Showing papers in "European Journal of Histochemistry in 2018"

Energy Dispersive X-ray (EDX) microanalysis: A powerful tool in biomedical research and diagnosis.

Abstract: The Energy Dispersive X-ray (EDX) microanalysis is a technique of elemental analysis associated to electron microscopy based on the generation of characteristic Xrays that reveals the presence of elements present in the specimens. The EDX microanalysis is used in different biomedical fields by many researchers and clinicians. Nevertheless, most of the scientific community is not fully aware of its possible applications. The spectrum of EDX microanalysis contains both semi-qualitative and semi-quantitative information. EDX technique is made useful in the study of drugs, such as in the study of drugs delivery in which the EDX is an important tool to detect nanoparticles (generally, used to improve the therapeutic performance of some chemotherapeutic agents). EDX is also used in the study of environmental pollution and in the characterization of mineral bioaccumulated in the tissues. In conclusion, the EDX can be considered as a useful tool in all works that require element determination, endogenous or exogenous, in the tissue, cell or any other sample.

What happens to an acellular dermal matrix after implantation in the human body? A histological and electron microscopic study

Abstract: Acellular matrices are used for various purposes and they have been studied extensively for their potential roles in regenerating tissues or organs. The acellular matrix generates physiological cues that mimic the native tissue microenvironment. Acellular dermal matrix (ADM) is a soft connective tissue graft generated by a decellularization process that preserves the intact extracellular skin matrix. Upon implantation, this structure serves as a scaffold for donor-side cells to facilitate subsequent incorporation and revascularization. In breast reconstruction, ADM is used mainly for lower pole coverage and the shaping of a new breast. It helps control the positioning of the implant in the inframammary fold, and prevent the formation of contractile pseudocapsule around the breast implant. In this study, we provide a comprehensive histological description of ADM used for human breast reconstruction over the course of several months following implementation. Using immunohistochemical methods (a panel of 12 antibodies) coupled with optical and transmission electron microscopy, we confirmed that the original acellular dermal matrix became recolonized by fibroblasts and myofibroblasts, and also by various other free cells of the connective tissue (lymphocytes, macrophages and multinucleated giant cells, granulocytes, mast cells) after implantation into the patient’s body. Within the implanted ADM, there was a relatively rapid ingrowth of blood vessels. Lymphatic vessels were only detected in one case 9 months after the implantation of the ADM. These results suggest that lymphangiogenesis is a longer process than angiogenesis.

Cancer stem cell markers ALDH1 and CD44+/CD24- phenotype and their prognosis impact in invasive ductal carcinoma.

Abstract: Breast cancer is a very heterogeneous disease. The intrinsic molecular subtypes can explain the intertumoral heterogeneity and the cancer stem cell (CSC) hypothesis can explain the intratumoral heterogeneity of this kind of tumor. CD44+/CD24- phenotype and ALDH1 expression are the major CSC markers described in invasive breast cancer. In the present study, 144 samples of invasive breast carcinoma, no special type were distributed in 15 tissue microarrays (TMA) and then evaluated for expression of the CD44+/CD24- phenotype and ALDH1 to understand the importance of these CSC markers and the clinical aspects of breast cancer. The samples were classified into four molecular subtypes according to clinicopathological criteria: Luminal A, Luminal B, HER2, and Basal-like. A statistical association was found between the molecular subtypes and the CSC markers, with HER2 the most frequent subtype for both markers. ALDH1 was also associated with other poor prognostic variables, such as a high histological grade and larger tumors, but it was not associated with the patients' prognosis in this sample and nor was the CD44+/CD24- phenotype in a multivariate analysis. There are still many controversies about the role of these markers in breast cancer molecular subtypes. The identification of these populations of cells, through immunohistochemical markers, can help to better understand the CSC theory in clinical practice and, in the near future, contribute to developing new target therapies.

Low ozone concentrations promote adipogenesis in human adipose-derived adult stem cells.

Abstract: Ozone is a strong oxidant, highly unstable atmospheric gas. Its medical use at low concentrations has been progressively increasing as an alternative/adjuvant treatment for several diseases. In this study, we investigated the effects of mild ozonisation on human adipose-derived adult stem (hADAS) cells i.e., mesenchymal stem cells occurring in the stromal-vascular fraction of the fat tissue and involved in the tissue regeneration processes. hADAS cells were induced to differentiate into the adipoblastic lineage, and the effect of low ozone concentrations on the adipogenic process was studied by combining histochemical, morphometric and ultrastructural analyses. Our results demonstrate that ozone treatment promotes lipid accumulation in hADAS without inducing deleterious effects, thus paving the way to future studies aimed at elucidating the effect of mild ozonisation on adipose tissue for tissue regeneration and engineering.

