Immunocytochemistry is a laboratory technique used to visualize the presence and location of specific proteins or antigens in horse tissue or cell samples. This method employs antibodies that bind to target proteins, which are then detected using various markers, such as fluorescent dyes or enzymes. In equine research, immunocytochemistry is applied to study cellular processes, disease mechanisms, and tissue pathology. It allows researchers to observe the distribution and expression of proteins within horse tissues, aiding in the understanding of equine diseases and conditions. This page compiles peer-reviewed research studies and scholarly articles that explore the application, methodology, and findings of immunocytochemistry in equine science.
Chan G, Morgan WH, Yu DY, Balaratnasingam C.Astrocytes are critical for the maintenance of retinal ganglion cell (RGC) axonal function and viability, and form a key component of the functional neurovascular unit. Recently, we described the quantitative properties of astrocytes in relation to the capillary distributions in optic nerve laminar regions. Here, we provide a quantitative analysis of astrocytes and RGC axons in longitudinal sections of optic nerve tissue. Histological and immunocytochemical techniques are used to demonstrate the density of astrocytes, RGC axons and glia-neuron ratios across the pre laminar, lamina cribrosa and...
Tarko A, Fabová Z, Kotwica J, Valocký I, Alrezaki A, Alwasel S, Harrath AH, Sirotkin AV.The influence of environmental contaminant toluene and of plant fennel (Foeniculum vulgare Mill.) on reproduction are reported, but the mechanisms of their action and the protective effect of fennel on contaminant influence remain to be elucidated. In this study, we hypothesized that toluene and fennel directly affects basic ovarian cell functions, and that fennel can be used as an appropriate natural protective agent against the potential adverse effects of toluene. This study aimed to examine the action of toluene (20 μg/mL) and fennel extract (0, 1, 10, 100 μg/mL), and assess their comb...
Yang Q, Lopez MJ.The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning e...
Yang Q, Pinto VMR, Duan W, Paxton EE, Dessauer JH, Ryan W, Lopez MJ.Damage to an ectodermal-mesodermal interface like that in the equine hoof and human finger nail bed can permanently alter tissue structure and associated function. The purpose of this study was to establish and validate culture of primary progenitor cell isolates from the ectodermal-mesodermal tissue junction in equine hooves, the stratum internum, with and without chronic inflammation known to contribute to lifelong tissue defects. The following were evaluated in hoof stratum internum cell isolates up to 5 cell passages (P): expansion capacity by cell doublings and doubling time; plasticity ...
Jaworska J, Janowski T.Retained fetal membranes (RFM) is a prevalent postpartum disease in mares. During parturition, various proinflammatory cytokines are released in the placenta, which further facilitates uterine contractions, expulsion of the fetus and fetal membranes. We hypothesized that an altered inflammatory response in mares results in retained fetal membranes. Samples of the endometrium and the allantochorion were collected from 15 mares with RFM and from 29 control mares within 2 h of foal delivery. Next, the mRNA expression of IL-1β, IL-6 and TNFα proteins was analyzed by Real Time PCR and Western b...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Duran MC, Starrak G, Dickinson R, Montgomery J.After physical examination, ultrasonographic evaluation of thorax and abdomen, and peritoneal fluid analysis, gastrointestinal neoplasia with suspected diffuse peritoneal metastasis was diagnosed in a 17-year-old Arabian gelding. The owner elected euthanasia and declined postmortem examination. Immunocytochemistry analysis of the peritoneal fluid resulted in a diagnosis of B-cell lymphoma. Immunocytochimie du liquide péritonéal utilisée pour le diagnostic d’un cas possible de lymphome gastro-intestinal à cellules B. Après un examen physique, une évaluation échographique du thorax et d...
Fülber J, Maria DA, da Silva LC, Massoco CO, Agreste F, Baccarin RY.Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank. Methods: Expression of the markers CD90, CD105, CD44, and ...
Merkl M, Ertl R, Handschuh S, Aurich C, Schäfer-Somi S.In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen ...
Kim JT, Jung HJ, Song H, Yoon MJ.Acrosin Binding Protein (ACRBP) is specifically localized in the acrosome of germ cells of several species, including mice, pigs, guinea pigs, and humans. The main objective of this study was to investigate ACRBP patterns in the germ cells of stallions at different reproductive stages and seminiferous tubule stages using Western blot, immunohistochemistry, and immunocytochemistry techniques. The stallion reproductive stages were classified as follows: pre-pubertal and post-pubertal stages based on the presence/absence of lumen opening in the seminiferous tubules and full spermatogenesis. The p...
