Regional Structural and Functional Specializations in the Urethra of the Female Rat: Evidence for Complex Physiological Control Systems

This study characterizes the complex structural and functional elements of the female rat urethra that may be involved in controlling urethral closure and continence. Urethras were dissected from female Sprague‐Dawley rats (N = 12) euthanized by pentobarbital overdose. Tissues were fixed (4% paraformaldehyde), frozen, and sectioned (8 μm) for light microscopy and immunohistochemistry. Antibodies were used to detect immunoreactivity to calcitonin gene related peptide, nitric oxide synthase, vesicular acetylcholine transporter, and tyrosine hydroxylase. Measurements of urethral wall compliance were taken along its length and in different axes using a closed ended catheter with a circular aperture. The bladder neck and proximal urethra are characterized by a highly folded epithelium and lamina propria. A smooth muscle layer is apparent but not pronounced. Distal to this region the smooth muscle layer thickens and forms the body of the internal sphincter, which has a complex innervation. In the mid urethra, the smooth muscle is thickened resulting in a luminal protrusion, producing an occlusion of the lumen. The structure of the distal urethra is different. The epithelium has few folds and, immediately below the lamina propria large thin walled vascular lacunae can be found. Measurements of the urethral wall compliance demonstrate distinct regional differences with proximal and distal specialisations. These variations, which correlate with muscular and vascular elements, suggest the operation of discrete systems, hence effecting urethral closure during filling. An understanding of these systems may yield insights into urethral pathology and direct approaches to develop pharmacological interventions to promote continence. Anat Rec, 301:1276–1289, 2018. © 2018 Wiley Periodicals, Inc.     

胆碱能神经在正常人胃壁的分布

为了给临床病理状态下胃溃疡神经分布提供参照，更好地为溃疡病的防治提供形态学依据，本文采用Ｋａｒｎｏｖｓｋｙ－Ｒｏｏｔｓ法，观察了４例正常人胃壁胆碱能神经纤维分布特点。结果：胃壁各层均有乙酰胆碱酯酶（ＡＣｈＥ）阳性纤维，但上皮和固有层上部未见ＡＣｈＥ阳性纤维；ＡＣｈＥ阳性纤维有４种形态：终末纤维、终末前纤维、神经束、神经干，胃壁各层细小动脉壁也存在神经末梢分布，在含非特异胆碱酸酶反应中，可见胃粘膜上皮下固有膜内，存在分支的神经末梢交互联成网状，可能是传入性质，对其功能意义做了讨论     

Dual innervation of the mammalian urinary bladder A histochemical study of the distribution of cholinergic and adrenergic nerves

The pattern of autonomic innervation of the urinary bladder was studied in the cat, dog, rabbit and rat, using specific histochemical technics for acetylcholinesterase and norepinephrine. Cholinergic and adrenergic ganglion cells exist in all layers throughout the bladder wall. Large cholinergic and adrenergic nerve trunks coursing in the adventitial coat and deep lamina propria branch into the muscularis. The terminal cholinergic ramifications form a neuroterminal plexus which surrounds every smooth muscle cell in the bladder wall. The terminal adrenergic fibers are less abundant, do not form a plexus, and show regional variations in number at different levels and depths of the muscularis. These variations suggest that two regions of the bladder, namely the base and body, may be distinguished on the basis of differences in muscular innervation. In the lamina propria cholinergic and adrenergic nerves are grouped as deep and superficial subepithelial nerves. The latter form networks of variable complexity and supply fibers to the epithelium. Throughout the bladder wall, the blood vessels have a dual cholinergic and adrenergic perivascular plexus from which fibers extend into the media. Although the basic pattern of innervation is similar in the species studied, certain variations exist in the relative abundance and arrangement of epithelial and subepithelial nerves. The muscularis has a uniform cholinergic and a variable adrenergic innervation in different species.     

