Bile Microinfarcts in Cholestasis Are Initiated by Rupture of the Apical Hepatocyte Membrane and Cause Shunting of Bile to Sinusoidal Blood.

Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.

Developmental stages and growth of the proventriculus of post-hatching Muscovy duck: Light and electron microscopic study.

The present study was implemented to provide comprehensive information on the developmental sequence of the proventriculus of Muscovy ducks by gross examination, macro-micrometric analysis and by using light microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). Samples from 55 healthy post-hatching Muscovy ducks of both sexes ranging from 1 to 60 days old. The proventriculus began cranially opposite to the cranial end of the liver at 1-15 days old, but in front this level at 30 and 60 days old. Morphometrically, the length of the proventriculus was increased by about four folds while weight by 19 folds at 60 days old when compared with those at one day old. Scanning electron microscopy of the proventricular lumen at one day old exhibited numerous small mostly rounded irregularly distributed openings of the glands, but uniformly distributed and surrounded by closely packed concentrically arranged mucosal folds resembling a rosette shape at the older ages. Histologically, in all studied stages, rounded, elongated oval or polymorphic shaped lobules of the proventricular glands were occupied within the muscularis mucosa. The tubuloalveolar secretory units lined with secretory (oxyntico-peptic) cells with variable shapes had secretory granules increased by the development. Numerous argyrophilic endocrine cells were demonstrated away from the glandular lumen at older ages. Transmission electron microscopy revealed that the cytoplasm of the secretory cells contained homogeneously electron-dense granules at a young age, but two types of these granules could be recognized at 60 days old. In conclusion, this study provides a wide difference in the morphometric and the structure of the proventriculus from one day to 60 days old. This difference between the examined age-stages may be related to the feeding strategy (behavior) and the functional adaptations from the young to the older ages.

Influence of Liver Fibrosis on Lobular Zonation.

Little is known about how liver fibrosis influences lobular zonation. To address this question, we used three mouse models of liver fibrosis, repeated CCl4 administration for 2, 6 and 12 months to induce pericentral damage, as well as bile duct ligation (21 days) and mdr2-/- mice to study periportal fibrosis. Analyses were performed by RNA-sequencing, immunostaining of zonated proteins and image analysis. RNA-sequencing demonstrated a significant enrichment of pericentral genes among genes downregulated by CCl4; vice versa, periportal genes were enriched among the upregulated genes. Immunostaining showed an almost complete loss of pericentral proteins, such as cytochrome P450 enzymes and glutamine synthetase, while periportal proteins, such as arginase 1 and CPS1 became expressed also in pericentral hepatocytes. This pattern of fibrosis-associated 'periportalization' was consistently observed in all three mouse models and led to complete resistance to hepatotoxic doses of acetaminophen (200 mg/kg). Characterization of the expression response identified the inflammatory pathways TGFß, NFκB, TNFα, and transcription factors NFKb1, Stat1, Hif1a, Trp53, and Atf1 among those activated, while estrogen-associated pathways, Hnf4a and Hnf1a, were decreased. In conclusion, liver fibrosis leads to strong alterations of lobular zonation, where the pericentral region adopts periportal features. Beside adverse consequences, periportalization supports adaptation to repeated doses of hepatotoxic compounds.

Hair histology as a tool for forensic identification of some domestic animal species.

Animal hair examination at a criminal scene may provide valuable information in forensic investigations. However, local reference databases for animal hair identification are rare. In the present study, we provide differential histological analysis of hair of some domestic animals in Upper Egypt. For this purpose, guard hair of large ruminants (buffalo, camel and cow), small ruminants (sheep and goat), equine (horse and donkey) and canine (dog and cat) were collected and comparative analysis was performed by light microscopy. Based on the hair cuticle scale pattern, type and diameter of the medulla, and the pigmentation, characteristic differential features of each animal species were identified. The cuticle scale pattern was imbricate in all tested animals except in donkey, in which coronal scales were identified. The cuticle scale margin type, shape and the distance in between were characteristic for each animal species. The hair medulla was continuous in most of the tested animal species with the exception of sheep, in which fragmental medulla was detected. The diameter of the hair medulla and the margins differ according to the animal species. Hair shaft pigmentation were not detected in all tested animals with the exception of camel and buffalo, in which granules and streak-like pigmentation were detected. In conclusion, the present study provides a first-step towards preparation of a complete local reference database for animal hair identification that can be used in forensic investigations.

