Immunohistochemical characteristics of neurons supplying the porcine bulbourethral gland

In the male pig, the bulbourethral gland (BG) is a particulary well developed accessory genital gland (AGG) which produces complex secretion contributing to the fluid component of semen. The secretory and motor function of AGGs is thought to be under the autonomic nervous system control. Although relatively much is known about the innervation of the prostate gland and, to a lesser degree, of the seminal vesicle, the paucity of data dealing with the innervation of BG is striking. Therefore, combined retrograde tracing and double-labelling immunofluorescence have been used to investigate the distribution and immunohistochemical properties of autonomic and primary afferent neurons projecting to this gland in the pig. BG-projecting neurons were found in some ipsilateral (I) and contralateral (C) sympathetic chain ganglia (SChG), the caudal mesenteric ganglion (CaMG), pelvic ganglia (PG) and some dorsal root ganglia (DRG). Immunohistochemistry revealed that the vast majority of CaMG and SChG BG-projecting neurons contained tyrosine hydroxylase (TH) and dopaminebeta-hydroxylase (DbetaH), and some neuropeptides including neuropeptide Y (NPY), somatostatin (SOM) and galanin (GAL). Three subpopulations of PG neurons supplying BG could be distinguished: 1) cholinergic neurons [vesicular acetylcholine transporter (VAChT)-positive] which also contained vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), SOM and NPY, 2) adrenergic neurons (TH-positive) which also stained for NPY, GAL or leu5-enkephalin (LEU), and 3) non-adrenergic, non-cholinergic neurons (NANC). DRG BG-projecting neurons contained mostly substance P (SP) and/or calcitonin gene-related peptide (CGRP) which sometimes colocalized with GAL. The possible functional significance of the substances found within the neurons is discussed.     

Gender differences in the morphometric properties of muscle fibres and the innervation ratio of motor units in rat medial gastrocnemius muscle

With 2 figures and 2 tables SUMMARY: Previous studies of motor unit contractile properties in the rat medial gastrocnemius revealed that these units generate higher forces in males than in females. Therefore, in the present study the number and morphometric parameters of muscle fibres and the innervation ratio of motor units in the medial gastrocnemius muscle were studied in male and female Wistar rats. The study additionally aimed at determining reasons of gender differences in motor unit force parameters, i.e. the number and diameter of muscle fibres, and mean values of the motor unit innervation ratios. Following staining of reticuline fibres by silver impregnation, the number, diameter and cross-section area of muscle fibres were determined on microscopic images of transverse muscle sections. In males, the muscles were approximately 1.5 times larger by mass and contained about 11 800 muscle fibres, whereas in females the muscles contained around 8000 fibres. In addition, the mean diameter and mean cross-section area of muscle fibres were 14 and 29% larger in males, respectively. Based on previously determined numbers of motoneurons innervating the medial gastrocnemius muscle in male and female rats, the mean innervation ratio, i.e. the number of muscle fibres innervated by one motoneuron, was estimated. This ratio was approximately 26% greater in males compared to females, with values of 207 and 153 fibres per motoneuron, respectively. Therefore, the differences in muscle fibre morphometric parameters and in the innervation ratio are responsible for higher forces of motor units in male muscles.     

Immunohistochemical aspects of anti-microbial properties in goat submandibular glands

Salivary glands are known as the principal source of anti-microbial substances, which are considered to be essential components of saliva. The distribution of anti-microbial substances in the submandibular glands of Japanese miniature (Shiba) goat was studied using immunohistochemical methods as performed by physical development procedures. In the goat, anti-microbial substances such as lysozyme, IgA, lactoferrin and β-defensin were demonstrated to be immunolocalized in the submandibular glands, especially in the serous demilunes and duct cells. The results obtained are discussed with regard to the specific functions of the saliva. The presence and secretion of anti-microbial substances suggest that they participate in the maintenance of oral health among the elements of saliva.     

Immunohistochemical study of caveolin-1 and -2 in the rat retina

The expression of caveolin-1 and -2 in the retina was examined; Western blot analysis showed that both were present. Immunohistochemistry indicated that caveolin-1 was expressed in the majority of retinal layers, including the ganglion cell layer, inner plexiform layer, outer plexiform layer, and in the vascular endothelial cells of the retina. Caveolin-2 was primarily immunostained in the vessels, but in a few other elements as well. This is the first demonstration of caveolin differential expression in the retina of rats, and suggests that caveolin plays an important role in signal transduction in glial cells and neuronal cells.     

Changes in the muscle fibre properties of the mouse temporal muscle after weaning

To clarify changes in the muscle fibre properties of the temporal muscle related to the start of masticatory movement, we immunohistochemically investigated myosin heavy chain (MyHC) isoform protein expression using pre-weaning and post-weaning mice. In addition, we examined the expression of a gene coding for those MyHC proteins. Immediately after weaning, isoforms with fast and potent contractility were frequent. This suggests that the temporal muscle plays an important role in a marked functional change in the oral cavity from lactation to mastication, contributing to oral function in cooperation with other masticatory muscles.     