Histology of the canine urethra. I. Morphometry of the female urethra

Urinary bladders and urethrae were collected from six adult and two juvenile female dogs. Five urethral regions and the neck and body of the bladder were sampled. Volume fractions for connective tissue including elastic fibers, smooth and striated muscle, and epithelium were obtained by projecting section images onto an array of points and computing the number of points overlying a tissue constituent per total points overlying the tissue section. Smooth muscle occupied approximately half the volume of the bladder wall, one-third the volume of the vesical neck, and one-fourth the volume of the proximal urethra. Striated muscle was present in the distal half of the urethra, where the total muscle coat occupied about one-third of the urethral wall volume. Smooth muscle was practically absent in the terminal urethra, where the striated urethralis muscle encircles urethra and vagina in common. Epithelial area and lumen perimeter were not significantly different along the length of the urethra except that urethral epithelium was significantly thicker adjacent to the vesical neck. In terms of histological proportions, the vesical neck was intermediate between the body of the bladder and the proximal urethra.     

Sensor cells and nerve terminals in some gut-derived organs: a review

The intestinal mucosa is supplied with intraepithelial sensor paraneurons and with nerve fibers restricted in the lamina propria. The present study compares some gut-derived and -related organs in regard to their neuroparaneuronal elements deviated in structure and function from those in the gut. At the light microscopic level the urethra contains intraepithelial sensor paraneurons with ramified cell base containing serotonin and sensory nerves penetrating the epithelium to reach the lumen. The trachea and bronchi contain, besides sensor paraneurons, numerous sensory nerves within the basal layer of the epithelium. These nerves are proposed to form a sensory complex, being associated with the epithelial cells. The respiratory portion of the nasal mucosa lacks intraepithelial paraneurons, while revealing numerous sensory nerves passing through the epithelium to directly detect stimulants in the air.     

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve terminals in the whole mount preparations of the dog urethra.

To visualize the entire shape of the intraepithelial nerve fibers, whole mount preparations of the dog urethra were produced and immunostained with an antiserum against CGRP, one of the predominant substances contained in the nerves. The immunoreactive nerves in the lamina propria were smooth (non-beaded) in appearance and weak in immunoreaction. Within the epithelium, they displayed typical beaded profiles and were intense in immunoreaction. The intraepithelial fibers branched and wound into an extensive network with wide meshes ("reticular terminal"). The bead-like swellings included large ones resembling Herring bodies in hypophyseal neurosecretory fibers. Another type of nerve terminal, consisting of fine and weakly immunopositive fibers, was also found in the epithelium. These branched in dendritic or in dense bouquet-like fashion, occupying smaller areas ("bouquet-like terminals). Vesicular swellings often characterized these terminals, though they were smaller and more uniform in size and far less in their amount of immunoreactive substance than were the swellings in the reticular terminals. Both types of nerve terminals originated from the same nerve trunk. The connection between the reticular and bouquet-like terminals, which may presumably represent secretory and receptive parts, respectively, morphologically supports the possible occurrence of an axon reflex in the urethral CGRP neurons. Our whole mount preparations, when doubly stained with CGRP and serotonin antibodies, further revealed the CGRP-positive reticular terminals being closely associated with serotonin- or CGRP-immunoreactive paraneurons dispersed in the epithelium.     

The Distribution of Muscles Fibers and Their Types in the Female Rat Urethra: Cytoarchitecture and Three‐Dimensional Reconstruction

