Morphology and histomorphometry of the bladder and urethra in ovariectomized rats after long-term use of tibolone.

BACKGROUND: This study aimed to evaluate the effect of long-term high-dose tibolone on the bladders and urethras of ovariectomized rats. METHODS: Bilateral ovariectomy was performed in 14 young adult rats randomly divided into 2 groups. Experimental rats (n = 9) received 1 mg/day of tibolone orally; control rats (n = 6) received a placebo. After 150 days, the bladders and urethras were removed. Bladder cell proliferation was analyzed by Ki-67 immunohistochemistry. A histomorphometric analysis was performed for epithelial thickness and the percent areas of collagen fibers and blood vessels. Data were compared using a Mann-Whitney test (significance level at p < 0.05). RESULTS: Urothelial thickness and the percent area of collagen fibers and blood vessels were not significantly different between the tibolone and control groups in the bladder and urethra. In addition, urothelium cell proliferation in the bladder showed a low immunopositivity in both groups. Furthermore, the glycogen and glycoprotein contents in urethral epithelium were slightly modified by tibolone and no change was observed in the bladder. CONCLUSION: Long-term administration of tibolone has no effect on urothelial trophism, collagen fibers, the number of vessels, or cell proliferation in the urethra and bladder of the ovariectomized rat.

Urethral sensation: basic mechanisms and clinical expressions.

A prerequisite for conscious bladder control is adequate sensory input to the central nervous system, and it is well established that changes in sensory mechanisms can give rise to disturbances in bladder function. Impulses related to the desire to void are believed to course through the pelvic nerves, and those for sensation of a full bladder course through the pudendal nerves. The sense of imminent micturition most probably resides in the urethra, and the desire to void comes from stretching the bladder wall. In addition, a variety of structures play an important role in terms of urethral closure (such as the urethral epithelium, vasculature and smooth muscle) that are necessary to maintain continence. This overview will discuss mechanisms related in part to the urethra involved in activation of bladder reflexes and sensation with a discussion on the mucosa (urothelium and underlying lamina propria) and underlying cellular structures.

3-Dimensional morphometric analysis of murine bladder development and dysmorphogenesis.

BACKGROUND: Disorders of the urinary tract represent a major cause of morbidity and impaired quality of life. To better understand the morphological events responsible for normal urinary tract development, we performed 3-D reconstructive analysis of developing mouse bladders in control, mgb-/-, and Fgfr2(Mes-/-) mice. RESULTS: Detrusor smooth muscle differentiation initiated in the bladder dome and progressed caudally with the leading edge extending down the right posterior surface of the bladder. Gender-specific differences in detrusor smooth muscle development were observed during early embryonic development. Bladder trigone morphology transitioned from an isosceles to equilateral triangle during development due to the preferential lengthening of the urethra to ureter distance. The primary defect observed in mgb-/- bladders was a significant reduction in detrusor smooth muscle differentiation throughout development. Deviations from normal trigone morphology correlated best with VUR development in Fgfr2(Mes-/-) mice, while alterations in intravesicular tunnel length did not. CONCLUSIONS: Multivariate morphometric analysis provides a powerful tool to quantify and assess urinary tract development.

Exogenous testosterone and estrogen affect bladder tissue contractility and histomorphology differently in rat ovariectomy model.

