Catherine Whitbeck

Functional changes in bladder tissue from type III collagen-deficient mice

Objective: Collagen fibers impart tensile strength and transfer tension from bladder smooth muscle cells. We have previously shown that fibrotic bladders are characterized by an increased type III:type I collagen ratio. To determine the effect of decreased type III collagen on bladder function, type III collagen-deficient mice (COL3A1) were studied physiologically.Methods: Bladders from wild-type (+/+) and heterozygous (+/−) COL3A1 mice were biochemically characterized to determine total collagen (hydroxyproline analysis) and collagen subtype concentration (cyanogen bromide digestion and ELISA). Alterations in collagen fiber diameter were assessed by electron microscopy. Bladder muscle strips were used to assess physiologic function. Results: Hydroxyproline content decreased in heterozygous bladders, which had 50% less type III collagen. Wild-type bladders had a biphasic distribution of collagen fiber sizes, whereas heterozygous bladder collagen fibers spanned a broad range. Physiologically, there were no differences in contractile responses between wild-type and heterozygotes when stimulated with ATP, carbachol or KCl, indicating normal contraction via purinergic and muscarinic receptors, and in response to direct membrane depolarization. In contrast, tension generation in heterozygotes was decreased after field stimulation (FS), indicating decreased synaptic transmission. Length–tension studies showed that the heterozygote muscle strips generated less tension per unit length, indicating that they were more compliant than wild-type controls. Conclusions: Critical levels of type III collagen appear to be a requirement for normal bladder tension development and contraction. Our data show that a decrease in the type III:type I collagen ratio, and altered fiber size, results in a more compliant bladder with altered neurotransmitter function.

The effects of cyclical oestrogen on bladder and urethral structure and function

To determine the effects of cycling oestrogen in rabbits, as oestrogen is essential for physiological maintenance and integrity of the female urogenital tract.

Effects of in vivo ischemia on contractile responses of rabbit bladder to field stimulation, carbachol, ATP and KCl.

Rabbits were subjected to bilateral ischemia for 3, 6, or 18 h then euthanized and their bladders excised. Isolated strips of rabbit bladder detrusor were incubated in normal Tyrode’s solution and contractile responses to FS, carbachol, ATP, and KCl measured. Maximal contraction, maximal rate of tension generation, and length of time to maximal contraction were determined. These studies revealed that contractile responses to FS (neurogenic stimulation) were most affected by ischemia. Contractile responses to carbachol, ATP and KCl were all similarly sensitive to ischemia.

Effect of Oral Kohki Tea on Bladder Dysfunction Induced by Severe Partial Outlet Obstruction

PURPOSE Extracts of the leaves of Engelhardtia chrysolepis, a subtropical plant that grows wild in southern China, have been used medicinally in east Asia for hundreds of years. A standard extract named Kohki tea (Maruzen Pharmaceuticals, Onomichi City, Japan) is sold over the counter in Japan as a sweet tea shown to confer many beneficial effects on general health and well-being. The tea contains strong antioxidants, including several dihydroflavonol glycosides. The results of previous studies show that natural products with antioxidant activities provide protective effects on the bladder of rabbits with partial outlet obstruction. We determined in vivo and in vitro whether oral pretreatment of rabbits with Kohki tea protects the bladder from dysfunction induced by partial outlet obstruction. MATERIALS AND METHODS A total of 28 New Zealand White rabbits were separated into 4 groups of 7 each. Rabbits in groups 1 and 2 were treated by gavage with 100 mg./kg. Kohki tea daily in distilled water, while those in groups 3 and 4 were given distilled water. After 4 weeks of daily oral administration each rabbit was sedated, the bladder was catheterized and cystometry was performed at a filling rate of 1 ml. per minute. At the completion of cystometry the rabbits were immediately anesthetized. Moderate outlet obstruction was created in groups 1 and 3, and sham surgery was performed in groups 2 and 4. Treatment was continued for an additional 4 weeks, when each rabbit was sedated and cystometry was repeated. After cystometry the bladder was exposed through a midline incision, excised, weighed and 4 strips of bladder body were cut for contractility studies. The balance of the bladder was separated between smooth muscle and mucosa by blunt dissection, frozen in liquid nitrogen and stored at -70C for biochemical analyses. RESULTS Partial outlet obstruction stimulated similar increases in the bladder weight of all obstructed rabbits. Partial outlet obstruction resulted in a significant decrease in bladder compliance in all obstructed animals. However, the bladder of obstructed rabbits given Kohki tea were significantly more compliant than those given water. Voiding pressures in the control group and the obstructed group given distilled water were approximately equal, while obstructed rabbits given Kohki tea showed significantly higher maximal voiding pressure. The contractile responses to all forms of stimulation were reduced by obstruction to a significantly greater degree in the rabbits not given tea than in those given tea. Sarcoplasmic reticulum Ca2+-adenosine triphosphatase enzyme activity of the bladder was significantly reduced in obstructed rabbits given vehicle but activity was not reduced in obstructed rabbits given Kohki tea. CONCLUSIONS Kohki tea had a significant protective effect on bladder function, contractile responses and bladder biochemistry in rabbits with moderate to severe partial outlet obstruction.

