An immunohistochemical study of ovarian innervation in the emu (Dromaius novaehollandiae)

The present study investigated the distribution of nerves in the ovary of the emu. The neuronal markers, protein gene product 9.5, neurofilament protein and neuron specific enolase demonstrated the constituents of the extrinsic and intrinsic ovarian neural systems. The extrinsic neural system was composed of ganglia in the ovarian stalk, as well as nerve bundles, which were distributed throughout the ovary. Isolated neuronal cell bodies, in the medulla and cortex, formed the intrinsic neural system. An interesting finding of the study was the presence of nerve bundles, circumscribed by lymphocytes, in the ovarian stalk. The findings of the study indicate that the distribution of nerve fibres and neuronal cell bodies in the emu ovary is similar, but not identical to that of the domestic fowl and ostrich.     

The fine structure of the Harderian gland in the ostrich (Struthio camelus)

The Harderian gland of the ostrich (Struthio camelus) is a tubuloalveolar gland containing holocrine secreting epithelial cells. The gland epithelium is composed of two different cell types, which can be classified as type I and type II. These cells contain dense secretory vesicles in their cytoplasm and they are connected laterally with desmosomes. At the basal site of these cells, myoepithelial cells are present. Plasma cells are observed in the subepithelial region of the gland. In the interlobular trabeculae, forming the gland stroma, fibroblasts, blood vessels and nerve fibres are included. Another important finding in the ostrich Harderian gland is the presence of homogeneous material.     

Has Active Immunization Against Gonadotrophin‐Releasing Hormone any Effect on Testis Innervation in the Pig? – An Immunohistochemical Study

The innervation of porcine testes was studied in intact animals and in boars undergoing active immunization against gonadotrophin-releasing hormone (GnRH) by means of immunohistochemistry using antibodies to tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), vasoactive intestinal polypolypeptide (VIP), neuropeptide Y (NPY), synaptosome-associated protein of 25 kDa (SNAP-25) and protein gene product 9.5 (PGP 9.5). Moreover, the distribution of luteinizing hormone (LH) receptors in clusters of Leydig cells was also investigated. To identify these cells easily, either the NADPH-diaphorase histochemical technique or the Mayer counter-staining procedure was applied. Differences in the distribution pattern and relative density of particular subsets of intratesticular nerve fibres were observed in immunized boars as compared to those found in the intact animals. In the testes of non-treated animals, only single TH-immunoreactive (TH-IR) nerve fibres were observed. However, many D beta H-IR nerve terminals surrounded blood vessels in the tunica albuginea and parenchyma. Very scarce VIP-IR nerves occurred only in the tunica albuginea, mainly in close vicinity to blood vessels. Immunoreactivity to NPY occurred in single nerve fibres. Immunoreactivity to SNAP-25 and PGP 9.5 was found in single nerve fibres distributed mainly in the tunica albuginea. The interstitial cells were heavily stained for LH-receptors and NADPH-diaphorase. In the testes of immunized animals, only single TH-IR nerve fibres, scattered mainly in the tunica albuginea, were observed. Some TH-IR nerve terminals were also encountered in the parenchyma of the organ, where they were always associated with blood vessels. D beta H-IR nerve fibres formed a dense network distributed throughout the testis in association with the capsule, vasculature and interstitium. Some fibres were observed to run between seminiferous tubules. VIP-IR nerve fibres were located in the neighbourhood of blood vessels in the tunica albuginea and parenchyma. Only single VIP-IR nerves were found between seminiferous tubules. Numerous NPY-IR nerve fibres occurred in the tunica albuginea and parenchyma of the organ. SNAP-25-IR and PGP 9.5-IR nerve terminals formed a dense network distributed throughout the testis and many fibres were observed between seminiferous tubules. Interstitial cells were very weakly stained for LH receptors or NADPH-diaphorase.     

Cholinergic innervation of the oviduct in rabbits]

Distribution of cholinergic nerves in the oviduct wall in rabbits was investigated by a neurohistochemical method after El Badawi and Schenk (1967). Higher density of cholinergic nerve fibres was confirmed to occur mainly in the isthmic part, but in contrast with literary data it was also found in the ampullar part of oviducts. In the basal parts of oviduct mucosa there were only few cholinergic nerve fibres unlike their density in woman's oviduct. A major part of cholinergic nerves are concentrated in a thick muscular tissue; this is probable to be related with its influence on oviduct motility. In the course of pregnancy, progesterone reduces the number of cholinergic nerves in rabbit oviducts, the number is largely reduced in all layers. These results are considered as preliminary with respect to the number of test rabbits; they indicate that the rabbit oviduct is not any ideal model to study the development of cholinergic innervation in dependence on oestrous cycle phase and pregnancy duration and that they should not be applied to changes in the functional neuromorphology of tuba uterina in woman.     