INTRODUCTION: Changes in sex hormone levels may play a role in the etiology of lower urinary tract dysfunction of aging women where the possible role of testosterone is overlooked. AIM: To determine the effect of testosterone with/without estrogen replacement on histological and functional deterioration in ovariectomized rat bladder tissue. METHODS: A total of 54 female Sprague Dawley rats were divided into 6 groups. Except sham operated (control group), all others underwent bilateral ovariectomy. No further treatment was given to the ovariectomy-only group (OVX group). At the third week of ovariectomy treatments were started; vehicle agent (VA group), estradiol (E2 group), testosterone undecanoate (T group), and estradiol + testosterone undecanoate combination (E2 + T group) in physiological doses. Nine weeks after ovariectomy, bladder strips were harvested for isometric tension and histopathological studies. MAIN OUTCOME MEASURES: To assess the effect of testosterone/estradiol on ovariectomized rat bladder tissue function and histomorphology. RESULTS: OVX and VA groups showed statistically significant histological changes such as urothelial damage, inflammatory cell infiltration, increase in collagen fibers and muscular atrophy compared with the control group. Both E2 and T reversed these changes but best histomorphological restoration was observed in E2 + T group. In isometric tension studies, ovariectomy tended to increase contractile responses which were normalized after E2 treatment. In contrary to E2, T significantly increased contractile responses that were normalized with combination treatment. During relaxation studies statistically significant higher relaxation responses were observed in ovariectomized rats. Although both exogenous testosterone and estradiol tended to reverse this effect, a statistically significant difference was found only after testosterone treatment. CONCLUSION: Either estradiol or testosterone replacement alone or in combination prevents significant alterations in bladder tissue histology following ovariectomy whereas both affect the bladder tissue contractility. Thus, combination treatment appears to be the best method to restore both contractility and histomorphology of bladder tissue after ovariectomy.

Constructive remodeling of biologic scaffolds is dependent on early exposure to physiologic bladder filling in a canine partial cystectomy model.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate the constructive remodeling of several tissue types. Previous studies suggest that the ECM scaffold remodeling process is dependent on microenvironmental factors, including tissue-specific biomechanical loading. The objective of the present study was to evaluate the effects of long-term catheterization (LTC), with its associated inhibition of bladder filling and physiologic biomechanical loading, on ECM scaffold remodeling following partial cystectomy in a canine model. Reconstruction of the partial cystectomy site was performed using ECM scaffolds prepared from porcine small intestinal submucosa (SIS) or porcine urinary bladder matrix (UBM). Animals were randomly assigned to either a long-term catheterization (LTC) group (n=5, catheterized 28 d) or a short-term catheterization group (STC, n=5, catheterized 24 h), and scaffold remodeling was assessed by histologic methods at 4 and 12 wk postoperatively. By 4 wk, animals in the STC group showed a well-developed and highly differentiated urothelium, a robust vascularization network, abundant smooth muscle actin (SMA), and smooth muscle myosin heavy chain (smMHC) expressing spindle-shaped cells, and many neuronal processes associated with newly formed arterioles. In contrast, at 4 wk the scaffolds in LTC animals were not epithelialized, and did not express neuronal markers. The scaffolds in the LTC group developed a dense granulation tissue containing SMA+, smMHC-, spindle-shaped cells that were morphologically and phenotypically consistent with myofibroblasts, but not smooth muscle cells. By 12 wk postoperatively, the ECM scaffolds in the STC animals showed a constructive remodeling response, with a differentiated urothelium and islands of smooth muscle cells within the remodeled scaffold. In contrast, at 12 wk the scaffolds in LTC animals had a remodeling response more consistent with fibrosis even though catheters had been removed 8 wk earlier. These findings show that early exposure of site-appropriate mechanical loading (i.e., bladder filling) mediates a constructive remodeling response after ECM repair in a canine partial cystectomy model.

Antioxidant levels in the pig urinary bladder: distribution within the bladder wall and in the urothelium derived from different bladder regions.

This study was designed to determine the antioxidant levels in the urinary bladder wall layers as well as urothelium derived from different bladder regions. Samples of the urothelium, lamina propria, muscularis, and serosa were prepared from the pig's urinary bladder body, while samples used for regional mapping of the urothelium were prepared from trigone, ventral and dorsal middle bladder body, and apex region. Activities of superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase were determined. Concentrations of ascorbic acid and glutathione were also measured. Antioxidant activities, i.e. concentrations of superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione, were shown to be highest in the urothelium and progressively lower towards the serosa. Regional mapping of the urothelium singled out apex as the region with the lowest antioxidant activities, i.e. concentrations of glutathione peroxidase, ascorbic acid, and glutathione. The fact that antioxidants are concentrated in the urothelium implies that urothelium functions as a barrier against reactive species. The urothelium derived from the apex is the region with the lowest antioxidant levels and is therefore probably the region most liable to development of oxidative damage.

