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International Journal of Reproductive... May 2018Troxerutin is a flavonoid antioxidant that protect different organ against damage caused by ischemia-reperfusion
BACKGROUND
Troxerutin is a flavonoid antioxidant that protect different organ against damage caused by ischemia-reperfusion
OBJECTIVE
The aim of this study was to evaluate the effect of troxerutin in reducing the damages caused by ischemia-reperfusion in rat's testis.
MATERIALS AND METHODS
40 Male Wistar rats (2 month old) were divide to four groups (n=10). Group1 (sham), Group 2 (control, ischemia-reperfusion (I/R) without treatment), Group 3 (I/R+150 mg/kg of troxerutin), and group 4 (I/R+20 mg/kg of vitamin C). Treatment of group 3 and group 4 during torsion (twists 720 counter clock at 90 min) followed by 50 days detorsion. After 50 days, blood samples were collected and rats in all study groups were killed and their testes were removed, and fixed with Bouin's solution. Testis was stained with hematoxylin and eosin dye and the level of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured with ELISEA methods. TUNEL was employed to detect apoptosis. Epididymis caudal part was removed and total sperm count was determined. Johnson techniques were used for assessment of seminiferous tubules quality.
RESULTS
Troxerutin treated group has higher Johnson score's (p≤0.001), antiapoptotic properties (p≤0.001), sperm count (p=0.065), and higher LH (p≤0.001), FSH (p≤0.001) and testosterone (p=0.002) levels than control group. Vitamin C treated group showed increase level of testosterone but didn't show significant differences on the number of apoptotic cells, Johnson scores, LH, FSH and sperm count than control group.
CONCLUSION
Troxerutin has protective effects on testicular torsion induced injury and can ameliorate spermatogenesis in the torsion-detorsion models.
PubMed: 30027147
DOI: No ID Found -
Infection and Immunity Dec 2017Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated...
Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with -engineered strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent -labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria ( < 0.05). A greater incidence ( < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences ( > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased ( < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly ( < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.
Topics: Animals; Biofilms; Endometritis; Endometrium; Female; Genes, Reporter; Horse Diseases; Horses; Luciferases; Pseudomonas Infections; Pseudomonas aeruginosa
PubMed: 28970274
DOI: 10.1128/IAI.00332-17 -
Molecular and Cellular Biology Nov 2006CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of...
CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.
Topics: Acetic Acid; Animals; Apoptosis; CDC2 Protein Kinase; Calcium-Binding Proteins; Cell Proliferation; Epididymis; Fibroblasts; Formaldehyde; Infertility, Male; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Picrates; Recombination, Genetic; Spermatids; Spermatocytes; Spermatogenesis; Testis
PubMed: 16982698
DOI: 10.1128/MCB.01488-06 -
Environmental Health Perspectives Apr 1990There are three major epithelial types in the nasal mucosa, in addition to numerous accessory structures, some of which are species specific. Without careful and... (Review)
Review
There are three major epithelial types in the nasal mucosa, in addition to numerous accessory structures, some of which are species specific. Without careful and consistent processing of the nose tissue, histopathologic assessment of lesions in the nasal cavity may be compromised. While formalin fixation may be used for routine review of the nasal cavity, Bouin's fixation provides better histologic detail and fewer artifacts. Decalcification is not recommended for nasal tissues to be examined by transmission electron microscopy because of the detrimental effect of decalcifying solutions on sensory cells. Three levels of the nasal cavity may be used for routine histologic review of the nasal cavity, but four or five levels may be more appropriate for certain studies.
Topics: Acetates; Acetic Acid; Animals; Decalcification Technique; Epithelium; Fixatives; Formaldehyde; Histological Techniques; Male; Microscopy, Electron; Nasal Cavity; Nasal Mucosa; Picrates; Rats; Rats, Inbred F344
PubMed: 2200662
DOI: 10.1289/ehp.85-1568325 -
European Journal of Histochemistry : EJH Jun 2019Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are...
Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are "hard to hold onto" once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, the aim was the establishment of a reliable and reproducible protocol. Tissue samples were available from pig feeding experiments. In the present study, we focus on a fixation / staining procedure without making comparisons between differently fed pigs. Several fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section. Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin's solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with intact mucin containing vesicles within the cells, which was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was possible. In conclusion, reliable mucus quantification and assessment of mucus quality requires strictly tested procedures. According to our experience, the most important aim after cryosectioning is fast fixation of the mucosubstances, which requires a combination of different fixation steps.
Topics: Animals; Cytoplasmic Vesicles; Gastrointestinal Tract; Goblet Cells; Histocytochemistry; Mucins; Staining and Labeling; Swine
PubMed: 31232013
DOI: 10.4081/ejh.2019.3030 -
The American Journal of Pathology Jan 1998Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization...
Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.
Topics: Cells, Cultured; DNA, Viral; Female; Herpesvirus 4, Human; Hodgkin Disease; Humans; In Situ Hybridization; Papillomaviridae; RNA, Viral; Staining and Labeling; Tissue Fixation
PubMed: 9422521
DOI: No ID Found -
Veterinary Research 2001Monokines are glycoproteins, synthesised by macrophages, which exert various effects on the organism. The most important monokines are interleukin (IL)-1alpha, IL-1beta,...