Rapacz-Leonard A, Raś A, Całka J, Janowski TE.Fetal membrane retention can be a life-threatening condition and its incidence exceeds 50% in heavy draught mares. Although fetal membrane retention is commonly treated with repeated injections of oxytocin, based on the suggestion that it is caused mainly by secondary atony of the uterus, this treatment sometimes fails. This led us to ask if expression of oxytocin receptors differs in mares that retain fetal membranes due to secondary uterine atony. Objective: To determine whether expression of oxytocin receptors in equine placental tissues differs when heavy draught mares expel fetal membrane...
Miller L, Woodward EM, Campos JR, Squires EL, Troedsson M.The objectives of this study were to (i) verify localization of SP22 on fresh, cooled, and frozen/thawed equine spermatozoa and to (ii) determine SP22 mRNA and protein expression in equine testicular and epididymal tissues. Immunocytochemistry and Western blots were performed on the spermatozoa samples. Northern blots and Western blots were performed on the tissue samples. The immunocytochemistry revealed the presence of SP22 in all samples tested. The fresh spermatozoa stained predominantly over the equatorial segment as did the samples cooled for 1 and 2 days. The samples cooled for 3 days, ...
Donnelly KS, Giuliano EA, Sharm A, Mohan RR.To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Methods: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determi...
Jung HJ, Song H, Yoon MJ.Deleted in azoospermia-like (DAZL) is used as a germ cell marker in several species, including mice, rats, pigs, rhesus monkeys, bulls, and humans. Our objectives with this study were to investigate DAZL expression in stallion germ cells by using immunofluorescence, immunocytochemistry, and western blotting, and to determine the effects of reproductive stage and breeding season on the DAZL-positive cell population in seminiferous tubule cross sections. Testes were obtained during routine castration procedures at a large animal clinic and routine field service castration. The reproductive stage...
Gulati BR, Kumar R, Mohanty N, Kumar P, Somasundaram RK, Yadav PS.Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polyme...
Campos-Junior PH, Costa GM, de Avelar GF, Segatelli TM, Lacerda SM, Aponte PM, de França LR.Morphometry is a classical quantitative method often used in biology to provide a data basis for functional interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this specific and crucial microenvironment. Based on several recently ...
Maia L, Landim-Alvarenga FC, Da Mota LS, De Assis Golim M, Laufer-Amorim R, De Vita B, Barberini DJ, Listoni AJ, De Moraes CN, Heckler MC, Amorim RM.The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich ext...
Szober CM, Hauck SM, Euler KN, Fröhlich KJ, Alge-Priglinger C, Ueffing M, Deeg CA.The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, an...
Gautam A, Dubey JP, Saville WJ, Howe DK.Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. I...
Eberhardt C, Amann B, Stangassinger M, Hauck SM, Deeg CA.Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion techniqu...
Sonmez K, Gurel A, Takai S.The aim of the present study was to develop an immunocytochemical procedure for the early detection and demonstration of Rhodococcus equi in smears of tracheal aspirates taken from live foals in field conditions. Tracheal wash samples were collected from thoroughbred foals, aged 1-5 months and located in studs around Bursa and Istanbul, Turkey. Some foals were suspected of having R. equi infection on the basis of clinical examination (n=56) and others were unaffected control animals (n=54). Serum samples were also collected from each foal for testing for the presence of R. equi-specific antibo...
Buss DG, Sharma A, Giuliano EA, Mohan RR.Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Methods: An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFbeta1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion...
Mathes RL, Dietrich UM, Krunkosky TM, Hurley DJ, Reber AJ.To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation. Methods: Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immun...
Katavolos P, Ackerley CA, Viel L, Clark ME, Wen X, Bienzle D.Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horse...
Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF.To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Methods: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. Methods: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transdu...
Sabeur K, Ball BA.Carbohydrate-binding proteins are thought to be involved in a myriad of sperm functions including sperm-oviductal and sperm-zona interactions. Recent studies in our laboratory have characterized galactose-binding proteins on equine spermatozoa as possible candidate molecules for sperm adhesion to oviduct epithelial cells. In the current study, equine sperm membrane proteins were subjected to galactose-affinity chromatography, and bound proteins were eluted with excess galactose in a calcium-free buffer. The eluted fraction recovered after galactose-affinity chromatography was used for generati...
