John A. Gosling

Bladder Outflow Obstruction—A Cause of Denervation Supersensitivity

Eighteen Landrace pigs and 12 Göttingen mini-pigs were evaluated in a study of experimental bladder outflow obstruction. Twenty-two of the animals underwent partial bladder outflow obstruction for periods up to 12 months. The subsequent changes were assessed using cystometric, physiological and morphological means. There was a consistent increase in the voiding pressures and a concomitant reduction in the flow rates in all the obstructed animals. Seventy-seven per cent of the obstructed animals showed cystometric evidence of bladder instability. In vitro studies showed an increase in sensitivity to exogenously applied agonists and a reduction in sensitivity to intramural nerve stimulation. Morphological studies showed an inverse correlation between neuronal density and the duration of obstruction. These changes are typical of post-junctional supersensitivity secondary to partial denervation. These results suggest that agents capable of stabilising the bladder smooth muscle membrane may be useful in the treatment of detrusor instability secondary to bladder outflow obstruction.

CORRELATION BETWEEN THE STRUCTURE AND FUNCTION OF THE RABBIT URINARY BLADDER FOLLOWING PARTIAL OUTLET OBSTRUCTION

PURPOSE To understand the relationship between contractile and structural changes in the obstructed bladder, rabbit bladder was partially obstructed for up to 70 days and alterations in tension response to field stimulation and carbachol were compared with alterations in ultrastructure and innervation of detrusor smooth muscle (SM). The effect of partial outlet obstruction on the physiological responses to field stimulation (FS) (nerve mediated contraction) and carbachol (receptor mediated contraction) were correlated with the structure and innervation of the detrusor smooth muscle (SM) of the same animal during a 70 day period. MATERIALS AND METHODS 28 rabbits were subjected to 1 to 70 days of mild partial outlet obstruction. Sham operated rabbits were euthanized at 7, 14, 28, and 70 days post-obstruction. At each time period, isolated strips of bladder body were mounted in individual baths and the contractile response to FS and carbachol determined. Three additional strips from each bladder were fixed for electron microscopy. RESULTS Bladder mass increased rapidly during the first 7 days after obstruction, was constant for the next 7 days, and then continued to increase gradually. Dysfunction of the contractile response to FS was noted as early as 3 days and progressively increased over the 70-day study period. The decrease in the response to FS increased at a significantly faster rate than the decrease in the contractile response to carbachol. In ultrastructure studies, at 3 and 7 days post-obstruction the majority of SM cells displayed the characteristics of hypertrophy. At 28 days some SM cells displayed loosely packed myofilaments and an irregular distribution of sarcoplasmic dense bodies. At 70 days swollen mitochondria were present in all cell types of the bladder wall. Evidence of axonal degeneration was first observed at 7 days post-obstruction and became more extensive thereafter. No evidence of mitotic figures, nerve growth cones or regenerating SM cells was observed. CONCLUSIONS Prolonged partial bladder outflow obstruction is accompanied by a progressive decrease in contractility of SM. The present study describes the structural damage that occurs in the bladder wall in response to partial outlet obstruction and correlates these observations with the contractile dysfunction with which it is associated. Furthermore, mitochondrial damage in vessels and fibroblasts is suggestive of bladder wall ischemia.

Autonomic innervation of the human cardiac conduction system: changes from infancy to senility--an immunohistochemical and histochemical analysis.

In order to study the changes in the pattern of autonomic innervation of the human cardiac conduction system in relation to age, the innervation of the conduction system of 24 human hearts (the age of the individuals ranged from newborn to 80 years), freshly obtained at autopsy, was evaluated by a combination of immunofluorescence and histochemical techniques. The pattern of distribution and density of nerves exhibiting immunoreactivity against protein gene product 9.5 (PGP), a general neural marker, dopamine β‐hydroxylase (DBH) and tyrosine hydroxylase (TH), indicators for presumptive sympathetic neural tissue, and those demonstrating positive acetylcholinesterase (AChE) activity, were studied. All these nerves showed a similar pattern of distribution and developmental changes. The density of innervation, assessed semiquantitatively, was highest in the sinus node, and exhibited a decreasing gradient through the atrioventricular node, penetrating and branching bundle, to the bundle branches. Other than a paucity of those showing AChE activity, nerves were present in substantial quantities in infancy. They then increased in density to a maximum in childhood, at which time the adult pattern was achieved and then gradually decreased in density in the elders to a level similar to or slightly less than that in infancy. In contrast, only scattered AChE‐positive nerves were found in the sinus and atrioventricular nodes, but were absent from the bundle branches of the infant heart, whereas these conduction tissues themselves possessing a substantial amount of pseudocholinesterase. During maturation into adulthood, however, the conduction tissues gradually lost their content of pseudocholinesterase but acquired a rich supply of AChE‐positive nerves, comparable in density to those of DBH and TH nerves. The decline in density of AChE‐positive nerves in the conduction tissues in the elders was also similar to those of DBH and TH nerves. Our findings of initial sympathetic dominance in the neural supply to the human cardiac conduction system in infancy, and its gradual transition into a sympathetic and parasympathetic codominance in adulthood, correlate well with the physiologic alterations known to occur in cardiac rate during postnatal development. The finding of reduction in density of innervation of the conduction tissue with ageing is also in agreement with clinical and electrophysiological findings such as age‐associated reduction in cardiac response to parasympathetic stimulation. Finally, our findings also support the hypothesis that, in addition to the para‐arterial route, the parafascicular route of extension along the conduction tissue constitutes another pathway for the innervation of the conduction system of the human heart during development. Anat Rec 264:169–182, 2001.