Immunohistochemical Localization of Galanin in Bovine Reproductive Organs

The vagina, uterus and oviduct were shown to receive galanin immunoreactive (GAL-IR) nerve fibres, the number of which varied between particular organs. In the ovary, GAL-IR nerves were absent. A small number of these nerves were located in the layers of the oviduct. A moderate number of GAL-IR nerves were situated in the body and uterine horns, whereas the uterine cervix and vagina wall contained a large number of GAL-IR nerve fibres, evenly distributed throughout particular membranes of the organs. GAL-IR nerves were found to contain, simultaneously, either vasoactive intestinal polypeptide (VIP), substance P (SP) or Leu⁵-enkephalin (ENK). Many of the GAL-IR nerves contained tyrosine hydroxylase (TH). A group of GAL-IR nerves that did not possess immunoreactivity to VIP, SP, ENK or TH was also observed.     

Immunohistochemical Localization of Glial and Neuronal Cell Markers in the Developing Bovine Brain*

In the present immunohistochemical study, the distribution and differentiation of glial and neuronal cells in bovine fetal brains (age range: between1–2 and7–8 months) was examined using antibodies against nervous system-specific proteins, i. e., glial fibrillary acidic protein (GFAP), vimentin, neuron specific enolase (NSE) and a neurofilament protein subunit (NF 200 kD).     

Morphology of the intermandibular gland of the Lesser mouse deer, Tragulus javanicus

The morphology of the intermandibular gland of the Lesser mouse deer (Tragulus javanicus), which plays an important function in marking area and territory and in the reproductive behaviour of the animal, was examined using immunohistochemistry, lectin histochemistry and scanning electron microscopy. The gland was composed of sebaceous and apocrine glandular material. Sebaceous glands occupied a greater area of the total gland and consisted of many large lobules with polyhedral cells having a pale cytoplasm. The sebaceous gland, being holocrine, possessed no special secretory ducts. The apocrine gland was lined by cuboid cells and the secretory products were often seen in the apical portions of the cells. Myoepithelial cells contained actin filaments lining the basal membranes of the apocrine gland and were surrounded by nerve fibres which immunostained with protein gene product 9.5. The secretion of the gland appears to be a mixture of larger amounts of lipid material from sebaceous glands, and glycoconjugates secreted by both sebaceous and apocrine glands. Lectin histochemistry detected these as galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-mannose and D-glucose. The male gland was larger in size and contained more N-acetyl galactosamine and N-acetyl glucosamine in its secretion than the gland of the female. This implied the presence of sexual differences in secretions in the intermandibular gland of the Lesser mouse deer.     

A canine peripheral nerve sheath tumor including peripheral nerve fibers

Peripheral nerve sheath tumor was found in a 7-year-old male mongrel dog. The tumors were located in the right cheek subcutis and oral submucosa. Histologically, neoplastic cells were arranged in streaming bundles, occasionally interlacing bundles or whorls of elongated and spindle cells. Cellular atypia was poor and mitotic figures were rarely observed. Ultrastructurally, neoplastic cells had basement membrane, typical of Schwann cells. One bundle of normal peripheral nerve fibers and some myelinated axons were seen within the tumor tissues. Immunohistochemically, neoplastic cells reacted to vimentin, glial fibrillary acidic protein, S-100 protein and neuron specific enolase. In addition to the above immunoreactions, the included nerve fibers were positive for myelin basic protein and neurofilament protein. This paper also discusses immunohistochemical findings on differential diagnosis in comparison with those of canine hemangiopericytomas reported hitherto.     

Immunhistochemical Demonstration of Lumbar Intervertebral Disc Innervation in the Dog

Low back pain is a common ailment in dogs, particularly in specific breeds as for instance the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplate, and meninges have been associated with this condition. However, in a substantial proportion of all cases, the origin of pain remains obscure. A further structure often being involved in vertebral column disorders is the intervertebral disc. However, the presence of nerves is a precondition for pain sensation and, consequently, structures lacking sensory innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of intervertebral discs in man, rabbit and rat. With regard to the dog, however, extent of intervertebral disc innervation still is disputed. The goal of the present study, therefore, was to assess previous findings based on silver impregnation. Protein gene product (PGP) 9.5 was used as a marker for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This technique revealed nerve fibres to be confined to the periphery of the intervertebral discs. Thus, even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.     

