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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.

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]

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.

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.

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.

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.

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.

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: 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]

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]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. D-Serine, an endogenous coagonist of N-methyl-D-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/ familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the D-amino acid oxidase (DAO) gene, encoding a D-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and o-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the D-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through D-serine regulatioh and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing D-serine or controlling DAO activity in ALS should be tested in future studies. [ABSTRACT FROM AUTHOR]

Abstract: Widely used in the past against termites and soil insects, the chlorinated insecticide heptachlor (H) is a toxic contaminant which represents a risk for both terrestrial and aquatic organisms. Like many organochlorine pesticides, heptachlor and heptachlor epoxide (HE), with oxidation products synthesized by many plant and animal species, degrade slowly since many of the derived compounds are persistent. This increases the status of heptachlor as a hazardous pollutant. In the present experimental study we exposed specimens of Rana kl. esculenta, from the tadpole stage through to their complete metamorphosis, to three different concentrations of heptachlor (4, 40 and 400ppb). Mortality and HE bioaccumulation were evaluated on all the experimental groups. Since amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the toxic effects on the ventral epidermis of both tadpole and adult samples by employing such histo-cytopathological biomarkers as ultrastructural morphology, certain enzyme activities (acid and alkaline phosphatases, AcPase, and AlkPase; succinic dehydrogenase, SDH; α-naphtyl butyrate esterase, ANBE; nitric oxide synthase/NADPH diaphorase, NOS/NADPHd). Also, the levels of reactive oxygen species (ROS) in the different conditions were evaluated. The results obtained were of ecological relevance, in particular as regards the effects of this environmental toxicant on the samples of tadpole epidermis. Severe morphological alterations were observed in the larval epidermal cells (apical and skein cells), whereas the cell epidermis (keratinocytes and mitochondria-rich cells) of the adult survivors showed changes in enzyme activities, particularly those involved in the protective response to xenobiotic injury. In general, morpho-histochemical studies, analysis of HE bioaccumulation and mortality showed a relation to the H doses employed. [Copyright &y& Elsevier]

Whole mount enzyme histochemistry to localize lacZ-bearing transgenes (lacZ-WMH) also detects endogenous ß-galactosidases. The experiments reported here evaluated lacZ-WMH as a potential tool for transgene expression analysis during high-throughput rodent necropsies. A lacZ-WMH survey of organs from adult, wild-type, male and female mice (C57BL/6, FVB/N) and female rats (Sprague-Dawley) performed at the optimal pH (> 7.0) for bacterial lacZ yielded intense endogenous staining in the gonads, kidney, male accessory sex organs, salivary glands, submucosal glands in the duodenum, and thyroid. Substantial staining occurred in the adrenal cortex, lymph nodes, and linings of the gastrointestinal tract, the urinary bladder and uterus, and (for rat only) in the adenohypophysis, bone marrow, thymus, and trigeminal ganglia. Endogenous galactosidases were distributed similarly in sections of flash-frozen organs used for slide-based lacZ histochemistry (lacZ-SBH) at pH < 5.0 (optimal for eukaryotic enzymes). Cerebral neurons were labeled only by lacZ-SBH. At pH 7.4, endogenous but not specific lacZ activity was abolished for lacZ-SBH, while endogenous activity was not halted without reducing specific activity for lacZ-WMH. These data demonstrate that lacZ-WMH is feasible during rodent necropsies for many but not all organs if species-, strain-, and sex-specific divergence in endogenous galactosidase activity is considered and special fixation (3% paraformaldehyde for 3 hours at 4°C) is used. [ABSTRACT FROM PUBLISHER]