Detection of occult right ventricular dysfunction in young Egyptians with type 1 diabetes mellitus by two-dimensional speckle tracking echocardiography.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a common chronic disorder of childhood and adolescence. T1DM induced cardiomyopathy has a different entity than T2DM as it relies on different pathophysiological mechanisms, and rarely coexists with hypertension and obesity. Evaluation of right ventricular (RV) function in diabetic patients has been neglected despite the important contribution of RV to the overall cardiac function that affects the course and prognosis of diabetic cardiomyopathy (DCM). OBJECTIVE: To assess RV myocardial performance in asymptomatic T1DM using speckle tracking and standard echo parameters and correlate it with functional capacity using treadmill stress test. PATIENTS AND METHODS: Thirty-nine patients with TIDM (Group 1, mean age 18.2±1.7y, BMI=26.2±3.9kg/m2), without cardiac problems and 15 apparently healthy matched subjects as a control group (Group 2, mean age 18.8±2.3 y, BMI=22.8±3.3kg/m2) were enrolled. RV function was evaluated using conventional, tissue Doppler and 2D speckle tracking echocardiography (2D-STE). The peak RV global longitudinal strain (RV-GLS) was obtained. Functional capacity was assessed by treadmill exercise test and estimated in metabolic equivalent (METs). RESULTS: In this study; the diabetic group showed statistically highly significant decrease in the average RV-GLS (-14.0±6.9 in group 1 vs. -22.7±2.5 in group 2, P<0.001), significant decrease in RV S velocity (9.5±2.2 in group 1 vs. 11.5±1.8 in group 2, P<0.05), significantly reduced E/A ratio (1.0±0.2 in group 1 vs. 1.1±0.1 in group 2, P<0.05), and highly significant increased E/Em ratio (7.9±3.2 in group 1 vs. 5.2±0.7 in group 2, P<0.001). We did not found any significant differences between the two groups regarding the other echocardiographic or functional capacity parameters. CONCLUSION: In asymptomatic patients with T1DM, in addition to RV diastolic dysfunction, early (subclinical) RV systolic dysfunction is preferentially observed with normal RV and left ventricular (LV) ejection fraction (EF). 2D-STE has the ability to detect subclinical RV systolic dysfunction.

Neuropeptide AF Induces Piecemeal Degranulation in Murine Mucosal Mast Cells: A New Mediator in Neuro-Immune Communication in the Intestinal Lamina Propria?

Neuropeptides AF (NPAF), FF (NPFF) and SF (NPSF) are RFamide neuropeptides known to be widely expressed in the mammalian central nervous system, where they fulfill a wide range of functions with pain modulation being the most prominent one. Recent evidence indicates that RFamides act as mediators in mast cell-sensory nerve communications related to allergic disease. Previous work by our group has shown that the expression levels of some members of the Mas-related gene receptor (Mrgpr) family in both enteric neurons and mucosal mast cells change during intestinal inflammation. The Mrgpr subtypes C11 and A4 can be activated by NPAF, while A1 and C11 are triggered by NPFF. The aim of the present study was to investigate whether RFamides of the NPFF group are expressed in the gastrointestinal tract and to identify possible targets and receptors that might be involved in RFamide-associated mast cell modulation. To this end, the expression and distribution patterns of NPFF/AF receptors and the NPFF precursor protein were determined in bone marrow-derived mucosal mast cells (BMMCs) by immunocytochemistry and (RT-) PCR. BMMCs were found to express MrgprA4 and A1, and functional analysis of the effects of NPAF by means of a ß-hexosaminidase assay, mMCP-1 ELISA, electron microscopy and live cell calcium imaging revealed a piecemeal degranulation induced by NPAF. However, knock-out of MrgprA4 and A1 did not reduce the effect of NPAF, indicating that the BMMC response to NPAF was receptor independent. ProNPFF was expressed in neurons and BMMCs, suggesting that both cell types are potential sources of NPAF in situ. Our results show that the RFamide NPAF can be considered as a novel modulator of BMMC activity in the neuro-immune communication in the gastrointestinal tract, although the exact signaling pathway remains to be elucidated. Anat Rec, 00:000-000, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 301:1103-1114, 2018. © 2018 Wiley Periodicals, Inc.

