irradiation. SDH activity of the outer hair cells presented more declination than that of the inner hair cells. It was, therefore, concluded that a certain dosage of NFU irradiation at various frequencies could lead to metabolic changes in the basilar membrane and stria vascularis at different areas of cochlea. Moreover, these changes were found to be reversible or partially reversible. These changes also suggest that the cochlear hair cells located at different areas might be related to ultrasonic perception. [ABSTRACT FROM AUTHOR]

This study provides evidence for the Golgi-like activity of the multilayered interlaced network ( MIN) and new ultrastructural observations of the MIN in the sporoplasm of Anncaliia algerae, a microsporidium that infects both insects and humans. The MIN is attached to the end of the polar tubule upon extrusion from the germinating spore. It surrounds the sporoplasm, immediately below its plasma membrane, and most likely maintains the integrity of the sporoplasm, as it is pulled through the everting polar tube. Furthermore, the MIN appears to deposit its dense contents on the surface of the sporoplasm within minutes of spore discharge thickening the plasma membrane. This thickening is characteristic of the developmental stages of the genus Anncaliia. The current study utilizes transmission electron microscopy ( TEM), enzyme histochemistry, and high voltage TEM ( HVEM) with 3D tomographic reconstruction to both visualize the structure of the MIN and demonstrate that the MIN is a Golgi-related structure. The presence of developmentally regulated Golgi in the Microsporidia has been previously documented. The current study extends our understanding of the microsporidial Golgi and is consistent with the MIN being involved in the extracellular secretion in Anncaliia algerae. This report further illustrates the unique morphology of the MIN as illustrated by HVEM using 3D tomography. [ABSTRACT FROM AUTHOR]

Abstract: Odontogenic keratocyst (OKC, currently designated by the World Health Organization as a keratocystic odontogenic tumor) is a locally aggressive, cystic jaw lesion with a putative high-growth potential and a propensity for recurrence. Although it is generally agreed that some features of OKCs are those of a neoplasia, notably the relatively high proliferative rate of epithelial cells, controversies over the behavior and management of OKCs still exist. Although the question of whether the OKC is a cyst or a neoplasm is yet to be answered with certainty, recent advances in genetic and molecular research i.e. PTCH1 mutations and involvement of the Hedgehog signaling pathway have a facilitated better understanding of its pathogenesis, hinting at potential novel treatment options. While some surgeons suggest a more conservative approach to treating OKCs, notably marsupialization and decompression, future treatment strategies focus on molecular approaches that may eventually reduce or eliminate the need for aggressive surgical intervention. In addition to describing a case that was managed conservatively, we review the treatment of odontogenic keratocyst, briefly discussing recent trends that help make diagnosis and therapy more specific and effective. [Copyright &y& Elsevier]

Abstract: Objective: To explore the effect of AIBL on Oncomelania hupensis, the intermediate snail host of Schistosoma japonicum. Methods: The enzyme histochemical profiles of cholinesterase, cytochrome oxidase, lactate dehydrogenase, nitric oxide synthase, and succinate dehydrogenase in the soft tissues of Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum, were analyzed before and after treatment with the active ingredient of Buddleia lindleyana (AIBL), a potent and safe plant molluscicide. Results: Treatment with AIBL induced a notable decrease in the activities of the five enzymes (P<0.01). Conclusions: The results indicate that AIBL impairs the activities of the enzymes, thereby influencing the transfer of neurotransmitter and energy supply in Oncomelania hupensis and ultimately harming their various physiological functions, which are considered to cause death of the species. [Copyright &y& Elsevier]

Abstract: Objectives: To evaluate the effects of artificially decreased occlusal vertical dimension on the fiber phenotypes of the masseter muscle in the rat. Materials and methods: The experimental animals were divided into two groups: A decreased occlusal vertical dimension (DOVD) group and a sham-operated control group. Five rats from each group were sacrificed on days 0, 3, 7, and 14 after the operation. The extent of DOVD, the masseter muscle fiber composition, and fiber diameter were analyzed. Results: The average decrease in the vertical dimension was 1.1±0.5mm. The masseter muscle fiber composition of Type IIA and Type IIB muscle fibers shifted significantly in favor of Type IIA fibers on day 3 in the DOVD group. Toward the end of the observation period, shifting occurred back to the original composition. A significant decrease in the diameter of muscle fibers was seen on day 3 in the DOVD group. Conclusion: In this experiment, when the occlusal vertical dimension was artificially reduced, the masseter muscle atrophied and the muscle fiber type changed temporarily while adapting to the new position of the mandible. [Copyright &y& Elsevier]

Diet-related differences between the ratio of intestine length to body size and the enzymatic activity in the intestinal tract of wild and reared European eel ( Anguilla anguilla L.) were studied. Compared with reared eel, wild eel showed significantly shorter relative intestine length. For the purpose of histochemical examination, different parts (anterior, middle and posterior) of intestine proper were used. Activities of non-specific esterase, alkaline and acid phosphatase, and aminopeptidase were examined in each segment. All enzymes were present in the intestines of both wild and reared European eel. Fish from both groups showed similar enzyme distribution within the enterocytes, but distribution and intensity of enzyme activity along the intestine vary depending on the group. Generally, reared European eel showed highest enzymatic activity and wider distribution of enzymes throughout all parts of the intestine. These results suggest that different diets could be one of the reasons for observed changes. [ABSTRACT FROM AUTHOR]