Olmesartan ameliorates urinary dysfunction in the spontaneously hypertensive rat via recovering bladder blood flow and decreasing oxidative stress.

PURPOSE: As hypertension (HT) is one of the risk factors for lower urinary tract symptoms, we investigated the effect of an angiotensin II type I receptor blocker, olmesartan, on bladder dysfunction in the spontaneously hypertensive rat (SHR). MATERIALS AND METHODS: Twelve-week-old male SHRs were administered perorally with olmesartan (0, 1, or 3 mg/kg/day) or nifedipine (30 mg/kg/day) for 6 weeks. Wistar rats were used as normotensive controls. The effects of olmesartan or nifedipine on blood pressure (BP), bladder blood flow (BBF), urodynamic parameters, tissue levels of malondialdehyde (MDA), nuclear factor erythroid 2-related factor 2 (Nrf2), and nerve growth factor (NGF) were measured in the bladder. Localization of 4-hydroxy-2-nonenal (4-HNE), Nrf2, and NGF in the bladder was shown by immunohistochemistry. RESULTS: The SHRs showed significant increase in BP, micturition frequency, and expression of MDA, 4-HNE, Nrf2, and NGF when compared to the control Wistar rats. Conversely, there was a decrease in BBF and single voided volume in SHRs when compared to Wistar rats. Treatment with olmesartan and nifedipine significantly improved BP. However, only olmesartan significantly ameliorated urodynamic parameters and oxidative damage compared to the non-treated SHR. The immunoreactivities of 4-HNE, Nrf2, and NGF in SHR urothelium and blood vessels were increased compared to the control. Treatment with a high dose of olmesartan decreased the expressions of 4-HNE, Nrf2, and NGF in the bladder. CONCLUSION: Our data suggest that BP, BBF, and oxidative stress may be responsible for the functional changes in HT-related bladder dysfunction. Olmesartan significantly ameliorated this bladder dysfunction.

Characterization of silodosin and naftopidil in the treatment of bladder dysfunction in the spontaneously hypertensive rat.

PURPOSE: As increasing evidence suggest that α(1)-blockers prevent benign prostatic hyperplasia related overactive bladder and nocturia in the human, we investigated the effects of silodosin and naftopidil on hypertension-related bladder dysfunction in the spontaneously hypertensive rat (SHR) model. MATERIALS AND METHODS: Twelve-week-old male SHRs received no treatment or treatment with silodosin (100 µg/kg, p.o.) or naftopidil (10 or 30 mg/kg, p.o.) once daily for 6 weeks. Wistar rats were used as normotensive controls. After 6-week treatment, voiding functions were estimated by metabolic cages (dark- and light-cycle separately) and cystometric studies. Furthermore, the bladder blood flow (BBF) was measured employing the hydrogen clearance method. RESULTS: SHRs showed significant increases in micturition frequency, and decreases in BBF and single voided volume in both metabolic cages and cystometrograms compared to the Wistar group. Treatment with silodosin normalized the decreased BBF, and treatment with naftopidil increased the BBF in a dose-dependent manner in the SHR group. Although treatment with silodosin and the high dose of naftopidil significantly inhibited micturition frequency in one day, only treatment with the high dose of naftopidil significantly inhibited micturition frequency and urine production in the light-cycle compared to the non-treated SHRs. Although treatment with silodosin and the high dose of naftopidil significantly increased single voided volume, only treatment with silodosin significantly inhibited non-voiding contractions in the cystometrgrams. CONCLUSION: Our data suggest that both silodosin and naftopidil improve hypertension-related bladder dysfunction in the SHR, and naftopidil but not silodosin improves urinary frequency in the light-cycle due to inhibition of urine production.

Monomerization of TDP-43 is a key determinant for inducing TDP-43 pathology in amyotrophic lateral sclerosis.

The cytoplasmic aggregation of TAR DNA binding protein-43 (TDP-43), also known as TDP-43 pathology, is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism underlying TDP-43 cytoplasmic mislocalization and subsequent aggregation remains unclear. Here, we show that TDP-43 dimerization/multimerization is impaired in the postmortem brains and spinal cords of patients with sporadic ALS and that N-terminal dimerization-deficient TDP-43 consists of pathological inclusion bodies in ALS motor neurons. Expression of N-terminal dimerization-deficient mutant TDP-43 in Neuro2a cells and induced pluripotent stem cell-derived motor neurons recapitulates TDP-43 pathology, such as Nxf1-dependent cytoplasmic mislocalization and aggregate formation, which induces seeding effects. Furthermore, TDP-DiLuc, a bimolecular luminescence complementation reporter assay, could detect decreased N-terminal dimerization of TDP-43 before TDP-43 pathological changes caused by the transcription inhibition linked to aberrant RNA metabolism in ALS. These findings identified TDP-43 monomerization as a critical determinant inducing TDP-43 pathology in ALS.

