Layer-dependent role of collagen recruitment during loading of the rat bladder wall

In this work, we re-evaluated long-standing conjectures as to the source of the exceptionally large compliance of the bladder wall. Whereas these conjectures were based on indirect measures of loading mechanisms, in this work we take advantage of advances in bioimaging to directly assess collagen fibers and wall architecture during biaxial loading. A custom biaxial mechanical testing system compatible with multiphoton microscopy was used to directly measure the layer-dependent collagen fiber recruitment in bladder tissue from 9 male Fischer rats (4 adult and 5 aged). As for other soft tissues, the bladder loading curve was exponential in shape and could be divided into toe, transition and high stress regimes. The relationship between collagen recruitment and loading curves was evaluated in the context of the inner (lamina propria) and outer (detrusor smooth muscle) layers. The large extensibility of the bladder was found to be possible due to folds in the wall (rugae) that provide a mechanism for low resistance flattening without any discernible recruitment of collagen fibers throughout the toe regime. For more extensible bladders, as the loading extended into the transition regime, a gradual coordinated recruitment of collagen fibers between the lamina propria layer and detrusor smooth muscle layer was found. A second important finding was that wall extensibility could be lost by premature recruitment of collagen in the outer wall that cut short the toe region. This change was correlated with age. This work provides, for the first time, a mechanistic understanding of the role of collagen recruitment in determining bladder extensibility and capacitance.     

Localisation and measurement of VIP in the genitourinary system of man and animals

VIP is present in the genitourinary system of man and animals. In man the highest concentrations are found in the penis, the uterus and vagina and in the urinary bladder. VIP nerves heavily innervate the erectile tissue of the male external genitalia, the uterine smooth muscle and blood vessels, the seromucous glands of the cervix, and the lamina propria and vaginal epithelium. In the urinary bladder, VIP nerves are located beneath the transitional epithelium, in the lamina propria and in the smooth muscle. Other areas well innervated by VIP nerves include the prostate, seminal vesicles and vasa deferentia. Chemical (phenol- and 6-OHDA) or surgical (hypogastric or pelvic nerve section) extrinsic denervation fail to deplete the genitourinary system of its VIP content, supporting the view that VIP-containing nerves originate from local ganglion cells. Indeed, neuronal cell bodies containing VIP are seen in the paracervical ganglia of the female genitalia, the para- or intramural bladder ganglia and scattered through the base of the cavernosum body, the neck of the bladder and the prostate. The finding of elevated levels of VIP in the local circulation after induced penile erection in man and mammals and the ability of VIP to relax the detrusor muscle of the bladder suggests that the peptide may be involved in penile erection and bladder relaxation, as does the marked VIP depletion in the penis or bladder in patients suffering from diabetic impotence or bladder instability.     

Characterization of a fibroblast cell from the urinary bladder wall

Summary For the first time we report on the growth, culture, and matrix production characteristics of a cell type isolated from the lamina propria of the urinary bladder wall. A fibroblastlike cell was identified as distinct from bladder detrusor smooth muscle cells and urothelium based on morphology, growth characteristics, and immunohistochemical staining. Characterization of extracellular matrix synthesis by this cell type using³⁵S-methionine metabolic labeling demonstrated that these cells are capable of secreting components of the surrounding connective tissue, including several fibrillar collagens, a basement membrane collagen, and fibronectin.     

Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction

Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.     

Expression and distribution of ectonucleotidases in mouse urinary bladder

Background

Normal urinary bladder function requires bidirectional molecular communication between urothelium, detrusor smooth muscle and sensory neurons and one of the key mediators involved in this intercellular signaling is ATP. Ectonucleotidases dephosphorylate nucleotides and thus regulate ligand exposure to P2X and P2Y purinergic receptors. Little is known about the role of these enzymes in mammalian bladder despite substantial literature linking bladder diseases to aberrant purinergic signaling. We therefore examined the expression and distribution of ectonucleotidases in the mouse bladder since mice offer the advantage of straightforward genetic modification for future studies.