Concomitant use of heat-shock protein 70, glutamine synthetase and glypican-3 is useful in diagnosis of HBV-related hepatocellular carcinoma with higher specificity and sensitivity

Abstract: Hepatocellular carcinoma is the third leading cause of cancer-related death worldwide and late diagnosis is the main cause of death in HCC patients. In this study expression patterns of HSP70, GPC3 and GS and their relationships with pathogenesis of HCC in Iranian patients were investigated. The expression of HSP70, GPC3 and GS were determined by immunohistochemistry and quantitative real-time PCR (q-PCR) methods, using 121 cases from patients with HBV alone, HCC without HBV, HBV+HCC and 30 normal tissues as control group. HSP70, GPC3 and GS were expressed in higher levels in HBV-related HCC samples compared to HBV alone group. The results showed that the labeling index of HSP70, GPC3 and GS are correlated with immunohistochemical and molecular expressions of HSP70, GPC3 and GS. The sensitivity and specificity for HCC diagnosis were 43.4% and 89.7% for HSP70, 64.3% and 90.4% for GPC3, and 60.7% and 94.3% for GS, respectively. The sensitivity and specificity of the panels with 3, 2 and 1 positive markers, regardless of which one, were 21.6% and 100%, 51.3% and 100% and 93.4% and 80.5% respectively. The current study demonstrated an association between HSP70, GPC3 and GS expressions and HBV-related HCC in our population. It was concluded that HSP70, GPC3 and GS expressions could be useful biomarkers for increasing the specificity and sensitivity of HCC diagnosis to acceptable level. Also, proper combinations of these 3 markers could improve diagnostic accuracy.

Comparison of different histological protocols for the preservation and quantification of the intestinal mucus layer in pigs

Abstract: The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution After dehydration, methacarn fixed tissues were embedded in paraffin wax Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs

Regenerative potential of the Bichat fat pad determined by the quantification of multilineage differentiating stress enduring cells

Abstract: Published studies regarding Bichat fat pad focused, quite exclusively, on the implant of this adipose depot for different facial portions reconstruction. The regenerative components of Bichat fat pad were poorly investigated. The present study aimed to describe by an ultrastructural approach the Bichat fat pad, providing novel data at the ultrastructural and cellular level. This data sets improve the knowledge about the usefulness of the Bichat fat pad in regenerative and reconstructive surgery. Bichat fat pads were harvested form eight patients subjected to maxillofacial, dental and aesthetic surgeries. Biopsies were used for the isolation of mesenchymal cell compartment and for ultrastructural analysis. Respectively, Bichat fat pads were either digested and placed in culture for the characterization of mesenchymal stem cells (MSCs) or, were fixed in glutaraldehyde 2% and processed for transmission or scanning electron microscopy. Collected data showed very interesting features regarding the cellular composition of the Bichat fat pad and, in particular, experiments aimed to characterized the MSCs showed the presence of a sub-population of MSCs characterized by the expression of specific markers that allow to classify them as multilineage differentiating stress enduring cells. This data set allows to collect novel information about regenerative potential of Bichat fat pad that could explain the success of its employment in reconstructive and regenerative medicine.

The earthworm Dendrobaena veneta (Annelida): A new experimental-organism for photobiomodulation and wound healing

Abstract: Photobiomodulation (PBM) is a manipulation of cellular behavior using non-ablative low intensity light sources This manipulation triggers a cascade of metabolic effects and physiological changes resulting in improved tissue repair, of benefit in the treatment of tissue injury, degenerative or autoimmune diseases PBM has witnessed an exponential increase in both clinical instrument technology and applications It is therefore of benefit to find reliable experimental models to test the burgeoning laser technology for medical applications In our work, we proposed the earthworm Dendrobaena veneta for the study of non-ablative laser-light effects on wound healing In our preliminary work, D veneta has been shown to be positively affected by PBM New tests using D veneta were set up to evaluate the effectiveness of a chosen 808 nm-64 J/cm2-1W-CW laser therapy using the AB2799 hand-piece with flat-top bean profile, on the wound healing process of the earthworm Effective outcome was assimilated through examining the macroscopic, histological, and molecular changes on the irradiated posterior-segment of excised-earthworms with respect to controls Three successive treatments, one every 24 hours, were concluded as sufficient to promote the wound healing, by effects on muscular and blood vessel contraction, decrement of bacteria load, reduction of inflammatory processes and tissue degeneration D veneta was demonstrated to be a reliable experimental organism that meets well the 3Rs principles and the National Science Foundation statement Through their genetic and evolutionary peculiarity, comparable to those of scientifically accredited models, D veneta allows the effect of laser therapies by multidisciplinary methods, at various degree of complexity and costs to be investigated

Activation of the renin-angiotensin system in mice aggravates mechanical loading-induced knee osteoarthritis.