Morphology of the nerve endings in laryngeal mucosa of the horse

To discuss the significance of laryngeal sensation on various disorders of the horse, we studied the morphological and topographical characteristics of sensory structures in the laryngeal mucosa using immunohistochemistry and immunoelectron microscopy. Various sensory structures, i.e. glomerular endings, taste buds and intraepithelial free nerve endings, were found in the laryngeal mucosa by immunohistochemistry for protein gene product 9.5 (PGP 9.5) and neurofilament 200kD (NF200). Glomerular nerve endings were distributed mainly in the epiglottic mucosa; some endings were also found in the arytenoid region arising from thick nerve fibres running through the subepithelial connective tissue. Some terminals directly contacted the epithelial cells. Taste buds were distributed in the epithelium of the epiglottis and aryepiglottic fold. In the whole mount preparation, the taste buds were supplied by the terminal branching of the thick nerve fibres. In some cases, the taste buds were arranged around the opening of the duct of the epiglottic glands. The intraepithelial free nerve endings were found to be immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP). These nerve endings were surrounded by the polygonal stratified epithelial cells in the supraglottic region, and by the ciliated cells in the subglottic region. The density of the intraepithelial free nerve endings was highest in the corniculate process of the arytenoid region and lowest in the vocal cord mucosa. The densities of CGRP- and SP-immunoreactive nerve endings in the arytenoid region were (mean +/- s.d.) 30.6+/-12.0 and 10.0+/-4.9 per unit epithelial length (1 mm), respectively and in the vocal fold mucosa, 1.1+/-0.9 and 0.8+/-0.7, respectively. Approximately one half of the CGRP immunoreactive nerve endings were immunoreactive for SP, and most SP-immunoreactive nerve endings were also immunoreactive for CGRP. Well-developed subepithelial plexus with numerous intraepithelial fibres were observed in flat or round mucosal projections that existed on the corniculate process of the arytenoid region. In conclusion, the laryngeal mucosa of the horse seems to have morphology- and/or location-dependent sensory mechanisms against various endo-and exogenious stimuli.     

Immmunohistochemical study of the blood and lymphatic vasculature and the innervation of mouse gut and gut-associated lymphoid tissue

The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane-membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.     

Immunohistochemical study on the expressions of neuropeptides in the superficial and deep gland of the third eyelid of pigs

The superficial gland of the third eyelid (SGTE) and deep gland of the third eyelid (DGTE) are classified as accessory lacrimal glands. The aim of the present study was to immunohistochemically investigate the expression of vasoactive intestinal peptide (VIP), dopamine beta-hydroxylase (DβH), substance P (SP), galanin, neuropeptide Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin and calcitonin gene-related peptide (CGRP) in the porcine SGTE and DGTE. We demonstrated the distribution patterns of VIP, DβH, SP, NPY and galanin in the nerve fibres in the SGTE and DGTE. None of somatostatin-, PACAP- and CGRP-immunoreactive (IR) nerve fibres were found in the SGTE and DGTE. The majority of VIP- and DβH-IR nerves fibres were found near to glandular acini, tubules, secretory ducts and blood vessels in the SGTE and DGTE. VIP-IR nerve fibres were found in external connective tissue in SGTE and DGTE and only in interlobular connective tissue in the SGTE. DβH-IR nerve fibres were found in interlobular and external connective tissue in the DGTE but not in the SGTE. Single galanin-, SP- and NPY-IR nerve fibres were observed in close proximity to acini and tubules in the SGTE and DGTE. Single galanin-, SP-, NPY-IR nerve fibres were found in close proximity to the secretory ducts in the DGTE, however only SP-IR nerve fibres were found near to the secretory ducts in SGTE. In conclusion, our research aims to highlight some aspects of SGTE and DGTE innervation in pigs and may also be a source of basic knowledge for further studies.     

山羊黄体弥散性神经内分泌细胞的分布

为了探查山羊卵巢黄体中是否存在弥散性神经内分泌细胞,采用免疫组化链霉素抗生物素蛋白-过氧化物酶法对12~18月龄妊娠奶山羊卵巢中弥散性神经内分泌细胞标志物的分布进行了研究。结果显示:黄体组织中各区均含有催产素、神经元特异性烯醇化酶、S-100蛋白、突触素和5-羟色胺样免疫反应阳性细胞。这些细胞散在或成团分布,多呈圆形、卵圆形和三角形,一些细胞具有明显的突起。阳性细胞数由多至少顺序为:催产素、突触素、S-100蛋白、神经元特异性烯醇化酶、5-羟色胺。鉴于神经元特异性烯醇化酶、S-100蛋白、突触素和5-羟色胺是弥散性神经内分泌系统广谱的、共同的细胞标记物,结果提示山羊卵巢黄体中存在弥散性神经内分泌细胞。     

