Immunolocalization of nerve growth factor (NGF) and its receptors (TrkA and p75LNGFR) in the reproductive organs of Shiba goats

The objective of this study was to determine the immunolocalization of NGF and its receptors (TrkA and p75LNGFR) in the reproductive tract of the Japanese Shiba goats. Five adult goats were used in this study and sections of ovaries, uteri and oviducts were immunostained by the avidin-biotin-peroxidase complex method (ABC). The results showed that NGF and its receptors (TrkA and p75LNGFR) were expressed in granulosa cells, theca cells, interstitial cells and lutein cells in ovaries. Immunoreactions for NGF, TrkA and p75LNGFR were also detectable in epithelial cells and muscle cells of the ampulla and isthmus of the oviduct, and in epithelial cells and uterine glands of the uterus. These results strongly suggest autocrine and paracrine regulation of reproductive function by NGF in the reproductive tract of female Shiba goats.     

新生Wistar大鼠海马神经细胞的体外培养与鉴定

为探讨原代Wistar大鼠海马神经细胞的体外培养方法,本研究取新生24 h内的Wistar大鼠海马组织,无菌剪碎后采用胰酶消化法分离细胞,用含20%胎牛血清的DMEM/F12培养基培养,逐日在倒置相差显微镜下观察。结果发现,从海马组织中分离出的神经细胞具有增殖能力,细胞对数生长期为2~8 d,最长培养30 d;细胞经免疫荧光鉴定Nestin表达呈阳性;免疫组织化学结果显示,在传代培养细胞的胞体和突起均有NF阳性标记物,GFAP抗体和CD68抗体显色均为阴性。由此可见,分离培养的细胞是具有自我更新增殖和多分化潜能的神经元细胞。     

Immunolocalization of aquaporins in rat brain

The aquaporins (AQPs) are a family of homologous water channels expressed in many tissues. In this study, the expression and immunolocalization of different AQP subtypes in rat brains were investigated by RT-PCR, immunohistochemistry and immunofluorescence. The data showed that AQP1 was expressed in the subpial processes of astrocytes, choroid plexus and ependyma. AQP3, AQP5 and AQP8 had similar distribution patterns in piriform cortex, choroid plexus, hippocampus and dorsal thalamus. AQP4 and AQP9 were widely expressed in the rat brain and distributed in the subpial processes of astrocytes, ependyma, dorsal thalamus, hippocampus, white matter, suprachiasmatic nucleus (SCN) and supraoptic nucleus. AQP3, AQP4, AQP5, AQP8 and AQP9 were found in the Bergmann glial cells of cerebellum, cochlear nucleus and trapezoid nuclei. The distinct localization of various AQPs in cerebrum and the similarities of distribution patterns within cerebellum, cochlear nucleus and trapezoid nuclei suggest that AQPs may play an important role in maintaining the specific microenvironments of the brain.     

Seasonal changes in spermatogenesis and immunolocalization of inhibin/activin subunits in the wild male ground squirrel (Citellus dauricus Brandt)

The objective of this study was to investigate the seasonal changes in spermatogenesis and the immunolocalization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits during the breeding and non-breeding seasons in the wild male ground squirrel. The testicular weight and size and seminiferous tubule diameter were measured, and histological observations of testes were performed. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB during the breeding and non-breeding seasons. There were marked variations in testicular weight and size and seminiferous tubule diameter between the breeding and non-breeding seasons, and all types of spermatogenic cells, including spermatozoa, were found in the breeding season. In addition, immunoreactivity was also detected for the inhibin alpha, betaA and betaB subunits in Sertoli and Leydig cells during the breeding season, but immunostaining was only present for the inhibin alpha and inhibin/activin betaB subunits in Sertoli cells during the non-breeding season. These results suggest that seasonal changes in testicular weight and size and seminiferous tubule diameter of wild ground squirrels are correlated with changes in spermatogenesis, and the cellular localization of the inhibin/activin subunits showed season related changes in the breeding and non-breeding seasons.     