Protein networking in bladder cancer: immunoreactivity for FGFR3, EGFR, ERBB2, KAI1, PTEN, and RAS in normal and malignant urothelium.

A panel of markers, selected for the suspected bladder cancer relevance of their corresponding genes, were explored for their expression and subcellular location in urinary bladder tissue. The expression in normal urothelium, in non-metastasised transitional cell carcinomas (TCC), and in primary metastasised TCC with corresponding metastases was mapped. Potential associations between the proteins were identified. The observations were then combined in a set of hypotheses aimed at further hypothesis testing. Membranous ERBB4 and cytoplasmic p21RAS were downregulated in carcinoma cells compared with normal urothelium cells. FGFR3 was translocated from the cytoplasm to the nucleus. ERBB2 was translocated to the membrane and seemingly upregulated in one subgroup and conversely downregulated in another. EGFR, KAI1 and possibly PTEN revealed increased membranous immunoreactivity in non-metastasised tumours. The metastases showed decreased nuclear FGFR3 and membranous PTEN staining compared with corresponding primary tumours. EGFR expression was positively correlated with the expression of PTEN and FGFR3. The expression of ERBB2 was negatively correlated with p21RAS expression. According to our results, bladder carcinogenesis comprises FGFR3 translocation to the nucleus, upregulation of EGFR, ERBB2, KAI1 and PTEN; downregulation of p21RAS; and translocation of EGFR, ERBB2, and possibly PTEN to the membrane. Our results support the hypotheses regarding PTEN and KAI1 functioning as tumour suppressors in bladder cancer. EGFR and KAI1 may discriminate between non-metastasised and metastasised cancers. A complex network of associations between the factors is suggested.

Carcinoma urotelial infiltrante vesical, variedad "en nidos". Presentación de un caso / The nested variant of infiltrating urothelial bladder carcinoma. Case report

La variante en nidos del carcinoma urotelial infiltrante vesical es un tumor descripto en forma relativamente reciente. En esta comunicación se presenta un caso de esta entidad, un paciente de sexo masculino de 73 años de edad con compromiso de la región perimeatal izquierda. Histológicamente, las células neoplásicas se disponían en nidos poco definidos y tenían características nucleares de bajo grado. El tumor tenía un patrón de crecimiento infiltrativo. Se realizó cistectomía parcial pero el tumor recurrió tres meses después

The urothelium in overactive bladder: passive bystander or active participant?

The urothelium was once purported to be a passive membrane. However, a growing body of evidence indicates that the urothelium actively participates in sensory functions, expressing various receptors and ion channels, as well as releasing neurotransmitters in response to stimuli. Vanilloid receptors, P2X3 purinergic receptors, adenosine triphosphate, nitric oxide, and acetylcholine have been implicated in urothelial-neuronal interactions. Substances released from urothelial cells can alter the excitability of bladder afferent nerves acutely and chronically; these observations appear to be of particular importance in chronic bladder conditions and in the aging bladder. Evidence suggests that the involvement of the muscarinic receptor in bladder function extends beyond detrusor contractility and into afferent sensory functioning. These observations have significant implications for a more complete understanding of the effects of currently used drugs on these sensory mechanisms and for identifying potential targets for pharmacologic intervention in bladder disorders.

Profiles of the 2 INK4a gene products, p16 and p14ARF, in human reference urothelium and bladder carcinomas, according to pRb and p53 protein status.