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)

Effect of castration on male rabbit lower urinary tract tissue enzymes

AbstractObjectives The influence of testosterone on the prostate and corpus cavernosum have been studied extensively. However, the influence of testosterone on the lower urinary tract (bladder and urethra) have not been investigated to any great extent. The aim of this study was to determine whether androgen deprivation alters lower urinary tract metabolism. Methods A total of 16 rabbits were divided into four groups of four rabbits each. Each rabbit in groups 1–3 underwent surgical bilateral castration for duration of 1, 2 , and 4 weeks, and group 4 underwent sham operations. Sections of bladder body and base wall and mucosa, urethra and corpora were isolated, frozen, and stored under liquid nitrogen. The activities of citrate synthase-thapsigargin sensitive Ca2+ ATPase (Sarco/Endoplasmic Reticulum Ca2+ ATPase [SERCA]), and choline acetyl-transferase were examined as markers for mitochondrial function, sarcoplasmic reticular calcium storage and release, and cholinergic nerve function, respectively. Results The activity of SR function indicator, Ca2+ ATPase was significantly higher in the control corpora than in the control bladder or urethra. Castration resulted in decreased activity in the mitochondria specific enzyme, citrate synthase, the activity of which was greatest in the urethra and lowest in the corpora. Cholinergic nerve density indicator, choline acetyl-transferase activity was greatest in the bladder body and lowest in the urethra. Conclusions Our data indicate that (1) significant differences exist in the activities of all three enzymes in the various organs associated with the lower urinary tract; and (2) that castration results in significant alterations in the activities of all three enzymes in the bladder body, base, urethra, and corpora.

Protective effects of grape suspension on in vivo ischaemia/reperfusion of the rabbit bladder

To investigate the potential protective effect of a grape suspension in a rabbit model of in vivo bilateral ischaemia/reperfusion (I/R), which is a causal factor in obstructive bladder dysfunction.

Effect of bilateral in vivo ischemia/reperfusion on the activities of superoxide dismutase and catalase: response to a standardized grape suspension.

Purpose: Ischemia/reperfusion (I/R) is a major etiological factor in the bladder dysfunctions observed in men with lower tract obstruction, women with postmenopausal incontinence and with aging. A standardized grape suspension protects the rabbit urinary bladder from both the contractile dysfunctions and the morphologic changes mediated by I/R. Using a model of in vivo bilateral ischemia/reperfusion, the current study investigated the effect of this grape suspension on the endogenous antioxidant defense systems. Materials and methods: 24 NZW rabbits were separated into 6 groups of 4. Groups 1–3 were treated by gavage with aqueous grape suspensions; groups 4–6 received sugar-water vehicle. Groups 3 and 6 were controls. Groups 1 and 4 were subjected to bilateral ischemia for 2 h (I). Groups 2 and 5 underwent bilateral ischemia for 2 h and reperfusion for 1 week (I/R). For all rabbit bladders, the muscle and mucosa were separated by blunt dissection and analyzed separately. The effects of the various treatments on bladder antioxidant systems of cytoplasmic superoxide dismutase (Cu-Zn superoxide dismutase; SOD), and catalase (CAT) were evaluated. Results: The standardized grape suspension up-regulated both SOD and CAT activity of bladder muscle and mucosa in control animals. There were few differences in the grape suspension treated animals after ischemia, and in general the activities decreased following I/R. Conclusions: Increases of SOD and CAT activity in control animals as a result of grape suspension suggest a greater antioxidant capacity. This increase in the antioxidant defense system may explain the increased protection of grape suspension in the face of ischemia and I/R. However, the activities of both enzyme systems decreased in the smooth muscle subjected to I/R showing that reperfusion damages these systems probably via oxidation damage to the enzymes themselves.