Galanin-like immunoreactive neural elements in domestic ruminant pancreas

The distribution and ontogeny of the galanin-like immunoreactive (Gal-IR) neural structures in the pancreas of cattle, sheep and goat were investigated immunohistochemically. The present study confirmed the previous findings on the immunolocalization of galanin both in the neural elements and endocrine cells of cattle, and reported for the first time its exclusive localization in the neural elements of sheep and goat. The frequency of Gal-IR nerve fibers and nerve cell bodies was high in cattle and low in sheep and goat. Their first detection was at the first fetal trimester in cattle and third trimester in sheep and goat. In cattle, a marked increase in the frequency of Gal-IR nerve fibers was observed from the third trimester to early neonatal stage followed by a decrease after three months postnatal. In contrast to the non-preferential distribution pattem in sheep, the Gal-IR nerve fibers in cattle and goat pancreas were predominantly associated with the acini, excretory ducts and blood vessels, but rarely detected in the pancreatic islets. The Gal-IR nerve cell bodies were observed as isolated bodies in the intra- and interlobular connective tissues and as a group within the intrapancreatic ganglia. At the vicinity of the nerve cell bodies, Gal-IR nerve fibers were observed. The present findings may suggest that: (1) galanin regulates pancreatic function as neurotransmitter/neuromodulator in ruminants; (2) galanin plays a more important role in large than in small ruminants; and (3) particularly in cattle, it exerts its most dramatic effect during perinatal development.     

Immunolocalization of inhibin/activin subunits in the shiba goat fetal, neonatal, and adult testes

The objective of this study was to investigate the cellular immunolocalization of inhibin alpha and inhibin/activin (betaA and betaB) subunits in the fetal, neonatal and adult testes of Shiba goats. The testes were obtained from a fetus at 90 days, a neonate at 15 days, and two adult Shiba goats (both of 3 years old). The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA, and inhibin/activin betaB. Inhibin alpha and inhibin/activin (betaA and betaB) subunits were expressed in Leydig cells, but not in the Sertoli cells of the fetus with a weak immunostaining. An increase in the number of positive cells and a more intense immunohistochemical signal for inhibin alpha and inhibin/activin (betaA and betaB) subunits were observed in the Leydig cells of neonatal testes. Moreover, inhibin alpha, betaA, and betaB subunits were expressed in the Sertoli cells and Leydig cells of adult testes, respectively. These results suggest that Shiba goats testes have the ability to synthesize inhibins in the fetus, neonate, and adult, and the cellular localization of inhibin/activin subunits showed age-related changes in fetal, neonatal, and adult testes of Shiba goats.     

Immunohistochemical distribution of inwardly rectifying K+ channels in the medulla oblongata of the rat

The inwardly rectifying K+ channels, Kir1.1, Kir2.3 and Kir4.1-Kir5.1, are the candidate chemosensory molecules for CO2/H+. We determined the mRNA expression and immunohistochemical localization of these channels in the medulla oblongata of the rat. RT-PCR analysis revealed mRNAs of Kir1.1, Kir2.3, Kir4.1 and Kir5.1 were detected in the medulla. The immunoreactivities for Kir1.1, Kir2.3, Kir4.1, and Kir5.1 were observed in the medulla, and immunolabeling pattern was varied by the subunit. Immunoreactivities for Kir1.1 and Kir2.3 were observed in the nerve cell bodies and glial cells both in the chemosensory areas [nucleus tractus solitarius (NTS), nucleus raphe obscurus (RO), pre-B?tzinger complex (PreB?tC)] and non-chemosensory area [hypoglossal nucleus (XII), inferior olive nucleus (IO)]. Kir4.1 immunoreactivity was observed in the glial cells and neuropil, especially in XII and IO. Kir5.1 immunoreactivity was observed in the nerve cell bodies in the XII, RO, and PreB?tC, but not in the NTS or IO. In the NTS, a dense network of varicose nerve fibers showed immunoreactivity for Kir5.1. Our findings suggest that Kir channels may not act specific to the central chemoreception, but regulate the ionic properties of cellular membranes in various neurons and glial cells.     