Abstract: Epidemiological studies have shown an association between hypertension and knee osteoarthritis (OA). The purpose of this study was to investigate whether activation of the renin-angiotensin system (RAS) can aggravate mechanical loading-induced knee OA in mice. Eight-week-old male Tsukuba hypertensive mice (THM) and C57BL/6 mice were divided into running and non-running groups. Mice in the running group were forced to run (25 m/min, 30 min/day, 5 days/week) on a treadmill. All mice in the four groups (n=10 in each group) were euthanized after 0, 2, 4, 6, or 8 weeks of running or natural breeding. Cartilage degeneration in the left knees was histologically evaluated using the modified Mankin score. Expression of Col X, MMP-13, angiotensin type 1 receptor (AT1R), and AT2R was examined immunohistochemically. To study the effects of stimulation of the AT1R in chondrocytes by mechanical loading and/or Angiotensin II (AngII) on transduction of intracellular signals, phosphorylation levels of JNK and Src were measured in bovine articular chondrocytes cultured in three-dimensional agarose scaffolds. After 4 weeks, the mean Mankin score for the lateral femoral condylar cartilage was significantly higher in the THM running group than in the C57BL/6 running group and non-running groups. AT1R and AT2R expression was not detected at 0 weeks in any group but was noted after 4 weeks in the THM running group. AT1R expression was also noted at 8 weeks in the C57BL/6 running group. The expression levels of AT1R, COL X, and MMP-13 in chondrocytes were significantly higher in the THM running group than in the control groups. Positive significant correlations were noted between the Mankin score and the rate of AT1R-immunopositive cells, between the rates of AT1R- and Col X-positive cells, and between the rates of AT1R- and AT2R-positive cells. The phosphorylation level of JNK was increased by cyclic compression loading or addition of AngII to the cultured chondrocytes and was reversed by pretreatment with an AT1R blocker. A synergistic effect on JNK phosphorylation was observed between compression loading and AngII addition. Transgene activation of renin and angiotensinogen aggravated mechanical load-induced knee OA in mice. These findings suggest that AT1R expression in chondrocytes is associated with early knee OA and plays a role in the progression of cartilage degeneration. The RAS may be a common molecular mechanism involved in the pathogenesis of hypertension and knee OA.

Novel insights into pericarp, protein body globoids of aleurone layer, starchy granules of three cereals gained using atomic force microscopy and environmental scanning electronic microscopy.

Abstract: In this study, we applied Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy (ESEM-EDS) and Atomic Force Microscopy (AFM) analysis to three different cereal caryopses: barley, oat and einkorn wheat. The morphological structures, chemical elemental composition and surface characteristics of the three cereals were described. Regarding the morphology, barley showed the thickest pericarp, providing a strong barrier digestion and absorption of nutrients. The aleurone layer of each cereal type contained protein body globoids within its cells. Large type-A and small type-B starchy granules were revealed in the endosperm of barley and einkorn wheat, whereas irregular starchy granules were found in oats. The starchy granule elemental composition, detected by ESEM-EDS, was rather homogenous in the three cereals, whereas the pericarp and protein body globoids showed heterogeneity. In the protein body globoids, oats showed higher P and K concentrations than barley and einkorn wheat. Regarding the topographic profiles, detected by AFM, einkorn wheat starchy granules showed a surface profile that differed significantly from that of oats and barley, which were quite similar to one another. The present work provides insights into the morphological and chemical makeup of the three grains shedding light on the higher bio-accessibility of einkorn wheat nutrients compared to barley and oats, providing important suggestions for human nutrition and technological standpoints.

Seasonal expressions of androgen receptor, estrogen receptors and cytochrome P450 aromatase in the uteri of the wild Daurian ground squirrels (Spermophilus dauricus)

Abstract: The reproductive tissues including the uterus undergo dramatic changes in seasonal breeders from the breeding to non-breeding seasons Classically, sex steroid hormones play important roles in the uterine morphology and functions To clarify the relationship between sex steroid hormones and seasonal changes in the uterine morphology and functions, the wild Daurian ground squirrels (Spermophilus dauricus) were used as seasonal breeder model And the immunolocalizations and expression levels of androgen receptor (AR), estrogen receptors α and β (ERα and ERβ) and cytochrome P450 aromatase (P450arom) were investigated in the uteri of the wild Daurian ground squirrels in the breeding (April) and the non-breeding (June) seasons via immunohistochemistry, Western blot and RT-PCR Histologically, the uterine weight, the thickness of endometrium and the glandular density were significantly higher in the uteri of the breeding season than those of the non-breeding season In both seasons, the immunostaining of AR was only presented in stromal cells of the uteri; the positive staining of ERα and ERβ were localized in stromal cells and glandular cells; P450arom was merely immunolocalized in glandular cells The protein and mRNA expression levels of ERα, ERβ and P450arom were higher in the uteri of the breeding season than those of the non-breeding season; conversely, the expressions of AR were higher in the uteri of the non-breeding season comparing with those of the breeding season in both protein and mRNA levels The AR: ER ratio in the uteri of the non-breeding season exceeded the AR: ER ratio in the uteri of the breeding season in the wild Daurian ground squirrels These results suggested that seasonal changes in the expression levels of AR, ERs and P450arom might be correlated with the uterine morphology and histology changes, and estrogen may play an important autocrine/paracrine role in regulating the uterine functions of the wild Daurian ground squirrels