The INK4a/ARF locus encodes 2 cell cycle regulatory proteins: p16 and p14(ARF). P16 inhibits the activities of cdks, which maintain the retinoblastoma protein (pRb) in its active hypophosphorylated state. P14(ARF) blocks MDM2-induced p53 degradation and transactivational silencing. In this study, we investigated the expression of p16 and p14(ARF) in reference human urothelium and in 51 urothelial carcinomas (UCs) of all stages and grades, by reverse transcription-polymerase chain reaction (RT-PCR). Patterns of p14(ARF) and p16 expression were compared with each other and then with patterns of p53 and pRb protein expression, respectively, as determined by immunohistochemistry. P14(ARF) and p16 mRNAs were present at low levels or were undetectable in reference urothelia and in most superficial tumors, whereas they were present at high levels in a subset of tumors of advanced stage and high grade. The expression profiles of these 2 mRNAs were correlated in all but 4 cases, indicating that the 2 INK4a products may have nonredundant functions. Forty-six of the 51 tumors (90%) presented changes to or a lack of activation of the p14(ARF)-p53 pathway and were p53 positive (n = 10), p14(ARF) negative (n = 23), or both p53 positive and p14(ARF) negative (n = 13), suggesting that these 2 components of the pathway may be altered or nonactivated. Markedly high levels of p16 mRNA (n = 5) were associated with the absence of pRb expression, with the exception of 1 case in which the p16 gene contained a deletion mutation. A lack of p16 mRNA or low levels of this mRNA were associated with pRb detection in all but 1 case. In invasive UCs, the p16-pRb pathway, the p14(ARF)-p53 pathway, or in many cases both pathways were altered or not activated, demonstrating the involvement of these pathways in invasive bladder tumorigenesis.

[The relationship between uretero-cloacal structure in birds and sigmoidorectal pouch surgery]. / Verwandtschaft zwischen der ureterokloakalen Struktur bei Vögeln und der sigmoidorektalen Pouch-Chirurgie.

PURPOSE: The present study was planned to investigate morphological, functional and histological properties of the natural ureterocloacal for in birds as well as to provide an appropriate information which may contribute to rectosigmoidal pouch surgery and to decrease its complications. MATERIALS AND METHODS: Thirty chickens (Gallus domesticus, 26 broilers, 4 layers) with a mean weight of 1870 +/- 88 g were used for this study. Histological and morphological evaluation of the cloaca was performed. Urethral reflux was estimated using X-rays. RESULTS: We found that the cloaca had three parts from cranial to caudal: coprodeum, urodeum and proctodeum respectively. A coprourodeal fold (CUF) between the coprodeum and urodeum and a uroproctodeal fold between the urodeum and proctodeum were detected. Settling of the muscle clusters both in the ureter and urodeum near the opening area appeared to be a strong sphincter structure and the ureters are opened to a structure covered by a transitional epithelium, which is similar to bladder in humans (connecting two identical epithelia). According to X-rays following the administration of a radio-opaque substance, there was no ureteral reflux in any of them. CONCLUSIONS: The natural urointestinal structure in birds has a spontaneous ureterointestinal anti-reflux opening. The coprourodeal fold that prevents the mixing of faeces and urine, inhibits the effects of increased pressure and prevents the reflux of urine to the upper segments is provided with many of desired hinders properties for urointestinal diversion.

Validación histopatológica de la tecnología tissue-microarray de cáncer de urotelio. Nuestra experiencia / Histopatological validation of tissue-microarray technology in uroletial cancer. Our experience

INTRODUCCIÓN: La tecnología array ofrece: gran ventaja a los investigadores clínicos y básicos, facilita aplicar gran cantidad de técnicas (inmunohistoquímica, FISH, proteómica) para comprender los mecanismos moleculares del cáncer, ofrece economía de escala en los reactivos versus los procedimientos convencionales. Dado que la representación de la muestra es muy reducida, es exigible previamente validar el array. MATERIAL Y MÉTODOS: A partir de bloques de parafina de carcinomas de urotelio almacenados, cuya antigüedad oscilaba entre 5-7 años, se han seleccionado 52 casos consecutivos; se ha construido un array de tejido; los discos se colocaron en filas y columnas de manera aletoria, dibujando un topograma para guía de lectura. Se validó por otro patólogo ajeno a la selección de las muestras. RESULTADOS: Se han obtenido 87 laminillas. La número 1 se ha teñido con HE. Ha habido discrepancia en el 27% de las muestras en el estadiaje. No ha existido discrepancia en el diagnóstico histológico. En 11 puntos (17%) no hay representación de la muestra. DISCUSIÓN: Nuestros resultados ofrecen unos buenos resultados en la validación de las muestras. La antigenicidad del tejido está conservada. Las muestras seleccionadas en el array representan alrededor del 97%, similar a todo el conjunto de las secciones convencionales de la muestra problema (AU)