The distribution of vesicular acetylcholine transporter in the human male genitourinary organs and its co-localization with neuropeptide Y and nitric oxide synthase.

Because doubt still remains concerning the distribution of nerves that are unequivocally cholinergic in the human genitourinary organs, we have used a specific marker, namely, an antibody to vesicular acetylcholine transporter (VAChT), to immunolabel cholinergic axons and cell bodies in specimens of urinary bladder, seminal vesicle, vas deferens, and prostate gland obtained from neonates and children post mortem. In addition some sections were double‐immunolabeled with VAChT and either neuropeptide Y (NPY) or nitric oxide synthase (NOS). The results demonstrated a rich cholinergic innervation to the muscle coat of the bladder body with a much less prominent, but nonetheless significant, cholinergic innervation to the smooth muscle components of the seminal vesicle, vas deferens, and prostate. Small ganglia were scattered throughout the detrusor muscle of the urinary bladder, approximately 75% of the intramural neurons being VAChT immunoreactive, whereas approximately 95% contained NPY and approximately 40% contained NOS. VAChT immunoreactivity was observed in 40% of neurons in ganglia scattered throughout the pelvic plexus. Almost all these cholinergic neurons contained NPY and approximately 65% contained NOS. Almost all the cholinergic nerve fibers throughout the genitourinary organs also contained NPY. Although NOS was sparse in the cholinergic nerves of the bladder body, it occurred in the majority of cholinergic nerves at the bladder neck and was also present in a proportion of the cholinergic nerves in the other organs examined. VAChT‐immunoreactive nerves were also observed in a sub‐epithelial location in all the organs examined, the majority containing NPY, whereas a small proportion contained NOS. Although doubt remains about the function of sub‐epithelial cholinergic nerves in the urinary bladder, the majority of similar nerves in the seminal vesicle, vas deferens, and prostate gland are considered to be secretomotor. Collectively these findings demonstrate that the cholinergic innervation of the male genitourinary system is well established in the neonate and child. Neurourol. Urodynam. 19:185–194, 2000.

NITRIC OXIDE SYNTHASE AND TYROSINE HYDROXYLASE ARE COLOCALIZED IN NERVES SUPPLYING THE POSTNATAL HUMAN MALE GENITOURINARY ORGANS

PURPOSE The objective of this study was to examine the distribution of nitric oxide synthase (NOS) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in nerve fibers supplying the human neonatal male genitourinary organs. MATERIALS AND METHODS An indirect double label immunofluorescence technique was employed on specimens obtained from infants and children at postmortem examination. RESULTS Many nerve fibers immunoreactive for both NOS and TH were observed in the muscle coat of the vas deferens and the seminal vesicle, within the fibromuscular stroma of the prostate gland and at the bladder neck, and also formed perivascular plexuses in each of these organs. Double-labeled nerves occurred less frequently in the intramural ureters and superficial trigone while similar nerves in the bladder body were relatively sparse. Numerous nerves immunoreactive for NOS but not TH were observed at the base of the epithelium of each organ examined. Four types of autonomic ganglion cell were observed in nearby pelvic ganglia: those which contained NOS and TH, those which contained NOS alone, those which contained TH alone and those which contained neither NOS nor TH. CONCLUSION The results indicate that many of the noradrenergic nerves as well as non-noradrenergic nerves supplying the male genitourinary organs have the capacity to synthesize nitric oxide (NO) and that NO may play a significant role in the autonomic control of both the urinary and genital organs in the postnatal human male.