Marine bisindole alkaloid: A potential apoptotic inducer in human cancer cells.

Abstract: Marine organisms such as corals, sponges and tunicates produce active molecules which could represent a valid starting point for new drug development processes. Among the various structural classes, the attention has been focused on 2,2-bis(6-bromo-3-indolyl) ethylamine, a marine alkaloid which showed a good anticancer activity against several tumor cell lines. Here, for the first time, the mechanisms of action of 2,2-bis(6-bromo-3-indolyl) ethylamine have been evaluated in a U937 tumor cell model. Morpho-functional and molecular analyses, highlighting its preferred signaling pathway, demonstrated that apoptosis is the major death response induced by this marine compund. Chromatin condensation, micronuclei formation, blebbing and in situ DNA fragmentation, occurring through caspase activation (extrinsic and intrinsic pathways), were observed. In particular, the bisindole alkaloid induces a mitochondrial involvement in apoptosis machinery activation with Blc-2/Bcl-x down-regulation and Bax up-regulation. These findings demonstrated that 2,2-bis(6-bromo-3-indolyl) ethylamine alkaloid-induced apoptosis is regulated by the Bcl-2 protein family upstream of caspase activation.

Distribution of non-myelinating Schwann cells and their associations with leukocytes in mouse spleen revealed by immunofluorescence staining

Abstract: The nervous system and the immune system communicate extensively with each other in order to maintain homeostasis and to regulate the immune response. The peripheral nervous system (PNS) communicates specifically with the immune system according to local interactions, including the “hardwiring” of sympathetic/parasympathetic (efferent) and sensory nerves (afferent) to lymphoid tissue and organs. To reveal this type of bidirectional neuroimmune interaction at the microscopic level, we used immunofluorescent staining of glial fibrillary acidic protein (GFAP) coupled with confocal microscopy/3D reconstruction to reveal the distribution of non-myelinating Schwann cells (NMSCs) and their interactions with immune cells inside mouse spleen. Our results demonstrate i) the presence of an extensive network of NMSC processes in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath (PALS), marginal zone, trabecula, and red pulp; ii) the close association of NMSC processes with blood vessels (including central artries and their branches, marginal sinuses, penicillar arterioles and splenic sinuses); iii) the close “synapse-like” interaction/association of NMSC processes with various subsets of dendritic cells (DCs; e.g ., CD4 + CD11c + DCs, B220 + CD11c + DCs, and F4/80 + CD11c + DCs), macrophages (F4/80 + ), and lymphocytes (B cells, CD4 + T helper cells). Our novel findings concerning the distribution of NMSCs and NMSC-leukocytes interactions inside mouse spleen should improve our understanding of the mechanisms through which the PNS affects cellular- and humoral-mediated immune responses in a variety of health conditions and infectious/non-infectious diseases.

Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study.

Abstract: The aim of this post-mortem ultrastructural and immunohistochemical study is to explore the characteristics of acute myocardial ischemia in the context of sudden death, using the combination of two different methods, both more insightful than ordinary histology. Transmission electron microscope and immunohistochemistry, in addition to the traditional histology, were applied to study human heart specimens collected during forensic autopsies. The whole series was sub-grouped into cases (n=17) and controls (n=10). The control group consisted of unnatural death with a short agonal period (immediately lethal injuries). Heart samples of the two cohorts of subjects were prepared for electron microscopy. On the other hand, each specimen, formalin fixed and paraffin embedded, was stained with haematoxylin and eosin and immunoreacted with the following primary antibodies: antiFibronectin, antiConnexin-43, anti npCx43 (dephosphorylated form of Connexin43), antiZonula occludens-1. Immunopositivity of each marker in the myocardium was semi-quantitatively graded. Electron microscopy revealed a number of interesting differences between acute myocardial ischemia and controls, regarding the morphology of nucleus, mitochondria and intercellular junctions. By immunohistochemistry, fibronectin was found to be markedly increased in the extracellular matrix of the acute myocardial ischemia cases, with a remarkable difference in respect of controls. Connexin 43 staining disclosed a slightly increase in the cytoplasm of acute myocardial ischemia cases with respect to the controls, whereas no relevant differences were seen between cases and controls at intercellular junctions. Dephosphorylated form of Cx43 showed an evident difference of staining in cases compared to controls and overall this difference more evident in the cytoplasm. Zonula occludens 1, described as an important marker for functional modification of cardiac muscle fibers, resulted negative or very weak in the vast majority of both cases and controls. The present study attempts to simultaneously apply electron microscopy and immunohistochemistry, in order to figure out the morphological changes that might lead to pathological processes underlying the sudden, unexpected death due to acute myocardial ischemia, and consequently to find useful diagnostic markers of very early ischemic injury. Both methods showed significant differences between acute myocardial ischemia and controls, regarding, overall nuclei, mitochondria, and intercellular junctions.Â