An Immunohistochemical Observations on the Oviduct of the Goat

The oviduct has an important role in regulating transport of gametes and fertilization. The main role in these functions has a smooth muscle cells and ciliated epithelium lining the oviduct. All functions are under the influence of hormonal and nervous system. The objective of this study was immunohistochemically to examine the following structures: lining epithelium, smooth muscle cells, elastic fibres and nerve fibres. For this purpose, the following antibodies were used: cytokeratin 18, S‐100 protein, acetylated α‐tubulin, smooth muscle actin, desmin and elastin. Ciliary and secretory cells of the lining epithelium were positive for cytokeratin 18 and S‐100 protein. Cilia and the basal body‐associated structures of ciliary cells were positive to acetylated α‐tubulin. Smooth muscle cells (SMC) in mucosa and of the muscular layer were positive for α‐smooth muscle actin (SMA) and desmin. High density of nerve fibres positively reacted to acetylated α‐tubulin and S100 protein was present in the mucosa, muscular layer and serosa. Elastic fibres positive for elastin form a dense network at the base of the mucosal folds and in the muscle layer. A dense network of these fibres is accompanying the blood vessels. It is supposed that together with smooth muscle cells they are involved in the transport of ovum and in blood flow regulation.     

Immunohistochemical demonstration of lumbar intervertebral disc innervation in the dog

Low back pain is a common ailment in dogs, particularly in specific breeds such as the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplates and meninges have been associated with this condition. Yet, in spite of all diagnostic efforts, the origin of pain remains obscure in a substantial proportion of all cases. A further structure often being involved in vertebral column disorders is the intervertebral disc. The presence of nerves, however, is a precondition for pain sensation and, consequently, structures lacking innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of the intervertebral disc in man, rabbit and rat. With regard to the dog, however, the extent of intervertebral disc innervation is still being disputed. The goal of the present study, therefore, was to substantiate and expand current knowledge of intervertebral disc innervation. Protein gene product (PGP) 9.5 was used for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This general marker revealed nerve fibres to be confined to the periphery of the intervertebral discs. These results indicate that even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.     

Adrenergic and cholinergic innervation of the mammary gland in the pig

Adrenergic and acetylcholinesterase-positive (AChE-positive) innervation of the mammary gland in the sexually immature and mature pigs was studied using histochemical methods. Upon examining the adrenergic and cholinergic innervation, the adrenergic innervation was found to be much more developed. The majority of both sub-populations of the nerve fibres studied was localized in the subcutaneous tissue of the mammary gland. Adrenergic and AChE-positive nerve fibres also supplied structures of the nipple (subcutaneous tissue, blood vessels, smooth muscles fibres) and glandular tissue (blood vessels, lactiferous ducts). The glandular tissue contained the smallest number of adrenergic and AChE-positive nerve fibres. No distinct differences were observed in the adrenergic and AChE-positive innervation of the porcine mammary gland between the juvenile and non-pregnant adult animals.     

传染性支气管炎病毒感染对蛋雏鸡输卵管雌激素受体及孕激素受体表达的影响

以不同传染性支气管炎病毒（肾型IBV-河北分离株与呼吸型IBV-M41）分别感染蛋雏鸡,通过测定输卵管蛋白分泌部及子宫雌激素受体（Estrogen receptor,ER）和孕激素受体（Progesterone receptor,PR）的表达水平,来探讨IBV早期感染（10d）对抑制蛋鸡输卵管发育的作用机制。结果显示,IBV感染降低子宫和蛋白分泌部ER mR-NA、PR mRNA的表达水平,即致子宫ER mRNA和PR mRNA的表达低于检测水平;蛋白分泌部ER mRNA和PR mRNA的表达水平显著降低（P〈0.01）,且M41的作用幅度较肾型IBV更为显著（P〈0.01）。结果表明,IBV可通过降低ER和PR的表达水平,抑制输卵管对雌激素的应答能力而影响输卵管的发育。     

The effects of vagotomy on the abomasum in calves: radiography and protein gene product 9.5 immunohistochemistry.

Abomasal disorders of calves with total vagotomy, operated on at 1 week old, were investigated with radiography and protein gene product (PGP) 9.5 immunohistochemistry. Radiographic findings indicated abomasal atony with dilatation in all calves 2 weeks after vagotomy, while 4 weeks after vagotomy abomasal dilatation was detected in 2 calves and another 2 calves showed dilatation and impaction. The densities of PGP 9.5-immunoreactive nerves in the tunica muscularis decreased significantly in the corpus region of the greater curvature 2 weeks after vagotomy and in the pyloric region of the lesser curvature 4 weeks after vagotomy, and it was at its lowest 4 weeks after vagotomy in all regions examined. In conclusion, abomasal dilatation and/or impaction in vagotomized calves confirmed by radiography were related with a decreased frequency of nerves in the tunica muscularis of the abomasum.     