The Immediate Effect of Castration on Female Rabbit Bladder Blood Flow and Tissue Oxygenation

Background: The female urinary bladder is a target organ for estrogen. Reductions in circulating estrogen have been associated with urothelial and vaginal atrophy and bladder disorders including incontinence and increased incidence of bladder infections. We determined the effect of short-term ovariectomy on sex hormones, bladder blood flow, and tissue oxygenation in the rabbit model. Materials and Methods: Female New Zealand White rabbits were ovariectomized and evaluated on 1, 3, and 7 days after ovariectomy. Tissue oxygenation (pO2) and blood flow were measured with oxylab system of real time measurements. Serum estrogen and progesterone were determined at sacrifice. Tissue hypoxia was localized histologically using Hypoxyprobe-1 immunohistochemistry. Results: Short-term ovariectomy caused rapid decreases in serum estrogen and progesterone, significant decreases in urothelial oxygenation and blood flow. No significant decreases in blood flow or oxygenation were noted for the detrusor smooth muscle. Immunohistochemistry confirmed the presence of urothelial hypoxia at all times after ovariectomy. Bladder muscle did not demonstrate significant levels of hypoxia. Conclusion: The bladder urothelium is extremely sensitive to short-term ovariectomy, with significant urothelial hypoxia seen by post-ovariectomy day 1. Urothelial hypoxia may play a significant role in pelvic pain syndromes, incontinence, and increased susceptibility to bladder infection.

Normal detrusor is more sensitive than hypertrophied detrusor to in vitro ischemia followed by re-oxygenation.

Partial outlet obstruction results in marked metabolic as well as contractile alterations. Specifically, the ratio of anaerobic to oxidative metabolism is significantly greater in hypertrophied than normal bladder smooth muscle, lactate dehydrogenase (LDH) and lactic acid production are increased, and the contractile apparatus is altered to allow for metabolically more efficient tension generation. In addition, contractile responses of hypertrophied bladder are apparently more resistant than those of normal bladder to hypoxia. In the current experiment, we studied the effects of in vitro ischemia (hypoxia + substrate deprivation) followed by an in vitro model of reperfusion (re‐oxygenation + substrate replacement) on contractile responses of normal and hypertrophied urinary bladder strips. We used repetitive field stimulation (FS) during the hypoxic period as a model for hyperreflexia. The purpose of the current study was to compare the responses of normal and hypertrophied bladder smooth muscle to repetitive stimulation in the presence of in vitro ischemia followed by re‐oxygenation and substrate replacement. Thirty‐two rats were separated into four groups of eight each. The rats in groups 1 and 3 were subjected to partial outlet obstruction. Two weeks later, all rats were anesthetized; their bladders were isolated and cut into four strips. Each strip was mounted in an isolated bath, and after 1‐hour incubation in Tyrodes solution containing glucose (in the presence of O2), contractile responses to FS, carbachol, and KCl were determined. After this first set of stimulations, the strips were incubated without glucose and in the presence of N2 for 30 minutes and 1 hour (groups 1 and 2); and for 2 and 4 hours (groups 3 and 4). For groups 1 and 2, the tissues were stimulated at 5‐minute intervals with FS at 32 Hz, 1‐millisecond duration, 3‐second trains (in vitro model of hyperreflexia). For groups 3 and 4, no stimulations were performed during the ischemic period. At the end of the ischemic period, all strips were washed and incubated for 1 hour in the presence of O2 and with glucose. At the end of this incubation, all strips received a second set of stimulations. a) Partial outlet obstruction resulted in a significant increase in bladder weight. b) Responses to in vitro ischemia: After in vitro ischemia, contractile responses of both normal and hypertrophied tissues to FS were reduced to a significantly greater degree than were responses to carbachol and KCl. The rate of development of contractile dysfunction was significantly greater in normal bladder tissue strips than in hypertrophied bladder strips. c) Responses to repetitive stimulation: The rate of development of contractile dysfunction was significantly greater in all strips subjected to repetitive stimulation than in those not repetitively stimulated; in addition, normal bladder strips were more sensitive than hypertrophied strips to hypoxia and substrate deprivation‐induced contractile dysfunction.