Leptin affects filopodia and cofilin in NK-92 cells in a dose- and time-dependent manner.

Abstract: Hyperleptinemia, associated with obesity, is related with immune dysfunction and carcinogenesis. Natural Killer (NK) cells, a major component of the innate immune system are mediators of anti-tumor immunity and the most actively migrating cells among leukocytes. Actin rearrangement, promoted by cofilin plays a central role in cellular migration. Leptin affects the phosphorylation-dependent activity of cofilin and thus actin remodeling. We used human NK-92 cells to explore the in vitro effects of leptin on co-localization of cofilin and F-actin and on morphological changes in NK cells. NK-92 cells were incubated with different leptin concentrations (10 and 100 ng/mL) for 30 min and 24 h and immunocytochemically stained. Results demonstrate a dose- and time-dependent influence of leptin on cellular morphology. Utilizing confocal microscopy, we observed that the co-localization of cofilin-1 and F-actin was slightly influenced by leptin. In summary, the present study demonstrates an impact of a physiological leptin stimulation on the filopodia length, and a time-dependent effect on the co-localization of cofilin and F-actin in NK-92 cells.

Two histological methods for recognition and study of cortical microinfarcts in thick sections.

Abstract: Cortical microinfarcts are the most widespread form of brain infarction but frequently remain undetected by standard neuroimaging protocols. Moreover, microinfarcts are only partially detectable in hematoxylin-eosin-stained (H and E) 4-10 µm paraffin sections at routine neuropathological examination. In this short report, we provide two staining protocols for visualizing cortical microinfarcts in 100-300 µm sections. For low-power microscopy, the first protocol combines aldehyde fuchsine staining for detection of lipofuscin granules and macrophages with Darrow red counterstaining for Nissl material. The second protocol combines collagen IV immunohistochemistry with aldehyde fuchsine/Darrow red or with erythrosin-phosphotungstic acid-aniline blue staining for detailed study of the capillary network. In the first protocol, microinfarcts are recognizable as radially-oriented funnel-like accumulations of aldehyde fuchsine-positive macrophages. The second protocol recognizes microinfarcts and alterations of the capillary network, at whose center accumulations of dead neurons and aldehyde fuchsine-positive macrophages cluster. In addition, the second protocol permits visualization of abnormalities within the capillary network associated with more recent microinfarcts. Both protocols can be useful for comparing MRI datasets with cortical microinfarcts in corresponding whole brain sections of 100-300 µm thickness.

Expression of acetylated tubulin in the postnatal developing mouse cochlea

Abstract: Acetylation tubulin is one of the major post-translational modifications of microtubules. Stable microtubules are well known to contain acetylated tubulin. Here, we examined the spatiotemporal expression of acetylated tubulin in the mouse cochlea during postnatal development. At postnatal day 1 (P1), acetylated tubulin was localized primarily to the auditory nerve inside the cochlea and their synaptic contacts with the inner and outer hair cells (IHCs and OHCs). In the organ of Corti, acetylated tubulin occurred first at the apex of pillar cells. At P5, acetylated tubulin first appeared in the phalangeal processes of Deiters' cells. At P8, staining was maintained in the phalangeal processes of Deiters' cells. At P10, labeling in Deiters' cells extended from the apices of OHCs to the basilar membrane. Labeling was expressed throughout the cytoplasm of pillar cells. At P12, acetylated tubulin displayed prominent and homogeneous labeling along the full length of the pillar cells. Linear labeling was present mainly in the Deiters' cell bodies underlying OHCs. Between P14 and P17, acetylated tubulin was strongly expressed in inner and outer pillar cells and Deiters' cells in a similar pattern as observed in the adult, and labeling in these cells were arranged in bundles. In addition, acetylated tubulin was intensely expressed in stria vascularis, root cell bodies, and a small number of fibrocytes of the spiral ligament until the adult. In the adult mouse cochlea, immunostaining continued to predominate in Deiters' cells and pillar cells. Immunolabeling formed cups securing OHCs basal portions, and continued presence of acetylated tubulin-labeled nerve terminals below IHCs was shown. Our results presented here underscored the essential role played by acetylated tubulin in postnatal cochlear development, auditory neurotransmission and cochlear mechanics.