An attempt to explore urethral cytoarchitecture including the distribution of smooth muscles and fast and slow striated muscles of adult female Sprague Dawley rat—a popular model in studying lower urinary tract function. Histological and immunohistochemical stainings were carried out to investigate the distribution of urethral muscle fibers and motor end plates. The urethral sphincter was furthermore three‐dimensionally reconstructed from serial histological sections. The mucosa at the distal urethra was significantly thicker than that of other segments. A prominent inner longitudinal and outer circular layer of smooth muscles covered the proximal end of urethra. Thick circular smooth muscles of the bladder neck region (urethral portion) decreased significantly distalward and longitudinal smooth muscles became 2‐ to 3‐fold thicker in the rest of the urethra. An additional layer of striated muscles appeared externally after neck region (urethra) and in association with motor end plates ran throughout the remaining urethra as the striated sphincter layer. Most striated muscles were fast fibers while relatively fewer slow fibers often concentrated at the periphery. A pair of extraneous striated muscles, resembling the human urethrovaginal sphincter muscles, connected both sides of mainly the distal vagina to the dorsal striated muscles in the wall of the middle urethra. The tension provided by this pair of muscles, and in conjunction with the striated sphincter of the urethral wall, was likely to function to suspend the middle urethra and facilitates its closure. Comprehensive morphological data of urethral sphincter offers solid basis for researchers conducting studies on dysfunction of bladder outlet. Anat Rec, 296:1640–1649, 2013. © 2013 Wiley Periodicals, Inc.     

Adrenergic and Cholinergic Nerves of the Human Urethra and Urinary Bladder. A Histochemical Study

The occurrence and distribution of adrenergic and acetylcholine esterase (AChE) positive nerves in the human urethra and urinary bladder were studied histochemically with the fluorescence method of Falck and Hillarp, and the copper thiocholine method of Koelle and Friedenwald. Both types of nerves were mainly confined to the layers of smooth muscle cells in the walls of the organs. In all parts of the urethra, there was a scanty supply of adrenergic nerves. Few adrenergic nerves were also found in the urinary bladder, except in the trigone area, where they were abundant. AChE-positive nerves were uniformly and richly distributed in the urinary bladder. Throughout the urethra the distribution of AChE-positive nerve fibres was uniform, but the number was clearly less than in the urinary bladder. No intramurally located adrenergic or AChE-positive ganglion cells could be demonstrated.     

The variable fine structure of elastin visualized with verhoeff's iron hematoxylin

Verhoeff's iron hematoxylin (VIH) followed by lead citrate (LC) applied to epoxy thin sections stained the dense component of elastic fibers heavily and the peripheral microfibrillar component lightly in guinea pig trachea and mouse testis fixed with a glutaraldehyde-osmium tetroxide sequence. This method stained large fimbriated fibers beneath tracheal epithelium, small fibers and stacked aggregates thereof in the deep lamina propria, cartilage and adventitia of the trachea and large stacked fibers in the fibroelastic band of the trachea. Fibers of the fetus differed from those of the adult, especially in the subepithelial elastic lamina of the trachea. Elastic fibers were intimately associated with fibroblasts and particularly slender fibroblast processes in tracheal stroma and with chondrocytes in tracheal cartilage. Fibroblasts associated with elastic fibers in the tracheal subepithelial lamina propria were often closely bordered by eosinophils, mast cells, or monocytes. Occasional mast cells extended slender processes around elastic fibers in the subepithelial lamina propria. In mouse testis and in many regions of the trachea, small elastic fibers were identified which were below the limits of resolution for the light microscope and were not apparent at the ultrastructural level in routinely stained thin sections.     

The variable fine structure of elastin visualized with Verhoeff's iron hematoxylin.

Verhoeff's iron hematoxylin (VIH) followed by lead citrate (LC) applied to epoxy thin sections stained the dense component of elastic fibers heavily and the peripheral microfibrillar component lightly in guinea pig trachea and mouse testis fixed with a glutaraldehyde-osmium tetroxide sequence. This method stained large fimbriated fibers beneath tracheal epithelium, small fibers and stacked aggregates thereof in the deep lamina propria, cartilage and adventitia of the trachea and large stacked fibers in the fibroelastic band of the trachea. Fibers of the fetus differed from those of the adult, especially in the subepithelial elastic lamina of the trachea. Elastic fibers were intimately associated with fibroblasts and particularly slender fibroblast processes in tracheal stroma and with chondrocytes in tracheal cartilage. Fibroblasts associated with elastic fibers in the tracheal subepithelial lamina propria were often closely bordered by eosinophils, mast cells, or monocytes. Occasional mast cells extended slender processes around elastic fibers in the subepithelial lamina propria. In mouse testis and in many regions of the trachea, small elastic fibers were identified which were below the limits of resolution for the light microscope and were not apparent at the ultrastructural level in routinely stained thin sections.     