Arabidopsis as a molecular genetic model offers many advantages for the study of seed development, but these do not extend to biochemical and enzymatic studies, which are often compromised by the limited amount of material available from the small developing embryos. A set of assays based on the coupling of an enzymatic reaction to the reduction of NAD, NADP or FAD, and subsequent reduction and precipitation of a tetrazolium salt, have been adapted to investigate 18 enzyme activities associated with carbon metabolism in developing Arabidopsis embryos. The use of organelle-specific marker enzymes demonstrates the utility of the method for detection of activities in mitochondria, plastids and peroxisomes as well as the cytosol. The temporal staining patterns obtained allow classification of the activities into three main categories based on whether they peak in the early, intermediate or late stages of maturation. An interesting switch from ATP to pyrophosphate consuming pathways occurs at the onset of the maturation phase, which involves key steps in primary carbon metabolism such as phosphofructokinase. This spatiotemporal characterization of carbon metabolism has also been applied to various mutants disrupted in embryo development including gnom ( gn), acetyl-CoA carboxylase1 ( acc1), schlepperless ( slp), and wrinkled1 ( wri1). The data obtained demonstrate that the extent to which carbon metabolism is affected in mutants is not necessarily correlated to the severity of the mutation considered. Through the advanced characterization of trehalose-6-P synthase1 ( tps1) embryos, this approach finally provides new insight into the regulatory role played by trehalose metabolism in embryo development. [ABSTRACT FROM AUTHOR]

Abstract: Phosphatase enzymes cleave an inorganic phosphate from a substrate. Phosphatase enzyme histochemistry followed by flat-embedding in glycol methacrylate is extremely useful in studying retinal and choroidal vascular development and loss, since only viable blood vessels have these enzyme activities. Sites of occlusion and remodeling can be identified and analysed, resulting in new insights into the cause of occlusion. The phosphatase activities are elevated in neovascularization making possible high resolution analysis of neovascularization, the feeder vessels, and the retinal milieu in which angiogenesis occurs. Adenosine diphosphatase (ADPase) catalyzes ADP to an inorganic phosphate plus adenosine monophosphate, preventing accumulation of ADP, one of the most potent stimuli for platelet aggregation. The ADPase technique can be used in any species but this report highlights its use in dog and human retinas. The ADPase technique has yielded important insights into vaso-occlusive and vasoproliferative processes in retinopathy of prematurity, sickle cell and diabetic retinopathies. The alkaline phosphatase flatembedding technique is useful in evaluating dog, cat, and human choroidal vasculatures. It has permitted quantification of the loss of choriocapillaris in diabetic choroidopathy and of the RPE and choriocapillaris in geographic atrophy and exudative age-related macular degeneration. [Copyright &y& Elsevier]

Abstract: Considerations about back pain, its aetiology, pathogenesis and therapy often argue that low back pain is the “price” that humanity has to pay for the upright body posture and the bipedal mode of locomotion. In fact, there are only few species that have evolved an obligate bipedal locomotion (e.g. kangaroos). Surprisingly, there are only minor morphological adaptations in humans clearly connected to the upright body posture (e.g. the habitual lumbar lordosis). The overall organization of the body axis has been evolved in quadrupedal animals and was more or less unchanged suitable for the human bipedal mode of locomotion. Up to now, the assumed uniqueness of human''s trunk does not explain the frequency of back problems. Because of minor macroscopic differences between humans and other mammals, and even primates, we started to take a closer look at the paravertebral musculature. A three-dimensional investigation of muscle''s fibre type distribution was undertaken on laboratory rats. Serial sections from the caudal thoracic and lumbar regions of the back were analysed, and the fibre type distribution pattern in all paravertebral muscles was described. Comparisons to other species imply more general characters of the fibre type distribution in mammals. Established concepts of human back muscle function were exposed to be valid for quadruped mammals as well. Muscle properties predicted by a biomechanical model based on human''s anatomy (Wagner et al., this issue) were confirmed by results of the current study on a small mammal. Therefore, we propose only minor differences from the observed pattern in human back muscles. [Copyright &y& Elsevier]