Monokines are glycoproteins, synthesised by macrophages, which exert various effects on the organism. The most important monokines are interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-6. This paper reports on immunohistochemical techniques developed for the detection of IL-alpha, IL-1beta, IL-6 and TNF-alpha in fixed and paraffin-embedded pig tissues (spleen, lymph nodes, thymus, liver and kidney). Different fixatives (buffered formalin, acetic formalin, paraformadehyde-lysine-periodate and Bouin solution), and antigen unmasking techniques (permeabilisation with Tween 20, pronase enzymatic digestion and microwave-citrate buffer) were used. We describe different protocols for detection of monokines using polyclonal antibodies against the studied monokines. No signal was obtained with monoclonal antibodies against pig-TNF-alpha and human IL-1alpha. Bouin solution was shown to be the best fixative for immunohistochemical detection of IL-1alpha, TNF-alpha, and IL-6, using permeabilisation with Tween 20 as an unmasking antigen method. Acetic formalin was shown to be the best fixative for IL-1beta detection, not needing antigen retrieval techniques. Macrophages were identified as the main cytokine-producing cells, although other types of cells also stained positively to some cytokines. These techniques represent valuable tools for studies of the pathogenesis of viral and bacterial diseases, and of the immune system of the pigs.
Topics: Animals; Antibodies; Immunohistochemistry; Interleukin-1; Interleukin-6; Kidney; Liver; Lymph Nodes; Monokines; Spleen; Swine; Thymus Gland; Tumor Necrosis Factor-alpha
PubMed: 11777010
DOI: 10.1051/vetres:2001103 -
Veterinary Research Forum : An... 2017This study was designed to investigate the effects of applying 1 mT static magnetic field (SMF) during the vitrification process, on the viability of ovarian follicles...
This study was designed to investigate the effects of applying 1 mT static magnetic field (SMF) during the vitrification process, on the viability of ovarian follicles after vitrification-warming and autotransplantation. The study was conducted in two phases. In the first phase, ovaries of female NMRI mice (6 to 8 weeks old) were randomly divided into three groups: 1- Freshly isolated ovaries fixed in Bouin solution (control group), 2- Ovaries vitrified-warmed without exposure to magnetic field (V1 group) and 3- Ovaries exposed to magnetic field during equilibration step of the vitrification process (V2 group). In the second phase, the vitrified (V1 and V2 groups) and fresh ovarian tissues were autografted into the back muscles of the mice from which the ovaries were extracted. In both phases, morphological aspects and molecular characteristics of active-apoptotic caspase-3 antibody were evaluated. Results indicated the lower percentages of morphologically intact primordial, primary and antral follicles in the V1 group (67.6, 49.5 and 17.6%, respectively) than those of control (97.3, 85.4 and 42.1%, respectively) and V2 (94.1, 78.8 and 40.9%, respectively) groups. In addition, the mean percentages of morphologically intact follicles in the V1 group were statistically lower than those in other groups, after transplantation. The rate of apoptosis in preantral follicles of the V1 group was significantly higher than that in the other groups. It was concluded that exposure of mice ovaries to SMF during vitrification resulted in greater resistance to injuries.
PubMed: 29085613
DOI: No ID Found -
Human Reproduction (Oxford, England) Jun 2021Can ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin...
STUDY QUESTION
Can ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin (NBF) with 5% acetic acid (referred to hereafter as Form-Acetic)?
SUMMARY ANSWER
Fixation with Form-Acetic improved ovarian tissue histology compared to NBF in multiple species while still enabling histological molecular analyses.
WHAT IS KNOWN ALREADY
NBF fixation results in tissue shrinkage in various tissue types including the ovary. Components of ovarian tissue, notably follicles, are particularly susceptible to NBF-induced morphological alterations and can lead to data misrepresentation. Bouin's solution (which contains 5% acetic acid) better preserves tissue architecture compared to NBF but is limited for immunohistochemical analyses.
STUDY DESIGN, SIZE, DURATION
A comparison of routinely used fixatives, NBF and Bouin's, and a new fixative, Form-Acetic was carried out. Ovarian tissue was used from three different species: human (n = 5 patients), sheep (n = 3; 6 ovaries; 3 animals per condition) and mouse (n = 14 mice; 3 ovaries from 3 different animals per condition).
PARTICIPANTS/MATERIALS, SETTING, METHODS
Ovarian tissue from humans (aged 13 weeks to 32 years), sheep (reproductively young i.e. 3-6 months) and mice (10 weeks old) were obtained and fixed in 2 ml NBF, Bouin's or Form-Acetic for 4, 8, and 24 h at room temperature. Tissues were embedded and sectioned. Five-micron sections were stained with haemotoxylin and eosin (H&E) and the percentage of artefact (clear space as a result of shrinkage) between ovarian structures was calculated. Additional histological staining using Periodic acid-Schiff and Masson's trichrome were performed on 8 and 24 h NBF, Bouin's and Form-Acetic fixed samples to assess the compatibility of the new fixative with stains. On ovarian tissue fixed for both 8 and 24 h in NBF and Form-Acetic, immunohistochemistry (IHC) studies to detect FOXO3a, FoxL2, collagen IV, laminin and anti-Müllerian hormone (AMH) proteins were performed in addition to the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay to determine the compatibility of Form-Acetic fixation with types of histological molecular analyses.