Principal Findings

RT-PCR demonstrated that eight members of the ectonucleoside triphosphate diphosphohydrolase (NTPD) family, as well as 5′-nucleotidase (NT5E) are expressed in mouse bladder. NTPD1, NTPD2, NTPD3, NTPD8 and NT5E all catalyze extracellular nucleotide dephosphorylation and in concert achieve stepwise conversion of extracellular ATP to adenosine. Immunofluorescent localization with confocal microscopy revealed NTPD1 in endothelium of blood vessels in the lamina propria and in detrusor smooth muscle cells, while NTPD2 was expressed in cells localized to a region of the lamina propria adjacent to detrusor and surrounding muscle bundles in the detrusor. NTPD3 was urothelial-specific, occurring on membranes of intermediate and basal epithelial cells but did not appear to be present in umbrella cells. Immunoblotting confirmed NTPD8 protein in bladder and immunofluorescence suggested a primary localization to the urothelium. NT5E was present exclusively in detrusor smooth muscle in a pattern complementary with that of NTPD1 suggesting a mechanism for providing adenosine to P1 receptors on the surface of myocytes.

Conclusions

Ectonucleotidases exhibit highly cell-specific expression patterns in bladder and therefore likely act in a coordinated manner to regulate ligand availability to purinergic receptors. This is the first study to determine the expression and location of ectonucleotidases within the mammalian urinary bladder.     

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia     

Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency

Purinergic P2X receptors associated with the parasympathetic nerves supplying human bladder smooth muscle (detrusor) are implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalised with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder and in adults with the urodynamics findings of sensory urgency (SU) to determine how receptor distribution varied in patients with a small bladder capacity. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody (SV2) against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for haematuria (n=22, age 33–88 years) and adults with sensory urgency at cystoscopy/cystodistension (n=11, age 37–70 years). Normal adult specimens contained detrusor muscle innervated by parasympathetic nerves possessing large varicosities (1.2 m) distributed along their length. These mostly all showed colocalised patches of presynaptic P2X_1,2,3,5 subtypes while presynaptic subtypes P2X_4,6,7 were present in only 6–18% of varicosities. Detrusor nerve varicosities from SU patients revealed general loss of all presynaptic P2X subtypes with the proportion containing receptors reducing to only 0.5–5% depending on P2X subtype. The same loss was recorded from the sensory nerves in the surrounding lamina propria. This specific loss of P2X receptors may impair control of detrusor distension and contribute to the pathophysiology of sensory urgency.The study was funded by the National Health and Medical Research Council of Australia     

Anatomic Distribution of Nerves and Microvascular Density in the Human Anterior Vaginal Wall: Prospective Study

Background

The presence of the G-spot (an assumed erotic sensitive area in the anterior wall of the vagina) remains controversial. We explored the histomorphological basis of the G-spot.

Methods

Biopsies were drawn from a 12 o’clock direction in the distal- and proximal-third areas of the anterior vagina of 32 Chinese subjects. The total number of protein gene product 9.5–immunoreactive nerves and smooth muscle actin–immunoreactive blood vessels in each specimen was quantified using the avidin-biotin-peroxidase assay.

Results

Vaginal innervation was observed in the lamina propria and muscle layer of the anterior vaginal wall. The distal-third of the anterior vaginal wall had significantly richer small-nerve-fiber innervation in the lamina propria than the proximal-third (p = 0.000) and in the vaginal muscle layer (p = 0.006). There were abundant microvessels in the lamina propria and muscle layer, but no small vessels in the lamina propria and few in the muscle layer. Significant differences were noted in the number of microvessels when comparing the distal- with proximal-third parts in the lamina propria (p = 0.046) and muscle layer (p = 0.002).