Dynamic of lipid droplets and gene expression in response to β-aminoisobutyric acid treatment on 3T3-L1 cells.

Abstract: Research on adipobiology has recognized the browning process of white adipocytes as a potential therapeutic strategy for the treatment of obesity and related morbidities. Physical exercise stimulates the secretion of myokines, such as b-aminoisobutyric acid (BAIBA), which in turn promotes adaptive thermogenesis. White adipocyte conversion to brown cells involves dynamic changes in lipid droplet (LD) dimension and in the transcription of brown-specific marker genes. This study analyzes the effect of different doses of BAIBA and at different days of development on 3T3-L1 cells by evaluating morphological changes in LDs and the expression of browning gene markers. Results suggested that the highest concentration of BAIBA after 4 days of differentiation produced the most significant effects. The number of LDs per cell increased in comparison to control cells, whereas the surface area significantly decreased. Brown adipocyte markers were up-regulated, but the effect of treatment was lost at 10 days of differentiation. The thermogenic program induced by BAIBA may reflect a rapid adaptation of adipose tissue to physical exercise. This connection stresses the beneficial impact of physical exercise on metabolic health. The thermogenic program induced by BAIBA may reflect a rapid adaptation of adipose tissue to physical exercise. This connection stresses the beneficial impact of physical exercise on metabolic health.

Analysis of mineral apposition rates during alveolar bone regeneration over three weeks following transfer of BMP-2/7 gene via in vivo electroporation

Abstract: Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and in vivo electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bone-staining revealed that MAR of alveolar bone was as similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining non-viral BMP-2/7 gene expression vector and in vivo electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.

Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study.

Abstract: A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis. These results, if confirmed by in vitro studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease.

Overexpression of kynurenic acid and 3-hydroxyanthranilic acid after rat traumatic brain injury

Abstract: Using an immunohistochemical technique, we have studied the distribution of kynuneric acid (KYNA) and 3-hydroxyanthranilic acid (3-HAA) in a rat brain injury model (trauma). The study was carried out inducing a cerebral ablation of the frontal motor cortex. Two mouse monoclonal specific antibodies previously developed by our group directed against KYNA and 3-HAA were used. In control animals (sham-operated), the expression of both KYNA and 3-HAA was not observed. In animals in which the ablation was performed, the highest number of immunoreactive cells containing KYNA or 3-HAA was observed in the region surrounding the lesion and the number of these cells decreased moving away from the lesion. KYNA and 3-HAA were also observed in the white matter (ipsilateral side) located close to the injured region and in some cells placed in the white matter of the contralateral side. The distribution of KYNA and 3-HAA perfectly matched with the peripheral injured regions. The results found were identical independently of the perfusion date of animals (17, 30 or 54 days after brain injury). For the first time, the presence of KYNA and 3-HAA has been described in a rat trauma model. Moreover, by using a double immunocytochemistry protocol, it has been demonstrated that both metabolites were located in astrocytes. The findings observed suggest that, in cerebral trauma, KYNA and 3-HAA are involved in tissue damage and that these compounds could act, respectively, as a neuroprotector and a neurotoxic. This means that, in trauma, a counterbalance occurs and that a regulation of the indoleamine 2,3 dioxygenase (IDO) pathway could be required after a brain injury in order to decrease the deleterious effects of ending metabolites (the neurotoxic picolinic acid). Moreover, the localization of KYNA and 3-HAA in the contralateral side of the lesion suggests that the IDO pathway is also involved in the sprouting and pathfinding that follows a traumatic brain injury.

LOX-1 deficient mice show resistance to zymosan-induced arthritis.

Abstract: Recent data suggest that the lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1)/ox-LDL system may be involved in the pathogenesis of arthritis. We aimed to demonstrate the roles of the LOX-1/ox-LDL system in arthritis development by using LOX-1 knockout (KO) mice. Arthritis was induced in the right knees of C57Bl/6 wild-type (WT) and LOX-1 KO mice via zymosan injection. Saline was injected in the left knees. Arthritis development was evaluated using inflammatory cell infiltration, synovial hyperplasia, and cartilage degeneration scores at 1, 3, and 7 days after administration. LOX-1, ox-LDL, and matrix metalloproteinase-3 (MMP-3) expression in the synovial cells and chondrocytes was evaluated by immunohistochemistry. The LOX-1, ox-LDL, and MMP-3 expression levels in synovial cells were scored on a grading scale. The positive cell rate of LOX-1, ox-LDL, and MMP-3 in chondrocytes was measured. The correlation between the positive cell rate of LOX-1 or ox-LDL and the cartilage degeneration score was also examined. Inflammatory cell infiltration, synovial hyperplasia, and cartilage degeneration were significantly reduced in the LOX-1 KOmice with zymosan-induced arthritis (ZIA) compared to WT mice with ZIA. In the saline-injected knees, no apparent arthritic changes were observed. LOX-1 and ox-LDL expression in synovial cells and chondrocytes were detected in the knees of WT mice with ZIA. No LOX-1 and ox-LDL expression was detected in the knees of LOX-1 KOmice with ZIA or the saline-injected knees of both mice. MMP-3 expression in the synovial cells and chondrocytes was also detected in knees of both mice with ZIA, and was significantly less in the LOX-1 KO mice than in WT mice. The positive cell rate of LOX-1 or ox-LDL and the cartilage degeneration score showed a positive correlation. Our data show the involvement of the LOX-1/ox-LDL system in murine ZIA development. LOX-1-positive synovial cells and chondrocytes are potential therapeutic targets for arthritis prevention.