Singh Suri S, Janardhan KS, Parbhakar O, Caldwell S, Appleyard G, Singh B.Toll-like receptor (TLR) is a key component in launching innate immune response to microbial challenge. TLR4 and TLR2 are recognized as specific receptors for components of Gram-negative and Gram-positive bacteria, respectively. Horses are extremely sensitive to endotoxin-induced cardiopulmonary distress and mortality which causes significant economic losses. To date, there are no data on the expression of TLR4 and TLR2 in horse lungs. Therefore, we examined the expression of TLR4 and TLR2 in lungs from normal or Escherichia coli lipopolysaccharide (E. coli LPS; 50 ng/kg; iv) treated horses. W...
Soverchia L, Mosconi G, Ruggeri B, Ballarini P, Catone G, Degl'Innocenti S, Nabissi M, Polzonetti-Magni AM.Proopiomelanocortin (POMC) is a precursor protein that contains the sequences of several bioactive peptides including adrenocorticotropin (ACTH), beta-endorphin (beta-EP), and melanocyte-stimulating-hormone (MSH). POMC is synthesized in the pituitary gland, brain, and many peripheral tissues. Immunoreactive POMC-derived peptides as well as POMC-like mRNA have been evidenced in several nonpituitary tissues, thus suggesting that POMC is actively synthesized by these tissues. The present study was aimed at evaluating if also in the case of stallion POMC-derived peptide, beta-EP, is produced local...
Caminoto EH, Alves AL, Amorim RL, Thomassian A, Hussni CA, Nicoletti JL.To evaluate the effects of extracorporeal shock wave therapy (ESWT) on affected ligaments in the hind limbs of horses with experimentally induced suspensory ligament desmitis by use of ultrasonographic, ultrastructural, and immunocytochemical techniques. Methods: 10 horses. Methods: Suspensory ligament desmitis was induced in both hind limbs of each horse by use of 2 collagenase injections (administered 2 weeks apart) in each suspensory ligament. Two weeks after the second injection, the right hind limb of each horse was treated with ESWT (3 treatments at 3-week intervals); the left hind limb ...
The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min ...
Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF.To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Methods: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. Methods: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transdu...
Heinrichs M, Baumgärtner W, Capen CC.Adenomas of the pars intermedia from 19 horses and normal pituitary glands from seven horses were evaluated histologically and immunocytochemically for adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-endorphin (beta-END), proopiomelanocortin (POMC), prolactin, neuron specific enolase, and glial fibrillary acidic protein (GFAP). The 26 horses ranged in age from 7 to 31 years. Histologically, all adenomas had a uniform pattern characterized by cords of large columnar cells forming palisades and pseudoacini separated by a delicate fibrovascular stroma. I...
Donnelly KS, Giuliano EA, Sharm A, Mohan RR.To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Methods: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determi...
Fülber J, Maria DA, da Silva LC, Massoco CO, Agreste F, Baccarin RY.Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank. Methods: Expression of the markers CD90, CD105, CD44, and ...
Schnitzer J.The distribution of astrocytes has been studied in whole-mounted horse and monkey retinae by the immunocytochemical localization of glial fibrillary acidic protein (GFAP). In the horse, astrocytes were found to be restricted to a narrow zone close to the optic nerve head. This is also the only region of the horse retina that is vascularized. In the monkey, astrocytes were found ubiquitously in the nerve fiber layer of the retina, apart from the avascular fovea centralis which lacked astrocytes. These observations strongly suggest that retinal astrocytes co-occur with blood vessels, a feature w...
Katavolos P, Ackerley CA, Viel L, Clark ME, Wen X, Bienzle D.Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horse...
Kitamura N, Yamada J, Calingasan NY, Yamashita T.Endocrine cells immunoreactive for somatostatin, gastrin, glicentin, glucagon, secretin, cholecystokinin, motilin and neurotensin were identified immunocytochemically in the gastrointestinal mucosa of the horse. Somatostatin-, glicentin- and glucagon-immunoreactive cells were very numerous in the cardiac and fundic regions of the stomach, whereas most gastrin-immunoreactive cells were confined to the pyloric region. Somatostatin-immunoreactive cells also were detected in all portions of the small intestine while gastrin-immunoreactive cells were confined exclusively to the upper portion and gl...