INTRODUCTION: The array technology offers: a big advance to clinic and basic investigator, it provides a variety of technics (immunohystochemistry, FISH, proteomics) to undestand the molecular mechanisms of cancer. It offers scale economy in reagents versus the conventional methods. Array most be ratified because the sample is so reduced. MATERIAL AND METHODS: 52 consecutive cases have been cloosen from paraffin blocks of bladder and ureteral cancer which are 5-7 years old, a tissue array has been made; disks have been arranged in lines and columns, in an aleatory way, in order to guide it’s reading. It has been evaluated by a pathologist with any relation to specimen selection. RESULTS: 87 sheets ha been obtained. Number 1 has been dyed with HE. Has been discrepancy in 27% of sample’s stage. Has not been a discrepancy in hystopathologic diagnostic. There is no sample’s representation in 11 points (17%). DISCUSSION: Our results offer good results in sample’s validation. The sample’s antigenicity of tissue is conserved. Array sample’s represent a 97%, similary to all unit of conventional sections of the specimen (AU)

Identification of bladder wall layers by Raman spectroscopy.

PURPOSE: We explored the applicability of Raman spectroscopy to in situ investigation of bladder wall tissue. MATERIALS AND METHODS: Bladder wall tissue was obtained from a guinea pig model and frozen sections were used for Raman spectroscopic investigations. From each section 500 to 700 spectra were obtained in a 2-dimensional grid spanning the urothelium, lamina propria and muscle layer. The data set of spectra was subdivided into groups of similar spectra by a cluster analysis algorithm. With each group assigned a different color Raman maps of frozen sections were constructed based on group membership of measured spectra. These maps were then compared with histological and histochemical data obtained from hematoxylin and eosin and immunohistochemical staining for collagen I and III and for smooth muscle actin to correlate Raman spectral features with bladder wall structure and molecular composition. RESULTS: Urothelium, lamina propria and muscle layers could be clearly distinguished based on Raman spectra. Lamina propria spectra were dominated by signal contributions of collagen and the smooth muscle layer showed strong signal contributions of actin. The urothelium had a relatively strong lipid signal contribution. CONCLUSIONS: These results and the fact that Raman spectroscopy is rapidly evolving into a technology that can be applied in vivo by thin, flexible fiberoptic catheters indicate that prospects are good for in vivo analysis of the molecular composition of the normal and pathological bladder without biopsies.

An objective morphologic parameter to aid in the diagnosis of flat urothelial carcinoma in situ.

The diagnosis of carcinoma in situ (CIS) lacks objective criteria and is subject to misdiagnosis. We identified 20 bladder biopsy cases each of CIS, urothelial dysplasia, and normal urothelium according to the 1998 World Health Organization/International Society of Urological Pathology consensus classification of urothelial neoplasms. Lymphocytes from 10 bladder biopsy specimens were chosen as reference cells. Using an image analysis system, we measured the following nuclear features: area, diameter, roundness, ellipticity, and optical density (maximum, minimum, mean, median, standard deviation, and quartiles). We measured a mean of 75 urothelial nuclei/case and a total of 500 lymphocytes. Roundness and ellipticity were not useful in distinguishing among the 3 groups. The best discriminators were mean nuclear area and mean nuclear area of the 25% largest nuclei (upper quartile) of urothelial cells compared with lymphocytes. The mean nuclear area relative to lymphocytes was 1.8 times (1.2 to 2.5 times) in normal urothelium, 2.4 times (1.6 to 3.0 times) in urothelial dysplasia, and 3.6 times (2.8 to 5.7 times) in CIS. The mean upper quartile nuclear area relative to lymphocytes was 2.2 times (1.4 to 2.8 times) in normal urothelium (P <.0001), 2.9 times (1.8 to 3.6 times) in urothelial dysplasia (P <.0001), and 4.9 times (4.0 to 7.6 times) in CIS (P <.0001). The difference in optical density was statistically significant between CIS and the other 2 histologic categories (P <.0001). Nuclear area is an easy and objective morphologic parameter for the evaluation of bladder biopsy specimens. Pathologists can assess the size of urothelial nuclei without using an image analysis system and compare them with the size of nuclei of lymphocytes, which are almost always present in a bladder biopsy specimen. Dysplasia, which is a somewhat ambiguous lesion, overlaps in its measurements with those of benign urothelium. The most useful morphologic parameter is the mean nuclear area of the 25% largest nuclei; CIS nuclei are approximately 5 times the size of lymphocytes, whereas normal urothelial nuclei are only 2 times the size of lymphocytes.