GalNAc-T15 in gastric adenocarcinoma: Characterization according to tissue architecture and cellular location.

Abstract: Gastric cancer (GC) is the second most common cause of cancer-related deaths in the world. This study aims to investigate the differential tissue expression of ppGalNAc-T15 and to evaluate its possible association with clinical-pathological parameters and outcome of gastric adenocarcinoma patients. For these 70 patients were evaluated the expression by immunohistochemistry to ppGalNAc-T15. Our results showed that 33 (47.1%) patients were ppGalNAC-T15+ positive and 37 (52.9%) negative. Positive staining for ppGalNAc-T15 was significantly present in patients older than 60 years (P=0.0306) and submitted to total gastrectomy (P=0.0087). Also, some results remained at the limit of significance as surgical standing (P=0.0562) and histological grade (P=0.0549). Therefore, the ppGalNAc-T15 immunoreactivity can be useful to understand the prognosis of patients with gastric cancer.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost Cyprinus carpio.

Abstract: Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases.

Damaged muscle fibers might masquerade as hybrid fibers - a cautionary note on immunophenotyping mouse muscle with mouse monoclonal antibodies.

Abstract: We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serial-section-labeling, to identify damaged fibers. The quadriceps femoris muscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21 ± 0.21%, 98.34 ± 1.06%, 0.07 ± 0.01%, and 0.53 ± 0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.

Study of liver in HBV-related hepatocellular carcinoma: Stereology shows quantitative differences in liver structure.

Abstract: Hepatocellular carcinoma is one of the main consequences of liver chronic disease Hepatocellular carcinoma-related changes may be seen in patients with chronic hepatitis B The aim of the current study was to quantitate liver tissue elements by stereological technique in patients with hepatitis B-related cancer and compare the results with control and only hepatitis B group Needle liver biopsies from 40 patients with only chronic hepatitis B infection, from 41 patients with only early hepatocellular carcinoma, from 40 patients with early hepatitis B-related cancer and 30 healthy subjects (control group) were analyzed by stereological method using systematic uniform random sampling method Haematoxylin and eosin stained sections were used The numerical density of hepatocytes, hepatocyte volume, numerical density of Kupffer cells, volume density of the connective tissue in the portal space, and volume density of the connective tissue were assessed Quantitative analysis of liver samples indicated that there were statistically significant differences in the numerical density of hepatocytes, hepatocyte volume, numerical density of Kupffer cells, volume density of the connective tissue in the portal space, and volume density of the connective tissue between control and hepatitis B-related cancer and hepatitis B groups Quantitative, stereological technique is simple and reliable for evaluating HCC in chronic hepatitis B It is useful for assessing the liver tissue parameters Stereology is recommended for the diagnosis of people prone to cancer in patients with chronic hepatitis B

Fluorescent characterization of amyloid deposits in the kidneys of mdx mice.

Abstract: Amyloidosis is a group of diseases that occurs when amyloid proteins are deposited in tissues and organs. The traditional way of identifying amyloid in tissue sections is staining with Congo red. However, this method has a number of limitations including background staining (background fluorescence), low fluorescence intensity and false-positive staining. Therefore, a complex of fluorescence-based methods should be applied to characterize tissue localization of amyloid deposits. The aim of this study was to identify amyloid deposits in the kidneys of dystrophin-deficient mdx mice using different fluorescent dyes. We examined 8 cases of renal amyloidosis in aged mdx mice. In all cases, we used traditional methods for amyloid detection (Congo red and Thioflavin T), as well as a new fluorescent dye, disodium salt of 2,7- (1-amino-4-sulfo-2-naphthylazo) fluorene (DSNAF). In our study, we confirmed the amyloid structure of protein deposits in kidneys of aging mdx mice by several fluorescence-based staining methods. We found that fixation method has profound effects on downstream staining procedures, and demonstrated that the application of specific fixative, zinc-ethanol-formaldehyde (ZEF), instead of traditional NBF allow to reduce the background fluorescence. We also illustrated the usefulness of novel fluorescent dye DSNAF for detection of amyloid deposits in mouse tissues. Our results confirmed the strong affinity and high specificity of this dye for amyloid fibrils. The verification of DSNAF for detecting amyloid in human tissues will provide a conclusion on the applicability of the developed staining method in clinical research practice.