Histogenesis of the stomach of the pre-hatching quail: a light microscopic study.

The current study conducted a careful description of the histological events during the embryonic development of quail stomach. Daily histological specimens from the quail stomach from day 4 to day 17 post incubation were examined by light microscopy. The primitive gut tube of the embryonic quail appeared at day 4 post incubation. The gut tube consisted of an endodermal epithelium of pseudostratified type, surrounded by splanchnic mesenchyme. The prospective glandular epithelium invaginated at day 5 in the proventriculus and gradually developed to prospective proventricular glands. The muscular coat became distinguished at day 7 and day 8 in the proventriculus and gizzard, respectively. Transformation into simple columnar epithelium occurred in both proventriculus and the gizzard at day 12. The gizzard epithelium gave rise to tubular invaginations also at day 12. Canalization of the gizzard tubular glands was recognized at day 14. By day 15, the proventricular surface epithelium invaginated in a concentric manner around a central cavity to form immature secretory units that contained inactive oxyntico-peptic cells. The mucosal folding in the gizzard appeared at day 15 to form plicae and sulci. The wall of the proventriculus and gizzard at day 17 acquired histological features of post-hatching birds.

Long bone development in the Japanese quail (Coturnix coturnixjaponica) embryos.

The current study was undertaken to describe the main histological development stages of long bones (tibia and femur) from Japanese quail (Coturnix coturnix japonica) embryos. Whole Limbs or just tibia and femur of fifty Japanese quail embryos of different ages were fixed and embedded in paraffin or Spurr's resin. Paraffin and semi-thin, respectively, were undertaken and examined with light microscopy. Limb bud was established at day 5 of incubation. Mesenchymal cells differentiated into chondrocytes forming a cartilage template in the position of the future tibia and femur at day 6 of incubation. At day 7 of incubation, the cartilage template enlarged and had the shape and position of the future tibia and femur. At day 8, central chondrocytes underwent hypertrophy and were surrounded by a periosteal bone collar. Cellular and vascular invasion from the bone collar into the central zone of the cartilage template, cartilage resorption and formation of marrow tunnel and finally peripheral calcification was seen. Vascular cartilage canals penetrating the epiphysis were observed at day 9 and the canals gradually increased in thickness and number toward the hatching day. Articular epiphyseal growth cartilage with resting, proliferative and hypertrophic zones was clearly established by day 10 of incubation. After 17 days of incubation, the zonation of the articular epiphyseal cartilage were much clear, many cartilage canals were present within the epiphyses. In epiphyses of tibia but not femur, foci of chondrocytes hypertrophy were noticed close to the cartilage canals. The current study timed the main histological sequences of development of tibia and femur of embryonic quail.

Ossification of the femur and tibia of the post-hatching Japanese quail.

The current study aimed to describe the histological changes of the femur and tibia of the post-hatching quail. Femur and tibia from 1-day- to 6-weeks post-hatching quail were processed for light microscopy. Histological examination revealed that endochondral ossification was a delayed process in the development of femur and tibia preceded by periosteal ossification. Femur and tibia of 1-day-post-hatching quail consisted of growth cartilage enclosed in a tube of periosteal bone collar. The collar extended toward the epiphysis dividing it into articular cartilage proper and lateral articular cartilage. Down to the articular cartilage, there was a physeal growth cartilage, in which the chondrocytes were organized into resting, proliferative and hypertrophic zones. Focal areas of hypertrophic chondrocytes were observed in the epiphysis of the tibia but not of the femur, which acted as a nidus for formation of the secondary ossification centre after in 2-week-posthathcing quail. Primary ossification centre was seen in both femur and tibia after 2 weeks and ossification continued replacing the cartilage until the 6th week when only permanent articular cartilage remained. Cartilage canals were present in both femur and tibia starting from the day 1, but chondrified and completely disappeared after the 6th week. The current study suggests that the periosteal ossification preceded the endochondral ossification and plays an important role in quail long bones development.