Striated muscle and nerve fascicle distribution in the female raturethral sphincter

The anatomical basis for urinary continence depends on a thorough understanding of the tissues in the urethra. The objective of this study was to evaluate the morphology and neuroanatomy of urethral striated muscle, called the rhabdosphincter or external urethral sphincter, in normal female rats. Urethras from 12 female rats were dissected from the bladder, fixed, embedded in paraffin or epon, and sectioned every 1 mm. Striated muscle content was taken as the ratio of the striated muscle area to net urethral area. Nerve fascicles containing myelinated axons near the rhabdosphincter were counted and mapped. Both striated muscle content and number of nerve fascicles peak in the proximal third of the urethra, with a secondary peak at the distal end of the urethra. This secondary peak may correspond to an analog of the combined compressor urethrae/urethrovaginal sphincter located in the distal urethra in human. The rhabdosphincter has a variable distribution along the length of the urethra. In the middle and distal thirds of the urethra, the dorsal striated muscle fibers between the urethra and vagina become more sparse. The majority of nerve fascicles are contained in the lateral quadrants of the urethra, similar to the lateral distribution of somatic nerves in humans. In conclusion, this study demonstrates the normal distribution of the striated musculature and neuroanatomy in the urethra, with similarities to the human. It thus supports and extends the usefulness of the rat as an experimental model for studying urinary incontinence. Anat Rec 290:145–154, 2007. © 2007 Wiley‐Liss, Inc.     

Protein gene-product 9.5 in developing mouse circumvallate papilla: comparison with neuron-specific enolase and calcitonin gene-related peptide

The present study was made to investigate the ontogeny of protein gene-product 9.5 (PGP 9.5)-like immunoreactivity (-LI) in the developing mouse circumvallate papilla (CVP), and its distribution was compared to that of neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP). In adult CVP, PGP 9.5-LI was observed in the subgemmal nerve plexus; some thin PGP 9.5-like immunoreactive (-IR) nerve fibers penetrated taste buds and apical epithelium. PGP 9.5-LI was also observed in the spindle-shaped cells in taste buds, and a small number of round- or oval-shaped ganglionic cells in the lamina propria. The distribution of NSE-LI was comparable to that of PGP 9.5-LI. CGRP-LI was observed in the nerve fibers only; distribution of CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers, although the number of CGRP-IR nerve fibers was smaller than that of PGP 9.5-IR nerve fibers. At least six developmental stages were defined with regard to the developmental changes in the distribution of PGP 9.5-LI from embryonic day (E) 12 to adulthood: Stage I (E12–13) — a dense nerve plexus of PGP 9.5-IR nerve fibers was detected in the lamina propria beneath the core of newly-formed papilla. Stage II (E14–16) — thin PGP 9.5-IR nerve fibers penetrated the apical epithelium, and a few round-shaped cells in the apical epithelium also displayed PGP 9.5-LI. Stage III (E17–18) — thin PGP 9.5-IR nerve fibers penetrated the inner lateral epithelium of the trench. Stage IV [Postnatal day (P) 0–3] many PGP 9.5-IR nerve fibers penetrated the outer lateral epithelium of the trench; later in this stage, taste buds appeared. Stage V (P5–10) — a small number of PGP 9.5 IR cells in the taste buds appeared, and their number increased gradually. Stage VI (PI4-adult) — the number of PGP 9.5-IR taste cells increased and reached the adult level, while the number of PGP 9.5-IR nerve fibers decreased. The development of NSE-LI was similar to that of PGP 9.5-LI. CGRP-IR nerve fibers were detected at E12 in the lamina propria, and the development of the intraepithelial CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers. The present results indicate that invasion by nerve fibers of the epithelium of lingual papillae occurs in a complex manner, and that these nerve fibers may participate in the formation of the taste buds.     