The Distribution of Noradrenergic Nerves in the Human Lower Urinary Tract

The purpose of this presentation is to describe the distribution of noradrenergic nerves in the human genitourinary system. The techniques which have been employed include formaldehyde-induced fluorescence and immunocytochemical methods to demonstrate dopamine β-hydroxylase and tyrosine hydroxylase. These methods have been applied to human fetal, neonatal, infant, child and adult tissues removed either at post mortem examination or by surgical excision. The innervation of the fetal urinary bladder is well established by 13 weeks and, as in older specimens, the detrusor receives a sparse noradrenergic nerve supply. In contrast the smooth muscle of the terminal ureter is well supplied by this type of autonomic nerve. An additional incomplete muscle layer has been identified as a nomal component of the terminal ureter which is richly innervated by noradrenergic nerves. In some cases this muscle forms a complete collar which may be responsible for ureteric obstruction. By comparison with the detrusor, bladder neck smooth muscle receives a dense noradrenergic nerve supply particularly in the male. Unlike the detrusor, the structure and innervation of the vas deferens, seminal vesicle and prostate are poorly differentiated in the fetus. In the infant and child, the structure of the intramural smooth muscle of these organs remains immature although a rich noradrenergic nerve supply resembing the adult has been established in the fetus by 30 weeks. In the fetus, autonomic ganglia occur in association with noradrenaline rich paraganglia and surprisingly, with sensory nerve endings resembling pacinian corpuscles. Shortly after birth paraganglia are no longer associated with the autonomic ganglia of the genitourinary system. On the basis of size at least two types of autonomic neuron populate these autonomic ganglia. One type is relatively large and devoid of catecholamines but is closely associated with pericellular noradrenergic nerve fibres. The second type of neuron is small, contains noradrenaline and is arranged in clusters closely related to the capsule of the prostate gland. The significance of these observations will be considered with respect to the neurological control of the genitourinary system.

A double-label immunohistochemical study of intramural ganglia from the human male urinary bladder neck

Double‐label immunocytochemistry was used to investigate the colocalisation of various neuropeptides and the enzymes nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) in intramural ganglia of the human male urinary bladder neck and trigone. Postmortem specimens were obtained from 7 male infants and children ranging in age from 2 mo to 3 y who had died as a result of cot death or accidental trauma. On average 60% of the intramural neurons were non‐TH‐immunoreactive (‐IR) (i.e. presumptive cholinergic) and 40% were TH‐ and DbβH‐IR (i.e. noradrenergic). Within the non‐TH‐IR population, calcitonin gene‐related peptide (CGRP) was found in 65% of cells, neuropeptide Y (NPY) in 90%, nitric oxide synthase (NOS) in 45%, somatostatin (SOM) in 90%, and vasoactive intestinal polypeptide (VIP) in 40%. The corresponding values for the TH‐IR neurons were CGRP (54%), NPY (70%), NOS (58%), SOM (73%) and VIP (40%). All the observed bombesin (BOM)‐immunoreactivity was colocalised with TH while 90% of VIP and almost all the CGRP was colocalised with NPY. Less than 5% of neurons were immunoreactive for substance P (SP) or met‐enkephalin (m‐ENK) and some of these also contained TH. Varicose nerve fibres were seen in close proximity to some of the intramural neurons, the majority of such varicosities showing immunoreactivity to CGRP, VIP or TH. Less common were pericellular varicosities immunoreactive to NPY, SOM or SP. These results demonstrate the neurochemical heterogeneity of intramural neurons in the human bladder neck and provide indirect evidence for the complexity of the peripheral innervation of the human urinary bladder.

A Quantitative Histological Analysis of the Dilated Ureter of Childhood

A quantitative histological study of the dilated ureter of childhood was performed on 26 ureters. The specimens were from 15 male and 11 female patients 10 days to 12 years old (mean age 2.0 years). A color image analysis system was used to examine and compare collagen and smooth muscle components of the muscularis layers to normal control ureters of similar age. In comparing primary obstructed (12) to primary refluxing (14) megaureters and control ureters (6), there was a statistically different collagen-to-smooth muscle ratio (p < 0.001) between the primary obstructed and primary refluxing megaureter groups. For patients with primary refluxing megaureter there was a 2-fold increase in the tissue matrix ratio of collagen-to-smooth muscle when compared to patients with primary obstructed megaureter. In the primary obstructed megaureters the amount of collagen and smooth muscle was not statistically different from controls (p > 0.01). The increased tissue matrix ratio of 2.0 +/- 0.35 (collagen-to-smooth muscle) in the refluxing megaureter group compared to 0.78 +/- 0.22 in the obstructed megaureter group and 0.52 +/- 0.12 in controls was found to be due not only to a marked increase in collagen but also a significant decrease in the smooth muscle component of the tissue. Primary obstructed and normal control ureters had similar quantitative amounts of smooth muscle with 60 +/- 5% and 61 +/- 6%, respectively, while refluxing megaureters had only 40 +/- 5% smooth muscle. The percentage collagen was 36 +/- 5 in the obstructed megaureter group and 30 +/- 5 in controls, with refluxing megaureters having 58 +/- 5% collagen on analysis. Our findings emphasize the significant differences in the structural components (collagen and smooth muscle) of the dilated ureter of childhood, and provide us with further insight into the pathological nature of these dilated ureters and their surgical repair.