Effect of silodosin on detrusor overactivity in the male spontaneously hypertensive rat.

UNLABELLED: Recently, several studies have suggested that detrusor overactivity (DO) is the result of bladder ischaemia. Hypertension could affect pelvic arterial blood flow, resulting in loss of smooth muscle in the bladder with resultant loss of bladder compliance. Spontaneously hypertensive rats are considered a valuable tool for exploring the pathogenesis of DO. Some reports indicate that α(1) adrenoceptor antagonists improve chronic ischaemia of the lower urinary tract in patients with LUTS, with concomitant improvement of their symptoms as well as improvement of DO through an increased bladder blood flow (BBF) in the rat with bladder outlet obstruction. However, the mechanism of improvement of silodosin on storage or irritative symptoms is not well investigated and is still unclear. Silodosin prevents hypertension-related DO in the SHR via several possible mechanisms. One possible mechanism of the efficacy of silodosin to the DO includes the improvement of the BBF. OBJECTIVE: To investigate the effect of the α(1A) selective adrenoceptor antagonist, silodosin, on hypertension-related detrusor overactivity (DO) and its possible mechanism in spontaneously hypertensive rats (SHRs). MATERIALS AND METHODS: Twelve-week-old male SHRs received treatment with silodosin (100 µg/kg perorally) once daily for 6 weeks; vehicle-treated Wistar rats and vehicle-treated SHRs were used for our study. Six weeks after silodosin treatment, voiding functions were estimated by voiding behaviour and cystometric studies in all groups. The bladder blood flow was measured by the hydrogen clearance method, and tissue levels of nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the expressions of α1 adrenoceptor subtype mRNAs in the bladder were investigated by real-time PCR method. RESULTS: The SHRs showed significant increases in blood pressure, micturition frequency and tissue levels of NGF and CGRP in the bladder. Moreover, the SHRs showed significant decreases in bladder blood flow and single voided volume estimated by both voiding behaviour and cystometric studies compared with those in the Wistar rats. Six weeks of treatment with silodosin significantly ameliorated hypertension-related alterations of these variables with concomitant small changes of blood pressure. The expression levels of α1 adrenoceptor subtype mRNAs in the bladder were similar in all the groups and the rank order was α1A = α1D > α1B in all groups. However, there were no significant differences in the expressions of α(1A) adrenoceptor mRNAs between any groups. CONCLUSION: The data in the present study suggest that silodosin normalizes hypertension-related DO in SHRs, which could be related to its effect on the increased blood flow in the bladder.

Pharmacological preconditioning of ATP-sensitive potassium channel openers on acute urinary retention-induced bladder dysfunction in the rat.

UNLABELLED: What's known on the subject? and What does the study add? Acute urinary retention (AUR) and catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Previously, we reported that the induction of AURC significantly reduced contractile responses to both carbachol and KCl compared with a control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner; however, although we reported a possible beneficial effect of nicorandil and cromakalim on bladder dysfunction caused by AURC, its molecular mechanism is still unknown. Our study establishes that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both K(IR)6.1 and K(IR)6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder. OBJECTIVE: To investigate whether ATP-sensitive potassium (K(ATP)) channel openers prevent bladder injury after acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) in the rat. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were divided into five groups: a sham-operated control group, an AUR group, and three AUR groups treated with: nicorandil (10 mg/kg); cromakalim (300 µg/kg); or glibenclamide (5 mg/kg). AUR was induced by intravesical infusion of 3.0 mL of saline via cystostomy with simultaneous clamping of the penile urethra and, after 30 min of AUR, the bladder was allowed to drain for 60 min. After the experimental period, bladder function was assessed using organ bath techniques (carbachol and KCl), and by measuring tissue levels of 8-isoprostane, a marker of oxidative stress. The participation levels of K(ATP) channel pores were investigated using ELISA and real-time PCR methods, respectively. The degree of apoptosis was estimated using the TUNEL method in the bladder smooth muscle and epithelium. RESULTS: The AURC group showed significantly decreased contractile responses to carbachol and KCl, and significant increases in tissue 8-isoprostane levels and apoptosis index in the epithelium compared with the control group. Nicorandil and cromakalim, but not glibenclamide, significantly prevented these AURC-induced alterations. The expressions of K(IR)6.1 and K(IR)6.2 mRNAs were significantly up-regulated by the induction of AURC. Nicorandil and cromakalim, but not glibenclamide, significantly up-regulated expressions of K(IR)6.1 and K(IR)6.2 mRNAs in the bladder compared with the AUR group. CONCLUSION: Our data indicate that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via activation of K(ATP) channels, with a subsequent decrease in oxidative stress and decreased induction of apoptosis.