Sympathetic innervation of the urinary bladder and urethral muscle in the pig

Larsen , J.-J., J. Nordling and B. Christensen , Sympathetic innervation of the urinary bladder and urethral muscle in the pig. Acta physiol. scand. 1978. 104. 485–490. The sympathetic innervation of the lower urinary tract was studied in the female and male pig using a specific histochemical technique for visualization of noradrenaline containing nerves. In smooth muscle from the detrusor few evenly distributed adrenergic nerve terminals were found. A greater number of terminals were observed in the trigone, bladder neck and urethra. There was no sex difference. In smooth muscle specimens from pig detrusor and trigone sympathetic nerve terminals were more abundant than in corresponding tissue from humans.     

Histology of the canine uretha II. Morphometry of the male pelvic urethra

Urinary bladders and pelvic urethrae were collected from six adult and two juvenile male dogs. Within two vesical and six urethral sampling regions, volume densities were estimated for smooth and striated muscle, connective tissue and elastic fibers, stratum cavernosum, luminal epithelium, and prostate. The neck had significantly less smooth muscle and more connective tissue than the body of the bladder. In the prostatic urethra, smooth muscle was associated principally with trabeculae surrounding prostate lobules. Smooth muscle was sparse superficially in the prostatic capsule and practically absent in relation to the mid-prostatic urethra. Thus there was no mechanism for active closure of the middle prostatic urethra, and elastic fiber density was correspondingly high in this region. The smooth muscle sphincter needed to maintain urinary continuence and prevent semen reflux was primarily the vesical neck. Caudal to the body of the prostate, striated muslce comprised more than 50% of the urethral wall. Juvenile and adult postprostatic urethrae were similar except for a decreased quantity of stratum cavernosum in the pups.     

Changes in the extracellular matrix in periurethral tissue of women with stress urinary incontinence

Changes in structural support of the urethra and bladder neck have been proposed as important factors in the pathogenesis of stress urinary incontinence (SUI). In this context, we undertook an ultrastructural study on the periurethral connective tissue with an emphasis on incontinent women with normotonic and hypotonic urethras. Small specimens of periurethral connective tissue were obtained by dissection during a tension-free vaginal tape-implantation procedure in 34 stress urinary incontinent postmenopausal women with a normotonic urethra and 9 stress urinary incontinent postmenopausal women with a hypotonic urethra. In the samples taken from stress-incontinent women with a normotonic urethra, intact elastic fibers were closely connected with collagen fibers, smooth muscle cells and fibrocytes. In the samples taken from stress-incontinent women with a hypotonic urethra, we detected irregular fragmented distribution of the elastin within the tissue. We assume that these structural changes lead to functional consequences, such as diminished tissue extensibility and loss of stability surrounding the female urethra. These altered connective tissue properties may affect the mechanism of urethral closure under stress (e.g., coughing) and therefore contribute to the occurrence of SUI with a hypotonic urethra.     

Cholinergic innervation of the simian oxyntic gastric mucosa

The innervation of the oxyntic gastric mucosa was studied by light and electron microscopy in the rhesus monkey. An abundant net of acetylcholinesterase-positive nerves was seen in the lamina propria, with many slender fibers noted close to the basal lamina of the glands. Electron microscopic observation disclosed the presence of nerve fibers containing axons and varicosities in the lamina propria, often in very close proximity to the epithelial glandular cells. Nerve endings partly enveloped by a Schwann cell were occasionally seen in direct contact with a parietal cell.     