Singh Suri S, Janardhan KS, Parbhakar O, Caldwell S, Appleyard G, Singh B.Toll-like receptor (TLR) is a key component in launching innate immune response to microbial challenge. TLR4 and TLR2 are recognized as specific receptors for components of Gram-negative and Gram-positive bacteria, respectively. Horses are extremely sensitive to endotoxin-induced cardiopulmonary distress and mortality which causes significant economic losses. To date, there are no data on the expression of TLR4 and TLR2 in horse lungs. Therefore, we examined the expression of TLR4 and TLR2 in lungs from normal or Escherichia coli lipopolysaccharide (E. coli LPS; 50 ng/kg; iv) treated horses. W...
Raulo SM, Sorsa T, Tervahartiala T, Pirilä E, Maisi P.Gelatinolytic activity was analysed to study whether elevated activity previously found at the tracheal level of the respiratory tract of horses with chronic obstructive pulmonary disease (COPD) could also be found at the lower part of the respiratory tract. Furthermore, presence and significance of the gelatinolytic matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in respiratory secretions of healthy and COPD horses were determined. Elevated gelatinolytic matrix metalloproteinases were detected in bronchoalveolar and tracheobronchial secretions from COPD horses. The main pathologically elevat...
Chao TI, Grosche J, Friedrich KJ, Biedermann B, Francke M, Pannicke T, Reichelt W, Wulst M, Mühle C, Pritz-Hohmeier S, Kuhrt H, Faude F, Drommer W....Müller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the 'midperiphery' of retinae immunocytochemically labeled for vimentin, estimates of Müller cell densities per unit retinal surface area, and of neuron-to-(Müller) glia indices were performed. Müller cell densities were strikingly similar among the species studied (around 8000-11,000 mm-2) with the extremes of the horse ( or = 20,000 mm-2). By contrast, the number of neurons per Müller cell varied widely, being clustered at 6-8 (in retinae with many cones), at about 16, and at up t...
Gulati BR, Kumar R, Mohanty N, Kumar P, Somasundaram RK, Yadav PS.Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polyme...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Hess MF, Roser JF.The large amount of testicular estrogens produced by the stallion is unique compared to the amounts found in other domestic species. Although the cellular locale of the cytochrome P450 aromatase (P450arom) enzyme that converts C19 androgens to C18 estrogens has been identified in the Leydig cell of adult equine testis, the location in the immature equine testis is not known. The goal of this work was to localize the enzyme in colts and stallions during sexual development. Testes were obtained from prepubertal (n=7), pubertal (n=6), and postpubertal (n=8) colts and stallions during both the bre...
Jaworska J, Janowski T.Retained fetal membranes (RFM) is a prevalent postpartum disease in mares. During parturition, various proinflammatory cytokines are released in the placenta, which further facilitates uterine contractions, expulsion of the fetus and fetal membranes. We hypothesized that an altered inflammatory response in mares results in retained fetal membranes. Samples of the endometrium and the allantochorion were collected from 15 mares with RFM and from 29 control mares within 2 h of foal delivery. Next, the mRNA expression of IL-1β, IL-6 and TNFα proteins was analyzed by Real Time PCR and Western b...
Maia L, Landim-Alvarenga FC, Da Mota LS, De Assis Golim M, Laufer-Amorim R, De Vita B, Barberini DJ, Listoni AJ, De Moraes CN, Heckler MC, Amorim RM.The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich ext...
Raulo SM, Sorsa TA, Kiili MT, Maisi PS.To determine collagenase activity and evaluate matrix metalloproteinase (MMP)-8 and MMP-13 in horses with chronic obstructive pulmonary disease (COPD). Methods: 12 horses with COPD and 12 healthy control horses. Methods: Collagenase activity was determined by use of an assay for degradation of type-I collagen. Western immunoblot analysis was used to identify interstitial collagenases MMP-8 and MMP-13 in tracheal epithelial lining fluid (TELF). Immunocytochemistry and in situ hybridization were used to determine cellular expression of these 2 collagenases in cells in bronchoalveolar lavage flui...
Buss DG, Sharma A, Giuliano EA, Mohan RR.Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Methods: An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFbeta1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion...
Sandmann D, Boycott BB, Peichl L.The morphology of horizontal cells chiefly of the horse, but also of asses, mules, and a zebra, has been examined by Lucifer yellow injections into lightly fixed retinae and by immunocytochemistry. In common with other mammals, equids have a B-type horizontal cell, i.e., a cell with dendrites synapsing with cones and possessing a single axon synapsing with rods. Most mammalian retinae have a further type of horizontal cell, the A-type, also synapsing with cones but without an axon. The second type of horizontal cell in equids also has no axon; otherwise, it is most unusual. Compared with other...