Study of migration of neural crest cells to adrenal medulla by three-dimensional reconstruction

Adrenal medullary cells are derived from the neural crest. To study the formation process of the adrenal medulla in the embryonic period, we visualized chromaffin cells of rat embryos at 13 to 17 days of gestation using anti-tyrosine hydroxylase (TH) antiserum, and created three-dimensional images from serial tissue sections. Between 13 and 15 days of gestation, TH-positive cells (chromaffin cells) migrated from a group of TH-positive cells present dorsal to the adrenal primordium via the medial cranial end of the adrenal primordium into the adrenal primordium. At or after 16 days of gestation, the adrenal capsule was formed except on the ventral aspect of the cranial end of the adrenal gland, from which TH-positive cells penetrated into the adrenal gland. The reconstructed images showed that TH-positive cells were present contiguously from the sympathetic chain ganglia through a group of TH-positive cells ventral to the adrenal gland into the adrenal cortex, and that the group of TH-positive cells ventral to the adrenal gland communicated with the preaortic ganglion present ventral and caudal to the adrenal gland. These results suggest that neural crest cells use the same pathway to migrate to the sympathetic chain ganglia dorsal to the adrenal gland, to the adrenal gland, and to the preaortic ganglion.     

Calretinin immunoreactive nerve endings in the trachea and bronchi of the rat.

Nerve endings showing calretinin immunoreactivity were examined in the lower respiratory tract of the adult rat. Tree-like nerve endings were immunostained in the tracheal and bronchial smooth muscle layer. The endings that arose from thick nerve fibers and formed corpuscles composed of many arborized nerve terminals. A few of the nerve endings were also observed in the lamina propria of the tracheal mucosa, close to the epithelial layer. Immunoelectron microscopy revealed that the immunoreactive terminals were filled with mitochondria and scattered among the intermuscular collagen fibrils. Schwann cell sheath and collagen fibrils were intercalated between the smooth muscle cells and nerve endings. The calretinin immunoreactive nerve endings observed in the present study seem to be slowly adapting stretch receptors.     

Age-Dependent Immunolocalization of Fibronectin and Histological Changes in the Thymus of Rats

Age-dependent variations in the immunolocalization of fibronectin (FN) in the thymus were investigated in 1-, 6-, 12- and 20-month-old male and female Swiss albino rats (Rattus rattus) at the light-microscopic level and the changes with ageing in the histological structure of the thymus were also studied. There were no significant differences in the age-dependent variations in the immunolocalization of fibronectin or in the histological structure of the thymus between male and female rats of the same age but there were increases with ageing in the fibronectin content of the thymic capsule, the connective tissue between the lobules, around blood vessels, and in the medulla and cortex of thymus. The connective-tissue content between lobules, fat cells, Hassall's corpuscles, the thickness of capsule and the ratio of the medulla to the cortex of the lobules showed age-dependent increases in the thymus of rats of both sexes. Decreases in the organ weight/body weight ratio were also observed with ageing.     

Immunocytochemical Identification of Different Cell Types in Bovine Nasolabial Glands with Particular Emphasis on Cytoskeletal Protein Expression

Nasolabial glands are serous glands forming a thick subcutaneous layer in the bovine muzzle. In order to identify the different epithelial cell types, both immunofluorescent and immunoperoxidase techniques were employed on frozen and fixed sections using monoclonal antibodies to cytoskeletal proteins and S-100. Actin was also detected with phalloidin. The results show that four cell types can be identified on the ground of the different composition of the cytoskeletal filaments: acinar, basket, luminal duct and basal duct cells. Acinar, luminal duct cells and basal duct cells express different patterns of cytokeratins, as shown by the 12 anti-cytokeratin monoclonal antibodies used, and both basket cells and the basal cells of intercalated ducts are also reactive to phalloidin and anti-x-smooth muscle actin monoclonal antibody. The presence of actin supports the conclusion that basal duct cells are also contractile elements, i. e. myoepithelial cells. Vimentin, glial fibrillary acidic protein (GFAP), and S-100, molecules considered to be markers of myoepithelial cells by many AA., were not found. The intermediate filaments of the duct epithelium appear more complex and heterogeneous in comparison with those present in the acinar cells.     