Obstructive bladder dysfunction: Morphological, biochemical and molecular changes

Abstract Obstruction can cause changes in bladder structure and function, which may at a certain point in time become irreversible. Both the rabbit and the rat have proven to be excellent models to study the morphological, biochemical and molecular changes that occur in the bladder following obstruction. Similarities between partial outflow obstruction in animals and obstructive dysfunction in man include increased bladder mass, increased fibrosis, reduced compliance, increased incidence of detrusor instability and decreased contractile ability. Obstructed bladder function can remain relatively normal for prolonged periods of time, even though bladder mass is increased (compensated stage). If the obstruction is not relieved, bladder function destabilizes and then decompensates, with subsequent risk of serious complications. It is hypothesized that the shift from the compensated to the decompensated stage is related to cyclical periods of ischaemia followed by reperfusion (I/R). This initiates degenerative membrane effects, which supports the process of bladder decompensation. It seems that relief of obstruction during the compensated stage, at least in animals, can induce a rapid and full restoration of the bladder function. However, if the obstruction is relieved during the decompensated phase, bladder function only partially recovers. Irreversible bladder decompensation may be prevented by reducing increased bladder mass and/or by reducing ischaemia/increasing blood supply to the bladder. The antioxidant Vitamin E seems to reduce the progression of decompensation in rabbits. Treatment of rats with the α 1 -adrenoceptor antagonist doxazosin prior to partial outlet obstruction increases blood flow to the bladder, significantly decreases the effect of obstruction on bladder weight, and significantly protects the contractile function of the obstructed bladder. Pre-treatment of rabbits with the α 1 -adrenoceptor antagonist tamsulosin partly prevents the development of bladder wall hypertrophy due to obstruction. In conclusion, there is evidence that I/R plays an important role in the pathogenesis of obstructive bladder dysfunction. Therefore, I/R should be prevented or relieved. As I/R is the consequence of increased bladder mass following obstruction, therapies that prevent or reduce increased bladder mass (such as the α 1 -adrenoceptor antagonist tamsulosin) are likely to protect the bladder from (further) dysfunction. Therapies based on both the relief of I/R (increasing blood flow to the bladder, e.g. α 1 -adrenoceptor antagonists) and preventing I/R induced cellular damage (e.g. antioxidant activity) have been shown experimentally to significantly reduce the severity of bladder dysfunction secondary to partial outflow obstruction. Of course, these potentially useful and novel mechanisms of action of therapy should also be investigated in humans.

Co-localisation of tyrosine hydroxylase, nitric oxide synthase and neuropeptides in neurons of the human postnatal male pelvic ganglia

Double-label immunocytochemistry was used to investigate the co-localisation of neuropeptides and the enzyme nitric oxide synthase (NOS) with tyrosine hydroxylase (TH) in autonomic ganglia of the human postnatal male pelvic plexus. Postmortem specimens were obtained from six male infants and children ranging in age from 2 to 12 months who had died as a result of cot death or accidental trauma. On average, ganglia lying adjacent to the neck of the urinary bladder contained 45% of neurons which were TH-immunoreactive (-IR) while ganglia situated adjacent to the posterior and lateral aspects of the prostate gland contained 67% of neurons which were TH-IR. All the TH-IR neurons also contained dopamine beta-hydroxylase and were considered to be noradrenergic in type. On average, 61% of TH-IR neurons in bladder ganglia contained NOS, compared with 77% of non-TH-IR neurons (based on counts of over 1,000 cells in each case), while the percentages of TH- and non-TH-IR neurons containing neuropeptides were: calcitonin gene-related peptide (CGRP) (30%; 11%), neuropeptide Y (NPY) (66%; 92%), somatostatin (SOM) (70%; 29%), substance P (SP) (64%; 46%), vasoactive intestinal polypeptide (VIP) (64%; 83%). The equivalent values for TH- and non-TH-IR neurons in prostatic ganglia were NOS (38%; 59%), CGRP (55%; 18%), NPY (62%, 65%), SOM (14%, 20%), SP (13%, 8%) and VIP (42%; 82%). Varicose nerve fibers within the ganglia were seen forming pericellular arborizations around many of the ganglion cells, the most numerous containing TH-, CGRP-, NPY-, SOM- or VIP-immunoreactivity. Less common were pericellular varicosities containing SP-immunoreactivity while terminals containing NOS were not observed. No correlation could be detected between the peptide contents of the ganglion cells and of the associated pericellular terminals. However, the peptide content of the ganglion cells found in association with the urinary bladder and prostate gland correlates well with the previously documented coexistence of enzymes and neuropeptides in the intrinsic nerve fibers supplying these two regions of the human postnatal male genitourinary system.