Eberhardt C, Amann B, Stangassinger M, Hauck SM, Deeg CA.Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion techniqu...
Gamboa S, Ramalho-Santos J.Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobr...
Jung HJ, Song H, Yoon MJ.Deleted in azoospermia-like (DAZL) is used as a germ cell marker in several species, including mice, rats, pigs, rhesus monkeys, bulls, and humans. Our objectives with this study were to investigate DAZL expression in stallion germ cells by using immunofluorescence, immunocytochemistry, and western blotting, and to determine the effects of reproductive stage and breeding season on the DAZL-positive cell population in seminiferous tubule cross sections. Testes were obtained during routine castration procedures at a large animal clinic and routine field service castration. The reproductive stage...
Eisenhauer KM, McCue PM, Nayden DK, Osawa Y, Roser JF.Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Ser...
Caminoto EH, Alves AL, Amorim RL, Thomassian A, Hussni CA, Nicoletti JL.To evaluate the effects of extracorporeal shock wave therapy (ESWT) on affected ligaments in the hind limbs of horses with experimentally induced suspensory ligament desmitis by use of ultrasonographic, ultrastructural, and immunocytochemical techniques. Methods: 10 horses. Methods: Suspensory ligament desmitis was induced in both hind limbs of each horse by use of 2 collagenase injections (administered 2 weeks apart) in each suspensory ligament. Two weeks after the second injection, the right hind limb of each horse was treated with ESWT (3 treatments at 3-week intervals); the left hind limb ...
Merighi A, Kar S, Gibson SJ, Ghidella S, Gobetto A, Peirone SM, Polak JM.The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig. In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I-III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of i...
Merkl M, Ertl R, Handschuh S, Aurich C, Schäfer-Somi S.In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen ...
Thomas PG, Ignotz GG, Ball BA, Miller PG, Brinsko SP, Currie B.Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17 beta or a carrier control. All groups were subcultured three times. Epithelial cell morphology and fu...
Rapacz-Leonard A, Raś A, Całka J, Janowski TE.Fetal membrane retention can be a life-threatening condition and its incidence exceeds 50% in heavy draught mares. Although fetal membrane retention is commonly treated with repeated injections of oxytocin, based on the suggestion that it is caused mainly by secondary atony of the uterus, this treatment sometimes fails. This led us to ask if expression of oxytocin receptors differs in mares that retain fetal membranes due to secondary uterine atony. Objective: To determine whether expression of oxytocin receptors in equine placental tissues differs when heavy draught mares expel fetal membrane...
Bowker RM, Brewer AM, Vex KB, Guida LA, Linder KE, Sonea IM, Stinson AW.Two types of sensory receptors were located in the equine foot, using anatomic techniques. Histologic examination of stained hoof sections revealed lamellated corpuscles in the hoof dermis, which had many of the morphologic characteristics of Pacinian corpuscles. These sensory receptors were restricted to the palmar (caudal) aspects of the solar dermis of the heel. A second type of receptor was detected by use of immunocytochemistry, indicating apparently naked nerve endings containing the neuropeptide calcitonin gene-related peptide-like immunoreactivity in skin, solar dermal tubules, and the...
Kim JT, Jung HJ, Song H, Yoon MJ.Acrosin Binding Protein (ACRBP) is specifically localized in the acrosome of germ cells of several species, including mice, pigs, guinea pigs, and humans. The main objective of this study was to investigate ACRBP patterns in the germ cells of stallions at different reproductive stages and seminiferous tubule stages using Western blot, immunohistochemistry, and immunocytochemistry techniques. The stallion reproductive stages were classified as follows: pre-pubertal and post-pubertal stages based on the presence/absence of lumen opening in the seminiferous tubules and full spermatogenesis. The p...
Wooding FB, Morgan G, Fowden AL, Allen WR.The placenta is the only channel for transport of nutrients to the conceptus and the fetal nutrient demands increase exponentially to term. The 9 kDa calcium binding protein (calbindin, 9CBP) and the iron binding protein uteroferrin (UF) are proving to be reliable markers for epithelia that mediate active transcellular calcium and iron transport and the glucose transporter proteins (GT1 and GT3) for glucose transport by facilitated diffusion. Light and electron microscope immunocytochemistry have been used on perfusion fixed resin embedded material to establish the distribution of 9CBP, UF, GT...