Immunolocalization of ghrelin in the stomach of Sprague-Dawley rat

To investigate the distribution of ghrelin in different regions of stomach in Sprague-Dawley rat, and attempt to compare with those of humans and other mammalian species in this study, the stomach of rats was divided into five sections, cardia, fundus, greater curvature, lesser curvature and pylorus. Immunohistochemistry and Western blotting were performed to investigate the ghrelin-producing cells. The immunolocalization and protein levels of ghrelin differed significantly in different regions of stomach in rats. It was present at a high level in the greater curvature of the pars glandularis, and the lesser curvature. In the fundus and pylorus, no ghrelin immunoreactive cells were detected. In this study, we elucidated the distribution of ghrelin-producing cells in different regions of rat stomach in detail for the first time. It is further considered that the differences of ghrelin distribution in stomach of different species may induce different stimulatory effects on fat accumulation and metabolism.     

Immuno-histochemical and -cytochemical evidence suggesting the presence of Campylobacter jejuni and Campylobacter coli in cases of porcine intestinal adenomatosis

Antisera against a number of Campylobacter species were used in immuno-histochemical and -cytochemical studies on cases of porcine intestinal adenomatosis. Avidin-biotin-complex (ABC) and streptavidin immunoperoxidase methods were used on formalin-fixed, paraffin-embedded and frozen sections. Protein A gold method was used on formaldehyde fixed and frozen sections for immuno-cytochemistry. The antisera used were raised in rabbits by subcutaneous or intravenous injection of living or formalin treated organisms. Anti-sera against different serotypes of the thermotolerant, catalase positive campylobacters, Campylobacter jejuni and Campylobacter coli, gave positive reactions in the immuno-histochemical studies. The staining was found in intestinal epithelial cells both in the ileum and in the colon and was restricted to the apical cytoplasm of adenomatous epithelial cells. The staining had a granular pattern, the positive structures sometimes having the shape of Campylobacter. Epithelial cells in areas with normal differentiation of goblet cells did not stain. In contrast, no staining resulted with antisera against Campylobacter sputorum subsp. mucosalis and Campylobacter hyointestinalis. Immuno-cytochemistry, using antisera against Campylobacter jejuni, showed that the positive staining in altered epithelial cells were restricted to intracellular organisms having a structure resembling Campylobacter spp.     

Features of follicular dendritic cells in ovine pharyngeal tonsil: an in vivo and in vitro study in the context of scrapie pathogenesis

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion "reservoir" during the latency phase, thus playing a key role during the scrapie lymphoinvasion.     

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.     

Characterization of encephalitis in wild birds naturally infected by highly pathogenic avian influenza H5N1

During the outbreak of highly pathogenic avian influenza (HPAI) H5N1 in Sweden in 2006, disease and mortality were observed in a number of wild bird species. Encephalitis was one of the most consistent and severe findings in birds submitted for postmortem examination. However, the distribution and severity of the inflammation varied among individuals. This study characterized the encephalitis and the phenotype of the cellular infiltrate in brains of 40 birds of various species naturally infected with HPAI H5N1. Brain sections stained with hematoxylin and eosin and immunostained for influenza A viral antigen were evaluated in parallel to brain sections immunostained with antibodies against T lymphocytes (CD3+), B lymphocytes (CD79a+), macrophages (Lectin RCA-1+), and astrocytes expressing glial fibrillary acidic protein. The virus showed marked neurotropism, and the neuropathology included multifocal to diffuse areas of gliosis and inflammation in the gray matter, neuronal degeneration, neuronophagia, vacuolation of the neuropil, focal necrosis, perivascular cuffing, and meningitis. Broad ranges in severity, neuroanatomical distribution, and type of cellular infiltrate were observed among the different bird species. Since neurotropism is a key feature of HPAI H5N1 infection in birds and other species and because the clinical presentation can vary, the characterization of the inflammation in the brain is important in understanding the pathogenesis of the disease and also has important diagnostic implications for sample selection.     