Conclusions

Significantly increased density of nerves and microvessels in the distal-third of the anterior vaginal wall could be the histomorphological basis of the G-spot. Distal anterior vaginal repair could disrupt the normal anatomy, neurovascular supply and function of the G-spot, and cause sexual dysfunction.     

A Pacinian corpuscle in the human bladder lamina propria

The fortuitous finding of a complex Pacinian corpuscle within the lamina propria of the human urinary bladder is described. It consisted of a complex of encapsulated nerve endings within the areolar connective tissue of the lamina propria immediately adjacent to the inner aspect of the detrusor muscle. It showed no structural evidence of directional sensitivity and was associated on its outer aspect with small unmyelinated axons containing small clear and dense-cored vesicles. This appears to be the first report of an encapsulated nerve ending within the lining of the adult human urinary bladder.     

Morphometric and Quantitative Immunohistochemical Analysis of Disease-Related Changes in the Upper (Suburothelial) Lamina Propria of the Human Bladder Dome

The upper (suburothelial) lamina propria (ULP) is a distinct region in the human bladder with dense populations of interstitial cells (IC), fine vascular networks and variable development of muscularis mucosae (MM). It is more and more obvious that the ULP plays an important role in bladder physiology and bladder disease, and in the present study we have quantified changes in the cellular key players of the ULP in bladders from patients with carcinoma in situ (CIS), multiple sclerosis (MS) and bladder pain syndrome (BPS). Tissue samples for the different patient groups were obtained from radical cystectomy-specimens. Standardized immunohistochemistry with a panel of specific cell markers was used to characterise the ULP cellular structures, followed by digitalised morphometry and quantitative staining analysis. Alterations in the ULP area were most pronounced in MS bladders, but also present in BPS and CIS bladders. We observed an increased thickness and increased variability in thickness of the ULP IC area in MS and BPS bladders; a significantly increased development of MM in MS bladders; a changed organization of vascular plexuses in the lamina propria in most pathologic bladders and a changed phenotype of ULP IC: a significantly decreased expression of progesterone receptor in MS bladders and a trend towards decreased expression of alpha-smooth muscle actin in BPS bladders. We show here for the first time the presence of disease-specific changes in organisation and/or phenotype of the different key players of the ULP area in human bladder. The present findings further support the hypothesis that the ULP area is involved and altered in different bladder diseases.     

Histomorphometric study of the lower urogenital tract in pre- and post-menopausal women

In this study 22 histomorphometric parameters were used to quantify the histologic changes in the vagina, trigone of the bladder, and the proximal and distal urethra of 37 women aged between 18 and 82 years. The results of this quantitative study revealed a hormonally induced, age-independent atrophy of smooth muscle in the lamina propria of the mucosa and the longitudinal part of the muscular layer in the aforementioned anatomical sites in post-menopausal women. In the lamina propria of the mucosa of the lower urogenital tract in post-menopausal women hormonally induced quantitative changes of blood vessels were not observed. Senile atrophy of smooth muscles was only observed in the deeper part of the muscular layer of the vagina and the trigone of the urinary bladder and was less prominent than the hormonally induced smooth muscle atrophy.     

Neuronal galanin is widely distributed in the chicken respiratory tract and coexists with multiple neuropeptides

Summary The mammalian airways are known to be richly innervated by several types of peptide-containing nerve fibers. Galanin-containing fibers are, however, comparatively few. The results of the present immunocytochemical study indicate that the chicken airways receive a notably dense supply of galanin-storing fibers. Other major neuropeptides were neuropeptide Y, vasoactive intestinal peptide and substance P. Nerve fibers containing these peptides were distributed in the trachea, main bronchi, and the lungs. Minor nerve fiber populations contained calcitonin generelated peptide, enkephalin and gastrin-releasing peptide. In the trachea and main bronchi the majority of peptidecontaining nerve fibers was distributed beneath and sometimes also within the epithelium; fibers were fewer in the lamina propria. In the lungs they occurred both in association with the epithelium of small bronchi and in the septa. Adrenergic nerves (using tyrosine hydroxylase as marker) were predominantly distributed in the lamina propria among bundles of smooth muscle and blood vessels. In the nerve fibers associated with the epithelium and in nerve cell bodies in local ganglia of the tracheal wall, galanin was found to coexist with several other neuropeptides (neuropeptide Y, vasoactive intestinal peptide and substance P) suggesting co-expression of multiple neuropeptide genes in the same population of neurons.     

Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology.     

The distribution of noradrenergic nerves and small,intensely fluorescent (SIF) cells in the cat urinary bladder

Summary Fluorescence and electron microscopy have been used to study the distribution of noradrenergic nerves in the smooth muscle of the cat urinary bladder. Using the former technique, relatively few fluorescent noradrenergic nerves were observed in the body and fundus, while a rich plexus occurred adjacent to muscle cells of the bladder neck. The trigone could not be distinguished neuromorphologically from detrusor muscle in this region. Electron microscopy showed that the majority of noradrenergic terminals in the body and fundus were associated with presumptive cholinergic axons, while in the bladder neck noradrenergic terminals formed typical neuroeffector relationships with individual smooth muscle cells.Numerous ganglia occurred both in the adventitia and among the smooth muscle bundles, particularly in the bladder neck. The majority of the nerve cell bodies were non-fluorescent, although many contained bright orange autofluorescent granules, believed to be lysosomes. A small minority of ganglion cells were associated with fluorescent noradrenergic nerve terminals, thereby providing structural evidence for limited intraganglionic inhibition. In addition, occasional groups of small intensely fluorescent (SIF) cells were observed in some intramural ganglia and these were subsequently identified in the electron microscope. The possibility that these cells may provide a second inhibitory influence on bladder activity was considered.     

Vascular response of the rabbit bladder to chronic partial outlet obstruction

Partial outlet obstruction of the rabbit urinary bladder results, initially, in a rapid increase in bladder mass and remodeling of the bladder wall. Previously, it was shown that this response was characterized by serosal growth (thickening) which was apparent after 1 day of obstruction, before any visible vascularization was observed. After 1 week of obstruction, significant microvessel formation was seen in the transition region between the detrusor smooth muscle and the thickening serosa; after 2 weeks the entire serosa was vascularized.In this study we investigated the effect of chronic (4 week) partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium. Transverse sections of bladder wall were examined after 4 weeks of no surgery, sham surgery or partial obstruction.The microvessel density of the obstructed rabbit bladder mucosa and detrusor smooth muscle increased relative to augmentation of these compartments while new vessels appeared in the thickening serosa. Although vessel density did not change with obstruction a significant shift in mean vessel circumference to the left occurred indicating a significant increase in the number of microvessels and small vessels consistent with angiogenesis.     

Neuropeptide Y-like immunoreactivity in intramural ganglia of the newborn guinea pig urinary bladder

Immunoreactive neuropeptide Y (NPY) was demonstrated in neuronal elements in the urinary bladder wall of the newborn guinea pig. Numerous intramural ganglia were found lying among the smooth muscle bundles and in the submucosa, and NPY-like immunoreactive nerve cell bodies were demonstrated within all of these ganglia. Nerve fibres containing NPY were also richly distributed in the detrusor muscle, submucosa and around blood vessels. In dissociated cell cultures from newborn guinea pig detrusor muscle, a subpopulation (70-85%) of both mononucleate and binucleate intramural neurones was shown to contain NPY-like immunoreactivity. A low percentage (1-6%) of the intramural bladder neurones contained dopamine-beta-hydroxylase. In conclusion, while some NPY-containing nerve fibres in the wall of the bladder are of sympathetic origin, especially those supplying blood vessels, the results of this present study establish that many of these NPY-containing nerve fibres originate from non-adrenergic cell bodies within the intramural bladder ganglia.     