Age-related three-dimensional morphological changes in rat motoneurons innervating diaphragm and longissimus muscles

We investigated age-related morphological changes of rat motoneurons innervating diaphragm muscle (DI-MN) and lumber longissimus muscle (LL-MN) in which quite different activation patterns exist. In young (2-4 months) and old (24-26 months) rats, the motoneurons innervating both muscles were labelled retrogradely by intramuscular injection of cholera toxin B subunit. After a 4-day survival, horizontal slices of the spinal cord were processed with immunohistochemical staining (first antibody to cholera toxin B subunit and second antibody with Cy3) and observed with a confocal microscope. Three-dimensional reconstruction of labelled motoneurons was performed to examine soma and dendrite morphology. As compared to the soma volume in young rats, significantly smaller values were found in old rats in both motoneurons and the degrees of decline were 16.1% in DI-MN and 20.3% in LL-MN. Significant decreases in the thickness of primary dendrites were also found in both motoneurons, and the degrees of decline were 17.5% in DI-MN and 22.3% in LL-MN. Smaller changes were found in DI-MN than in LL-MN, indicating the possibility that increased activation by central drives can attenuate age-related morphological changes of the motor system in the spinal cord.     

Gender differences in morphometric properties of muscle fibres measured on cross-sections of rat hindlimb muscles

The study was aimed at demonstrating gender differences in the numbers, diameters and cross-section areas of muscle fibres for three hindlimb skeletal muscles responsible for locomotion and maintenance of body posture: soleus, tibialis anterior and flexor digitorum brevis in rats. The experiments were performed on five 6-month-old male and female Wistar rats. In both genders, all studied muscles of the right and left hindlimbs were isolated from surrounding tissues and excised for further procedures. The muscle transverse cross-sections taken from the muscle mid-belly were analysed. Following staining of reticular fibres by silver impregnation, the numbers, diameters and cross-section areas of muscle fibres were determined from microscopic images of muscle sections. The body mass of male rats was 80% higher than that of females. In addition, the muscle mass and the cross-section area were 53-82 and 26-45% higher in males, respectively. The number of muscle fibres was 11-42% higher in males than in females whereas the fibre diameters were 7-29% higher in males. The most conspicuous differences between males and females were found with respect to tibialis anterior, whereas the smallest differences were evident in soleus. The present study revealed that the gender morphometric differences in the studied rat hindlimb muscles were mainly owing to differences in number and size of muscle fibres and that the difference in muscle mass could be explained mainly from higher number of muscle fibres in males and to smaller degree from their larger diameters.     

严格供港肉禽的种质标准和供货集散方式

香港禽流感发生前后,美日及欧盟屡屡对中国禽产品的国际贸易制造壁垒。国人在据理抗争的同时,更注重禽产品的安全问题。本文作者的建议值得参考。     

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.     

Immunohistochemical detection of components of the insulin-like growth factor system during skeletal muscle growth in the pig

The insulin-like growth factor (IGF) system plays an important role in postnatal somatic and skeletal muscle growth in pigs. There is little information on the occurrence and distribution of components of the IGF system in postnatal porcine skeletal muscle. IGF-I, IGF receptor 1 (IGF1R) and the IGF-binding proteins IGFBP-1 and -3 in longissimus dorsi and triceps brachii were localized in muscle biopsies from 12 commercially crossbred pigs aged from 28 to 199 days as well as from the sire generation, by immunohistochemistry. Plasma IGF-I concentrations were also determined using radio-immunoassays. Unlike other species, IGF-I was localized in porcine skeletal muscle fibres. Staining intensity correlated with the highest plasma IGF-I levels and phases of intensive muscle growth from the 11th to 22nd week. The pattern of IGF1R immunostaining, which was strong, correlated with that of IGF-I, IGF1R was also localized in endomysial tissues. IGFBP-1 was not detected within muscle fibres, but was found in the endomysium and vessel walls, while IGFBP-3 was localized with IGF-1 and its receptor. Higher magnification revealed that IGF1R, IGFBP-3 and probably IGF-I appeared in the tubular system. Inhibitory as well as stimulating controls of IGFBP-1 and -3 on IGF functions are discussed, which may maintain a balance between autocrine growth promoting activities of IGF-I and IGF1R.     