ULTRASONOGRAPHIC ANATOMY OF THE TEMPOROMANDIBULAR JOINT IN HEALTHY PURE-BRED SPANISH HORSES

The objective was to describe the normal ultrasonographic appearance of the soft tissue and bony structures of the temporomandibular joint in Pure-Bred Spanish horses using frozen and plastinated sections performed as anatomical references. The temporomandibular joint of five healthy Pure-Bred Spanish horses and the head specimens from 12 Pure-Bred Spanish cadavers that were subjected to euthanasia for reasons other than temporomandibular joint problems were studied sonographically and anatomically. An 11 MHz linear-array transducer was used to obtain longitudinal ultrasonographic images of the temporomandibular joint from rostral, lateral, and caudal approaches. For anatomic study, a gross dissection was performed on six temporomandibular joints. The other six head specimens were first frozen at -30 degrees C for 48h and then, at -70 degrees C for a week. Three millimeter thick anatomic sections were collected in the same plane as the sonographic planes. These sections were plastinated, photographed, and compared with the corresponding ultrasonographic images. The articular surfaces, the articular cartilage, the disc, the intra-articular fat tissue, the joint capsule, and other structures were clearly visualized sonographically. Structures identified on the ultrasonographic images were in accordance with those identified on the corresponding anatomic sections. We confirmed the appearance of structures of the equine temporomandibular joint that have been described previously but we also identified the caudal fibrous expansion of the disc, the caudal compartment of the dorsal synovial pouch, and the lateral ligament. Our results define a reference that will aid in sonographic evaluation of the equine temporomandibular joint region.     

Intracoelomic teratoma in a domestic duck (Anas platyrhynchos domesticus): a case report including immunohistochemistry and electron microscopy

A female domestic duck (Anas platyrhynchos domesticus) suddenly died with abdominal distension and a large multilobulated mass within the coelomic cavity was found. Histologically and immunohistochemically, a benign mature tridermic teratoma was diagnosed and epithelial structures, cartilage, bone, myxoid tissue, adipocytes, muscle cells, cystic spaces lined by squamous epithelium, feather follicles, melanocytes and variable neural and glial differentiation were recognized. By electron microscopy, desmosomes, keratin bundles, dense core neurosecretory granules, aberrant Z-line material and Luse bodies were found. To our knowledge, this is the first report of tridermic benign intracoelomic teratoma of a duck in which an extensive immunohistochemical and electron microscopic examination has been performed and in which a common neural and glial differentiation has been demonstrated.     

Immunolocalization of sex steroid receptors in the epididymis and ductus deferens of immature and mature Japanese Quail, Coturnix Japonica

The goal of this study was to determine whether in the Japanese quail the male genital tract contains receptors for progesterone, androgen and estrogen (PR, AR and ER, respectively), which have significant roles in reproductive functions, and whether their localization changes during sexual maturation. The epididymis and ductus deferens (middle and ampulla regions) of immature (approximately 30-day-old) and mature male Japanese quail were collected and frozen sections of them were immunostained for PR, AR and ER. The immunoreaction products for AR and PR were found in the nuclei of epithelial cells in the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens of mature and immature birds. In the mature birds, the epithelial cells of the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens were positive for ER, although some of the cells in the ductus deferens were negative. The epithelial cells of the ductules in the epididymis stained positive for ER, but the immunoreactions were negligible in the ductus deferens of immature birds. These results suggest that the epididymis and ductus deferens in quail possesses PR, AR and ER receptors. Each receptor is expressed before sexual maturation, although enhancement of ER expression may occur during maturation.