Cytoplasmic lattices are not linked to mouse 2-cell embryos developmental arrest.

Abstract: Cytoplasmic lattices are important regulators of oocyte maturation. They store components of the protein synthesis machinery including ribosomes and, among others, they are involved in the regulation of microtubule dynamics in both mouse and human. Cytoplasmic lattices undergo dramatic reorganizations at crucial stages of oocyte maturation, where they are abundantly present in the cytoplasm of developmentally competent oocytes named SN (Surrounded Nucleolus) while they are rare in the cytoplasm of 2-cell stage-arresting NSN (Not Surrounded Nucleolus) oocytes, suggestive of a requirement of cytoplasmic lattices for development past the 2-cell stage. Here, to elucidate this requirement, 2-cell mouse embryos derived from SN and NSN oocytes were analyzed by transmission electron microscopy. Contrary to what had been proposed hitherto, cytoplasmic lattices are present in 2-cell embryos derived not only from SN, but also from NSN oocytes, irrespective of the embryo production system (intra cytoplasmic sperm injection, parthenogenesis). Hence our conclusion that cytoplasmic lattices do not count among the factor(s) responsible for the embryo arrest at this crucial stage of development.

Localization of CGRP and VEGF mRNAs in the mouse superior cervical ganglion during pre- and postnatal development.

Abstract: The neuropeptide calcitonin gene-related peptide (CGRP) mediates inflammation and head pain by influencing the functional vascular blood supply. CGRP is a well-characterized mediator of receptor-regulated neurotransmitter release. However, knowledge regarding the role of CGRP during the development of the superior cervical ganglion (SCG) is limited. In the present study, we observed the localization of CGRP and vascular endothelial growth factor (VEGF-A) mRNAs during prenatal development at embryonic day 14.5 (E14.5), E17.5 and postnatal day 1 (P1) using in situ hybridization. The antisense probe for CGRP was detected by in situ hybridization at E14.5, E17.5, and P1, and the highest levels were detected at E17.5. In contrast, the antisense probe for VEGF-A was detected by in situ hybridization in gradually increasing intensity from E14.5 to P1. The differences in the expression of these two markers revealed specific characteristics related to CGRP concentration and release compared to those of VEGF-A during development. The correlation between CGRP and VEGF-A may influence functional stress and the vascular blood supply during prenatal and postnatal development.

Hexavalents in spermatocytes of Robertsonian heterozygotes between Mus m. domesticus 2n 26 from the Vulcano and Lipari Islands (Aeolian Archipelago, Italy)

Abstract: The size and shape of the chromosomes, as well as the chromosomal domains that compose them, are determinants in the distribution and interaction between the bivalents within the nucleus of spermatocytes in prophase I of meiosis. Thus the nuclear architecture characteristic of the karyotype of a species can be modified by chromosomal changes such as Rb chromosomes. In this study we analysed the meiotic prophase nuclear organization of the heterozygous spermatocytes from Mus musculus domesticus 2n=26, and the synaptic configuration of the hexavalent formed by the dependent Rb chromosomes Rbs 6.16, 16.10, 10.15, 15.17 and the telocentric chromosomes 6 and 17. Spreads of 88 pachytene spermatocytes from two males were studied and in all of them five metacentric bivalents, four telocentric bivalents, one hexavalent and the XY bivalent were observed. About 48% of the hexavalents formed a chain or a ring of synapsed chromosomes, the latter closed by synapsis between the short arms of telocentric chromosomes 6 and 17. About 52% of hexavalents formed an open chain of 10 synapsed chromosomal arms belonging to 6 chromosomes. In about half of the unsynapsed hexavalents one of the telocentric chromosome short arms appears associated with the X chromosome single axis, which was otherwise normally paired with the Y chromosome. The cluster of pericentromeric heterochromatin mostly determines the hexavalent’s nuclear configuration, dragging the centromeric regions and all the chromosomes towards the nuclear envelope similar to an association of five telocentric bivalents. These reiterated encounters between these chromosomes restrict the interactions with other chromosomal domains and might favour eventual rearrangements within the metacentric, telocentric or hexavalent chromosome subsets. The unsynapsed short arms of telocentric chromosomes frequently bound to the single axis of the X chromosome could further complicate the already complex segregation of hexavalent chromosomes.