Horse lumbrical muscle: possible structural and functional reorganization in regressive muscle

An anatomical study of horse lumbrical muscle (Lm) was carried out by light and electron microscopy in combination with immunochemical and cytochemical methods. Paraffin sections were subjected to haematoxylin and eosin (H & E) and Masson's trichrome staining for morphometric analysis. Paraffin sections were also used for immunostaining by anti-PGP 9.5 for reaction with nerve-protein associated-structures, anti-heat-shock protein 70 (hsp 70) for detection of gene expression changes, anti-fast myosin for the determination of muscle fibre types, and for detection of apoptotic gene expression of muscle fibres by the TUNEL method. The distribution of muscle fibre types on frozen sections was also examined by assaying ATPase (pH 4.2). We found that the proximal end of the tendon of the unipennate-shaped Lm binds to the deep digital flexor tendon, and the distal end of the Lm tendon connects to the medial surface of the palmar annular ligament. The Lm was not always present, but when found it varied in length greatly, up to 8 cm (muscle part alone), and weighed less than 1 g. The Lm was white, pale, or reddish in colour depending on the ratio of muscle fibre and connective tissue contents. The semi-tendinized regressive Lm was composed of rich vasculature, peripheral nerves, and nerve-like organs similar to the neuromuscular spindle (NMS). The extrafusal muscle fibres (e-lm) that surround the NMS were replaced with a thick outer capsule of connective tissues (CT) in the Lm nerve-like organ, which we named the neurotendinous capsule (NTC) organ. NTC organs exist alone or as multiple structures (up to eight) surrounded by a common outer capsule at the outermost CT ring. The NTC possesses several intrafusal muscle fibres (ifm) just as the NMS does. That the ifm was associated with nerve endings was confirmed by anti-PGP 9.5 and electron microscopic observation. Some muscle fibres in ifm and e-lm reacted with anti-fast twitch myosin and with anti-hsp 70. The e-lm exhibited at least two fibre types, determined by ATPase (pH 4.2) assay. The ifm exhibited mainly type I (slow twitch) fibres. No apoptotic gene expression was detected in either ifm or e-lm, suggesting the Lm is a vital organ. The degenerating fibres observed in ifm and e-lm indicate that the turnover rate of cytoplasmic components is accelerated. We attribute this phenomenon to the necessity for adaptation to new environmental demands. The surprising finding of tubular aggregates (TAs) in ifm of the NTC organ suggests that the Lm is continuously adapting. Some results related to variation in diameter of the collagen fibrils, isolation of the NTC organ and the myofibrillar protein constituents are also discussed. In conclusion, the so-called regressive Lm has rich vasculature, many peripheral nerves, and newly described NTC organs. The induction of heat-shock protein, lack of apoptotic gene expression in ifm and e-lm fibres, and TA formation in ifm suggest that horse Lm responds to environmental stress through reorganization and/or remodelling of cell constituents. We hypothesize that the horse Lm has lost its original role as a contractile element and changed to another function, likely as a vital nerve organ.     

Immunohistochemical characterization of nonhuman primate ovarian sex cord-stromal tumors

This study evaluates the immunoreactivity of 12 sex cord-stromal tumors of nonhuman primates (11 granulosa cell tumors and 1 luteoma). The markers selected are used in the characterization of gonadal tumors in dogs and other species, including cytokeratins AE1/AE3, GATA-4, inhibin-α, neuron-specific enolase, protein gene product 9.5, and vimentin. A normal nonhuman primate ovary was used as a control and to optimize immunolabeling. Staining was graded as follows: 0 (nonstaining), 1+ (< 10% positive cells), 2+ (10%-50% positive cells), and 3+ (> 50% positive cells). Calretinin, GATA-4, neuron-specific enolase, and vimentin were the most consistently expressed markers (12 of 12). Cytokeratins AE1/AE3 were also consistently expressed (11 of 12). Inhibin-α and protein gene product 9.5 were expressed in 8 and 10 sex cord-stromal tumors, respectively. Results indicate that immunoreactivity of nonhuman primate sex cord-stromal tumors is similar to that observed in other species and that calretinin, GATA-4, and neuron-specific enolase are the most consistently expressed markers in nonhuman primate sex cord-stromal tumors.