Research projects focusing on biomaterials related factors; the bulk implant material, the macro-design of the implant and the microsurface roughness are routinely being conducted at our laboratories. In this study, we have investigated the bone tissue reactions to turned commercially pure (c.p.) titanium implants with various thicknesses of the oxide films after 6 weeks of insertion in rabbit bone. The control c.p. titanium implants had an oxide thickness of 17–200 nm while the test implants revealed an oxide thickness between 600 and 1000 nm. Routine histological investigations of the tissue reactions around the implants and enzyme histochemical detections of alkaline and acid phosphatase activities demonstrated similar findings around both the control and test implants. In general, the histomorphometrical parameters (bone to implant contact and newly formed bone) revealed significant quantitative differences between the control and test implants. The test implants demonstrated a greater bone response histomorphometrically than control implants and the osteoconductivity was more pronounced around the test implant surfaces. The parameters that differed between the implant surfaces, i.e. the oxide thickness, the pore size distribution, the porosity and the crystallinity of the surface oxides may represent factors that have an influence on the histomorphometrical results indicated by a stronger bone tissue response to the test implant surfaces, with an oxide thickness of more than 600 nm. [Copyright &y& Elsevier]

To elucidate interactions between the glucose transport system and hepatic glucose production in the tumour-bearing state, glycogen storage, expression of glucose transporter isoform 2 (Glut 2) and activities of glucose-6-phosphatase (G-6-Pase) and hexokinase were histochemically examined in hepatocytes of tumour-bearing rats. Five male F344 rats, subcutaneously inoculated with methylcholanthrene(MCA)-induced sarcoma cells were compared with five pair-fed animals and four ad libitum fed controls. Glycogen storage was markedly decreased in liver cells of tumour-bearing rats compared to in those of control animals. Glut 2 immunoreactivity was uniformly seen in the cellular membrane of hepatocytes from control animals. In rats bearing sarcoma, the staining intensity was significantly decreased, suggesting that Glut 2 with its bi-directional transport capacity was down-regulated in the tumour-bearing state. Positive staining for hexokinase activity was located in the perivenous area in livers from control animals and was more diffusely located and more intense in livers from tumour-bearing animals. G-6-Pase activity, limited to the peripheral area in livers from controls, extended to the intermediate area and had stronger reactivity in livers from tumour-bearing animals. In the tumour-bearing cachectic condition, glucose may be partially consumed by a futile cycle, hepatic metabolic zonation was disturbed, and the release of glucose from the liver may not be mediated by a facilitative glucose transporter-2. [ABSTRACT FROM AUTHOR]

Preneoplastic liver lesions were produced in female Wistar rats by oral administration of 2-acetyl-aminofluorene for 165 days succeeded by a carcinogen-free standard diet up to 420 days. During the treatment numerous altered hepatic foci (AHF) and hyperplastic nodules (HN) were detected histochemically by a focal decrease or lack of adenosine-5-triphosphatase and glucose-6-phosphatase (G-6-Pase) activities. In addition, the immunohistochemically demosntrable amount of L-type pyruvate kinase was clearly reduced. The histochemically demonstrated decrease of G-6-Pase was substantiated by microbiochemical determination of the enzyme activity in microdissected material. Moreover, during the experimental period a continuous decrease in glucokinase and an increase in hexokinase was detected microbiochemically within AHF and HN. These alterations indicate, a shift in the carbohydrate metabolism from gluconeogenesis to glucose utilization and pentose-phosphate-pathway for biosynthesis of nucleic acids. Beside other oncofetal markers, HK may be used as indicator of the early stages of liver carcinogenesis. [ABSTRACT FROM AUTHOR]

Early changes of cerebellar and cerebral blood flow, as well as subsequent infratentorial ischaemia, were evaluated in an experimental model of cerebellar haemorrhage. Eight anaesthetized male adult Sprague-Dawley rats received an injection of autologous arterial blood (50 μl) into the right cerebellar hemisphere. Eight animals were sham-operated and served as controls. Cerebellar blood flow, measured at regular time intervals in the hemispheric cortex ipsilateral to the lesion by the hydrogen clearance method, was significantly depressed in the experimental group as compared with the control animals. This drop in cerebellar blood flow was evident 5, 30, 60, 90, and 120 min postoperatively, with a return to pre-injection values recorded 180 min after surgery. No significant difference in supratentorial blood flow was detected over the entire period examined between the two groups of animals. Enzyme histochemistry demonstrated areas of ischaemia around the clot and within the brain stem in animals with an intracerebellar haemorrhage at the end of the experiment. These results provided evidence of ischaemic damage within the infratentorial compartment after the induction of experimental cerebellar haemorrhage. [ABSTRACT FROM AUTHOR]