MAIN RESULTS AND THE ROLE OF CHANCE
Fixation in Form-Acetic improved ovarian tissue morphology compared to NBF from all three species and either slightly improved or was comparable to Bouin's for human, mouse and sheep tissues. Form-Acetic was compatible with H&E, Periodic acid-Schiff and Masson's trichrome staining and all proteins (FOXO3a, FoxL2, collagen IV and laminin and AMH) could be detected via IHC. Furthermore, Form-Acetic, unlike NBF, enabled antigen recognition for most of the proteins tested without the need for antigen retrieval. Form-Acetic also enabled the detection of damaged DNA via the TUNEL assay using fluorescence.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
In this study, IHC analysis was performed on a select number of protein types in ovarian tissue thus encouraging further studies to confirm the use of Form-Acetic in enabling the detection of a wider range of protein forms in addition to other tissue types.
WIDER IMPLICATIONS OF THE FINDINGS
The simplicity in preparation of Form-Acetic and its superior preservative properties whilst enabling forms of histological molecular analyses make it a highly valuable tool for studying ovarian tissue. We, therefore, recommend that Form-Acetic replaces currently used fixatives and encourage others to introduce it into their research workflow.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the Oxford Medical Research Council Doctoral Training Programme (Oxford MRC-DTP) grant awarded to B.D.B. (Grant no. MR/N013468/1), the Fondation Hoffmann supporting R.A. and the Petroleum Technology Development Fund (PTDF) awarded to B.V.A.
Topics: Acetic Acid; Animals; Anti-Mullerian Hormone; Female; Fixatives; Formaldehyde; Humans; Immunohistochemistry; Mice; Ovary; Sheep; Tissue Preservation
PubMed: 33956944
DOI: 10.1093/humrep/deab075 -
The localization of estrogen receptor alpha and its function in the ovaries of postmenopausal women.Folia Histochemica Et Cytobiologica 2007The localization of estrogen receptor alpha (ERalpha) in the ovaries of postmenopausal women is a very up-to-date topic in the aspect of using estrogens therapy in the...
The localization of estrogen receptor alpha (ERalpha) in the ovaries of postmenopausal women is a very up-to-date topic in the aspect of using estrogens therapy in the clinical situations of different type. In ovaries of reproductive age women ERalpha is present in ovary stroma, theca and granulosa cells, ovary surface epithelium (OSE) and in corpus luteum. The ovaries of postmenopausal women are smaller than those of women at the reproductive age, the division into cortex and medulla gets blurred, the ovaries have no follicles any longer, and the stroma is mainly composed of fibrous connective tissue, corpora albicantia, nerves, and blood and lymphatic vessels. The aim of our study was to investigate the immunolocalization and immunoexpression of ERalpha in the ovaries of postmenopausal women. The study involved 50 postmenopausal women who had their ovaries removed by laparotomy due to non-neoplastic diseases of the uterus. The women were divided into 3 groups (A, B, and C) depending on the time that had passed since the last menstruation. Group A consisted of women who had their last menstruation no more than 5 years earlier, in group B menopause occurred 5 to 10 years earlier, group C was composed of patients who had the last menstruation over 10 years earlier. In all the patients concentrations of follicle stimulating hormone (FSH), luteinizing stimulating hormone (LH), estradiol (E2), testosterone (T), androstendione (A) and dehydroepiandrosterone sulphate (DHEAS) in blood plasma were measured. Ovarian tissue was obtained during surgery. For morphological studies, ovaries were fixed in Bouin;s solution and 4% formalin and embedded in paraffin. Morphological analysis was carried out after hematoxylin-eosin (HE) staining. Comparing to groups A and B, the ovaries in group C contained a small number of corpora albicantia located in the medullary part as well as thinned blood vessels and few lymphatic vessels and nerves. For immunoohistochemical expression of ERalpha paraffin-embedded specimens fixed in 4% buffered formalin were used. The sections were next incubated with monoclonal mouse anti-human ERalpha antibody (N 1575 Dako, Denmark). Immunohistochemical nuclear expression of ERalpha in OSE, in epithelial inclusion cysts, in stroma, and in group A also cytoplasmic expression of ERalpha in luteal and paraluteal cells of disappearing corpus luteum were revealed. Immunohistochemical expression of ERalpha seems to decrease in the ovaries of women after menopause.
Topics: Estradiol; Estrogen Receptor alpha; Female; Follicle Stimulating Hormone; Humans; Immunohistochemistry; Luteinizing Hormone; Middle Aged; Ovary; Postmenopause; Protein Transport; Testosterone
PubMed: 18165170
DOI: No ID Found