Adaptive changes of telocytes in the urinary bladder of patients affected by neurogenic detrusor overactivity

Urinary bladder activity involves central and autonomic nervous systems and bladder wall. Studies on the pathogenesis of voiding disorders such as the neurogenic detrusor overactivity (NDO) due to suprasacral spinal cord lesions have emphasized the importance of an abnormal handling of the afferent signals from urothelium and lamina propria (LP). In the LP (and detrusor), three types of telocytes (TC) are present and form a 3D‐network. TC are stromal cells able to form the scaffold that contains and organizes the connective components, to serve as guide for tissue (re)‐modelling, to produce trophic and/or regulatory molecules, to share privileged contacts with the immune cells. Specimens of full thickness bladder wall from NDO patients were collected with the aim to investigate possible changes of the three TC types using histology, immunohistochemistry and transmission electron microscopy. The results show that NDO causes several morphological TC changes without cell loss or network interruption. With the exception of those underlying the urothelium, all the TC display signs of activation (increase in Caveolin1 and caveolae, αSMA and thin filaments, Calreticulin and amount of cisternae of the rough endoplasmic reticulum, CD34, euchromatic nuclei and large nucleoli). In all the specimens, a cell infiltrate, mainly consisting in plasma cells located in the vicinity or taking contacts with the TC, is present. In conclusion, our findings show that NDO causes significant changes of all the TC. Notably, these changes can be interpreted as TC adaptability to the pathological condition likely preserving each of their peculiar functions.     

Pathophysiology of overactive bladder and urge urinary incontinence

Storage symptoms such as urgency, frequency, and nocturia, with or without urge incontinence, are characterized as overactive bladder (OAB). OAB can lead to urge incontinence. Disturbances in nerves, smooth muscle, and urothelium can cause this condition. In some respects the division between peripheral and central causes of OAB is artificial, but it remains a useful paradigm for appreciating the interactions between different tissues. Models have been developed to mimic the OAB associated with bladder instability, lower urinary tract obstruction, neuropathic disorders, diabetes, and interstitial cystitis. These models share the common features of increased connectivity and excitability of both detrusor smooth muscle and nerves. Increased excitability and connectivity of nerves involved in micturition rely on growth factors that orchestrate neural plasticity. Neurotransmitters, prostaglandins, and growth factors, such as nerve growth factor, provide mechanisms for bidirectional communication between muscle or urothelium and nerve, leading to OAB with or without urge incontinence.     

Ultrastructure of Cajal-like interstitial cells in the human detrusor

The aim of this ultrastructural study was to examine the human detrusor for interstitial cells of Cajal (ICC)-like cells (ICC-L) by conventional transmission electron microscopy (TEM) and immuno-transmission electron microscopy (I-TEM) with antibodies directed towards CD117 and CD34. Two main types of interstitial cells were identified by TEM: ICC-L and fibroblast-like cells (FLC). ICC-L were bipolar with slender (0.04 μm) flattened dendritic-like processes, frequently forming a branching labyrinth network. Caveolae and short membrane-associated dense bands were present. Mitochondria, rough endoplasmic reticulum and Golgi apparatus were observed in the cell somata and cytoplasmic processes. Intermediate filaments were abundant but no thick filaments were found. ICC-L were interconnected by close appositions, gap junctions and peg-and-socket junctions (PSJ) but no specialised contacts to smooth muscle or nerves were apparent. FLC were characterised by abundant rough endoplasmic reticulum but no caveolae or membrane-associated dense bands were observed; gap junctions and PSJ were absent and intermediate filaments were rare. By I-TEM, CD34 gold immunolabelling was present in long cytoplasmic processes corresponding to ICC-L between muscle fascicles but CD117 gold immunolabelling was negative. Thus, ICC-like cells are present in the human detrusor. They are CD34-immunoreactive and have a myoid ultrastructure clearly distinguishable from fibroblast-like cells. ICC-L may be analogous to interstitial cells of Cajal in the gut.