Morphological and histomorphological structure of testes of the catfish "Clarias gariepinus" from Egypt.

Knowledge of the normal reproductive biology of catfish is of a great importance not only for better understanding of the theory of fish development, but also to be used as a baseline for understanding the pathological changes results form exposure to harmful toxicants. Thus, the aim of the current study was to elucidate the gross anatomical and light microscopic features of the testes of the catfish "Clarias gariepinus" from Egypt. The present study was carried out on 36 mature male catfish (Clarias gariepinus) collected monthly during the spawning period (April-September) throughout the Nile River, crossing Sohag city in Egypt, in the year 2011. Samples were processed for light and electron microscopy. Each testis consisted of two regions, a fringed outer lateral region and a smooth sagittal-medial region. Histological examination revealed that the testis was covered with a highly vascular connective tissue capsule sending septa dividing the testis into seminiferous lobules separated by interstitial connective tissue containing steroid secreting Leydig cells. Seminiferous lobules were made up of spermatogenic cells and Sertoli cells; the spermatogenic cells located within cysts formed by the cytoplasmic projections of the Sertoli cells. According to the maturation stages, the seminiferous lobules were classified into three types. Spermatogenic seminiferous lobules were lined with different spermatogenic cells but had few or no spermatozoa. Pubertal seminiferous lobules were packed with spermatozoa with few spermatogenic cells. Spent seminiferous lobules contained remnants of spermatozoa and spermatogenic cells. In conclusion, the testis of catfish Clarias gariepinus from Egypt was similar in structure to other catfish and teleost species.

Periostin expression distinguishes between light and dark hypertrophic chondrocytes.

Hypertrophic chondrocytes exist in two forms detectable by electron microscopy, light and dark chondrocytes; the functional implications of the heterogeneous morphology are unknown. The aims of the study were to establish a method for separating light from dark hypertrophic chondrocytes and to identify genes differentially expressed between the two populations. Three-dimensional pellet cultures of chondrocytes from cartilage of neonatal rats were induced to undergo hypertrophy by treatment with triiodothyronine. Cultures were dissociated and subjected to density gradient centrifugation. The cell fraction with the lowest density comprised predominantly light hypertrophic chondrocytes, and the fraction with the highest density comprised predominantly dark hypertrophic chondrocytes. An Affymetrix GeneChip rat expression array was used to compare expression between dark cell-containing pellets and the light cell-enriched fraction. Genes identified on the array as putative dark cell-selective genes included genes encoding extracellular matrix proteins and enzymic modulators thereof. Expression of a subset of genes (Col1a1, periostin, osteoglycin, tPA/Plat, and Chst11) was confirmed as dark cell-selective using quantitative reverse transcriptase polymerase chain reaction. The most highly differentially expressed dark cell-selective gene was periostin. In immunocytochemical studies of light and dark cell-enriched fractions, periostin staining was detectable in dark, but not light hypertrophic chondrocytes. The results provide insight into molecular differences between light and dark hypertrophic chondrocytes.

Altered gene expression in early osteochondrosis lesions.

Osteochondrosis is a condition involving defective endochondral ossification and retention of cartilage in subchondral bone. The pathophysiology of this condition is poorly characterized, but it has been proposed that the fundamental defect is failure of chondrocyte hypertrophy. The aim of the current study was to characterize phenotypic changes in chondrocytes associated with the initiation of osteochondrosis. Early lesions were induced in an equine model of osteochondrosis by feeding foals a high energy diet for 8 or 15 weeks. Lesions in articular-epiphyseal growth cartilage were examined histologically and by quantitative PCR analysis of expression of a number of genes representative of pathways that regulate chondrocyte behavior during endochondral ossification. There were more cells present in clusters in the lesions compared to normal articular cartilage. Expression of matrix metalloproteinase-13, type I collagen, type X collagen, and Runx2 mRNA was significantly greater in the lesions compared to normal cartilage from the same joint. Expression of vascular endothelial growth factor, type II collagen, connective tissue growth factor, aggrecan, Sox9, and fibroblast growth factor receptor 3 mRNA was not significantly different in lesions than in control cartilage. These observations suggest that osteochondrosis does not result from failure of chondrocytes to undergo hypertrophy.