Comparative anatomical and neurohistological observations on the tongue of Japanese pika (Ochotona hyperborea yezoensis,kishida)

The tongue of the Japanese pika was investigated using the silver impregnation method. On the dorsum, three vallate papillae, arranged in a row transversely, and foliate papillae with 12–13 clefts were observed. The papillae are both covered with an upper thickened epithelium. The abundant nerve fibers are widely distributed in the subpapillary layer of the lamina propria to form the subpapillary nerve plexus, which is composed of non-myelinated and thick myelinated fibers, and ganglion cells. The taste buds, of an embryonic type with an indefinite border, are located as a mass in both walls of the papillae symmetrically, closely associated with a small number of the fine fibers from the subpapillary plexus. They are large in size and penetrate into the subgemmal connective tissue. Their aspect suggests a peculiar receptive apparatus surrounding the orifice of the serous gland-ducts. In the papilla regions the serous glands are plentiful deep in the muscles and their ducts open into the bottom of the trench and furrow. The dorsum is densely packed only with the filiform papillae which are covered with highly cornified, thickened stratified squamous epithelium and poor in nerve supply, but the smooth inferior surface is well supplied with sensory nerves ending free and is furnished with many taste buds. No apical glands are present. The tongue contains many ganglion cells alongside the nerve fiber bundles running toward the apex.     

Morphological analysis of the acute effects of overdistension on the extracellular matrix of the rat urinary bladder wall.

PURPOSE: To investigate the morphological effects of acute overdistension in the structure of the extracellular matrix of the bladder wall in rats. MATERIALS AND METHODS: The bladders of a group of 6 male Wistar rats were transurethrally overdistended for 3 hours. Another identical group (the control group) was only submitted to a sham operation. Specimens from the bladder dome were analyzed with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). RESULTS: LM--The control group bladders had a 4 to 5 layer urothelium, a lamina propria, and a smooth muscle layer with longitudinal and transversal fibers. The overdistended bladders presented an intense interstitial infiltrate in the lamina propria, and a less intense infiltrate among the smooth muscle fibers. TEM--The cells of the overdistended bladders had a significant amount of vacuoles, unlike the control bladders, where such vacuoles were scarce or absent. SEM--A delicate three-dimensional mesh of collagen fibrils was observed in the lamina propria of the bladder walls from the control group. Whilst for the control group this mesh consisted of distinct geometric structures, with mostly circular cellular spaces surrounded by the fibrils, the overdistended group showed evidence of distortion of the mesh, with flattened and elongated cellular spaces. CONCLUSIONS: Acute bladder overdistension induces structural modifications, altering the arrangement and interaction of collagen fibrils, as well as incipient tissue damage as edema in the lamina propria and smooth muscle layers.     

Analysis of leucocyte subsets in the canine intestine

Immunohistochemistry and computer-aided morphometric analysis were used to define populations of leucocyte subsets in the intestinal tract of an outbred population of dogs with no evidence of gastrointestinal disease. In the small intestinal lamina propria, B cells and plasma cells (IgA+, IgM+ and IgG+) were prominent in peri-crypt regions, with a significant trend for a reduction in the number of cells towards the villous tip (P < 0.0001). By contrast, lamina propria T cells (CD3+) and T-cell subsets (CD4+ and CD8+) were present in greatest numbers at the tip of the villus, with significantly decreasing numbers towards the crypt regions (P < 0.0001). In the lamina propria, CD4+ cells outnumbered CD8+ cells (P = 0.05), but the opposite was true of the epithelial compartment (P < 0.001). The distribution of CD5+ lymphocytes was similar to that of CD3+ cells, in both the lamina propria and epithelial compartments. The numbers and distribution of cells expressing MHC class II, L1 and CD45 were recorded. Numerous eosinophils were present in the lamina propria, and an intra-epithelial population was also noted, especially in the crypt epithelium. Mast cells, which were mainly found in the subepithelial lamina propria, were also present within muscle layers, and cells expressing IgE had a similar distribution. Similar populations of cells were recorded in the colonic lamina propria and epithelium. The quantitative and qualitative data from this study will enable comparisons to be made with dogs suffering from inflammatory bowel diseases.