Discriminatory immunohistochemical staining of urothelial carcinoma in situ and non-neoplastic urothelium: an analysis of cytokeratin 20, p53, and CD44 antigens.

Distinction of urothelial carcinoma in situ (CIS) from reactive atypia on the basis of morphology alone may be difficult in some cases. Because this distinction is therapeutically and prognostically critical, we attempted to determine if an immunohistochemical panel would help in this differential diagnosis. The immunoprofile of 21 cases of CIS and 25 non-neoplastic urothelia (15 urothelial biopsies with reactive atypia from patients without a history of bladder cancer and 10 normal ureter sections from nephrectomies performed for renal cell carcinoma) was determined using antibodies against cytokeratin 20 (CK20), p53, and CD44 (standard isoform). In the normal urothelium CK20 showed patchy cytoplasmic immunoreactivity in only the superficial umbrella cell layer and CD44 stained only the basal cells. Nuclear immunoreactivity to p53 varied from negative to weak and patchy. Reactive urothelium also showed CK20 immunoreactivity in only the umbrella cell layer in all 15 cases, and p53 nuclear staining was predominantly negative with occasional weak positivity in the basal and parabasal intermediate cells. CD44 was overexpressed in the entire reactive urothelium in 9 cases (60%) or focally positive in intermediate cells in 6 cases (40%). In contrast, CIS showed intense CK20 and p53 positivity (81% and 57%, respectively) in the majority (>50%) of malignant cells. CD44 staining revealed residual basal cells with membranous reactivity in 44% of the cases of CIS; however, the neoplastic cells were immunonegative in all cases. At least one positive immunomarker (CK20 or p53) was abnormally expressed in all cases of CIS. Abnormal expression of CK20 (increased), p53 (increased), and CD44 (decreased) in urothelial CIS, and increased expression of CD44 in reactive atypia allows more confident distinction of urothelial CIS from non-neoplastic urothelial atypias. From a differential diagnosis perspective, use of a panel of all three antibodies with morphologic correlation would be essential.

Everything you wanted to know about the bladder epithelium but were afraid to ask.

The mammalian urinary bladder epithelium (urothelium) performs the important function of storing urine for extended periods, while maintaining the urine composition similar to that delivered by the kidneys. The urothelium possesses four properties to perform this function. First, it offers a minimum epithelial surface area-to-urine volume; this reduces the surface area for passive movement of substances between lumen and blood. Second, the passive permeability of the apical membrane and tight junctions is very low to electrolytes and nonelectrolytes. Third, the urothelium has a hormonally regulated sodium absorptive system; thus passive movement of sodium from blood to urine is countered by active sodium reabsorption. Last, the permeability properties of the apical membrane and tight junctions of the urothelium are not altered by most substances found in the urine or blood. The importance of the barrier function of the urothelium is illustrated by infectious cystitis. The loss of the barrier function results in the movement of urinary constituents into the lamina propria and underlying muscle layers, resulting in suprapubic and lower back pain and frequent, urgent, and painful voiding.

The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee.

This WHO/ISUP system is an attempt to develop as broad a consensus as possible in the classification of urothelial neoplasms, building upon earlier works and classification systems. It is meant to serve as a springboard for future studies that will help refine this classification, thus enabling us to provide better correlation of these lesions with their biologic behavior using uniform terminology.