Immunohistochemical localization of transforming growth factor alpha in regenerating rat liver

Immunohistochemical localization of TGF-alpha and cell proliferation kinetics during liver regeneration after two-thirds partial hepatectomy (PH) were investigated. Twenty-four to 72 hr after PH, appreciable increase in the number of TGF-alpha-positive hepatocytes was observed in zones 1 and 2. At the peak at 36 hr, almost all positive cells were stained in their nuclei. Considerable increase in the BrdU labeling index was observed 24-36 hr after PH with a peak at 24 hr in zones 1 and 2. These results indicated an association between TGF-alpha expression and hepatocyte regeneration. It is suggested that immunohistochemical localization of TGF-alpha may be a useful marker of cell proliferation activity in rat liver.     

Immunohistochemical analysis of MCT1 and CD147 in equine skeletal muscle fibres

Monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147 facilitate efflux of lactate from the muscle. Expression of MCT1 and CD147 were studied with immunohistochemistry in type I, IIA, IIAB and IIB fibres of equine gluteal muscle. Staining intensity of MCT1 in the cytoplasm as well as in the membranes of fibre types decreased in the order I = IIA > IIAB > IIB and correlated with the oxidative capacity. Capillaries were pronounced in the MCT1 staining. CD147 antibody stained plasma membranes of all fibre types evenly, whereas the staining in the cytoplasm followed that of MCT1. In the middle gluteal muscle the expression of MCT1 follows the oxidative capacity of muscle fibres, but the expression of CD147 in sarcolemma does not vary among fibre types. The use of horse specific MCT1 and CD147 antibodies can in future studies help to evaluate lactate efflux from different muscle fibre types.     

Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period

β-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production. This study was conducted to determine the possible changes in proportions and localization of β-defensin 1-4 in the rat uterus at the 1st, 3th, 5th, 10th and 15th days of postpartum and at the period of diestrus using immunohistochemical techniques. In the present study, it was determined that β-defensin 1-4 were generally found in all structural components of the endometrium (luminal and glandular epithelium, stromal cells and blood vessels) in both the nucleus and the cytoplasm of cells during the involution period and diestrus. Suprisingly, immunoreaction of β-defensin 2 was also observed in the lateral membrane of the luminal and glandular epithelial cells on the 10th day of involution and immunostaining of β-defensin 4 was also localized in the apical membrane of the luminal and glandular epithelial cells. The current study demonstrated β-defensin 1-4 immunoreactivities in the endothelium of blood vessels were stronger throughout the involution period. Although β-defensins 2 and 3 were localized in both the nuclei and the cytoplasm of endothelial cells, β-defensins 1 and 4 were present in only cytoplasm. These results show that the most component of rat endometrium expresses human β-defensin 1-4 in a involution-dependent manner. Therefore it may be asserted that these molecules constitute a organised protection to prevent uterus from probable infections during the involution process.     

Immunohistochemical detection of cellular prion protein (PrPc) in the rat central nervous system

A simple conventional method of immunohistochemistry (i.e. fixing the frozen sections in cold methanol) was used to determine the immunolocalization of cellular prion protein (PrPc), with good results. In the rat cerebrum, the cytoplasm of neural cells in the cortex and corpus stratum, pia mater, membrane limitans gliae superficialis, choroid plexus and blood vessel wall were immunostained. The formation of network structures of immunostained neural and/or glial fibers in the cerebral cortex was also observed. These immunostained network structures of neural and/or glial fibers were also observed in cultured neural cells. The results suggest that fixation of frozen sections and cultured cells with cold methanol is a useful method for detecting the immunolocalization of PrPc and that PrPc exists in the various components of the central nervous system of the rat.     

Immunohistochemical study on the secretory host defense system of bactericidal peptides in rat digestive organs

To clarify the fundamental regulation mechanism against indigenous bacterial proliferation in the alimentary tract, we immunohistochemically examined the localization of 4 bactericidal peptides (BP) in the rat digestive exocrine glands. In the upper alimentary tract, lysozyme was detected in the gustatory, extraorbital lacrimal and parotid glands. Secretory phospholipase A2 (sPLA2) was detected in the extraorbital lacrimal glands. β-defensin1 was detected in the gustatory and extraorbital lacrimal glands. β-defensin2 was detected in the Harderian glands. In the stomach, β-defensins were detected in the gastric superficial epithelial cells. In the small and large intestines, only lysozyme and sPLA2 were detected in the Paneth cells. In the cecum, all 4 BP were detected in the middle to apical portions of the crypts, and only sPLA2 was detected in the basal portion. No BP were localized in other exocrine glands associated with the alimentary tract. In addition, all 4 BP were also detected in the columnar epithelial cells of the apical portions of intestinal villi. In the intestinal superficial epithelial cells, lysozyme and β-defensins were detected in the ascending colon, whereas only β-defensin1 was detected in the descending colon and rectum. These results suggest that BP are mainly secreted from exocrine tissues in the initial portion of the digestive tract and play a role in host defense against indigenous bacteria throughout the digestive tract. Part of the BP in the chyme might be absorbed by the epithelium at the most inner sites of mucosae in the small and large intestines.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.