Hydroxyfasudil ameliorates penile dysfunction in the male spontaneously hypertensive rat.

Hypertension represents a major risk factor for erectile dysfunction. Although the etiology of hypertension-induced erectile dysfunction is multifactorial and still unknown, Rho-Rho kinase pathway is one of the key factors. To investigate whether administration of hydroxyfasudil, a Rho kinase inhibitor could prevent dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the SHR (spontaneously hypertensive rat), twelve-week-old male SHRs were treated with hydroxyfasudil (3 or 10 mg/kg, i.p.) once a day for 6 weeks. Wistar rats and SHRs treatment with vehicle were used as age-matched controls. Penile cGMP concentrations and Rho kinase activities were determined, and penile function was estimated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The participation mRNA levels of eNOS and participation protein levels of eNOS and phosphorylated eNOS were investigated by quantitative real-time PCR methods and immunoblot analysis, respectively. The SHR showed significantly decreased cGMP concentrations, increased Rho kinase activities, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue. Treatment with hydroxyfasudil significantly improved the decreased penile cGMP concentrations, the increased Rho kinase activities, the increased norepinephrine-induced contractions, and the decreased acetylcholine-induced relaxation in a dose-dependent manner. Although there were no significant differences in expression protein levels of eNOS among any of the groups, down-regulation of eNOS mRNAs as well as phosphorylated eNOS were significantly ameliorated after treatment with hydroxyfasudil. Our data suggest that hydroxyfasudil ameliorates hypertension-associated dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle possibly via inhibition of the Rho-Rho kinase pathway and activation of NO-eNOS pathway in the SHR.

Nicorandil ameliorates hypertension-related bladder dysfunction in the rat.

AIMS: There is increasing evidence that ischemia is one of the main etiology in overactive bladder (OAB), and that nicorandil prevents OAB. We investigated the effect of nicorandil on hypertension-related bladder dysfunction in spontaneously hypertensive rats (SHRs). METHODS: Twelve-week-old SHRs received six-weeks treatment with nicorandil (0, 3, or 10 mg/kg, i.p. every day). Wistar rats were used for normotensive controls. Six weeks after nicorandil treatment, the bladder blood flow was estimated by hydrogen clearance method, and the bladder functions were estimated by voiding behavior studies and functional studies. Tissue levels of nerve growth factor (NGF) were measured by ELISA method. Furthermore, the participation levels of K(ATP) channel pores were investigated by real-time PCR. RESULTS: SHRs showed significant increases in blood pressure, micturition frequency, tissue levels of NGF and expressions of both K(IR) 6.1 and K(IR) 6.2 mRNAs, and a significant decrease in the bladder blood flow. The carbachol-induced contractile responses were similar in all groups. Although both doses of nicorandil failed to decrease the blood pressure, nicorandil significantly decreased the micturition frequency, tissue levels of NGF and increased the bladder blood flow in a dose dependent manner. The expressions of K(IR) 6.1 and K(IR) 6.2 mRNAs were slightly up-regulated by the low dose of nicorandil, whereas the high dose of nicorandil significantly up-regulated those expressions compared to non-treated SHRs. CONCLUSIONS: These data indicate that nicorandil prevents hypertension-related bladder dysfunction in the SHR, which may be related to its effect on the increased blood flow in the bladder.

Rhos and Rho kinases in the rat prostate: their possible functional roles and distributions.

As there is increasing evidence that Rho-Rho kinase (ROCK) pathway plays an important role in the proliferation and contraction in many tissues, we investigated the contractile role of a ROCK inhibitor, fasudil, and the distribution of RhoA, RhoB, RhoC, ROCK1, and ROCK2 in the rat prostate. Twelve-week-old Sprague-Dawley rat prostate was used in this study. Rat prostatic contractile responses induced by carbachol and norepinephrine were investigated in organ bath studies without or with 10(-7), 10(-6), and 10(-5) M of a non-selective ROCK inhibitor, fasudil. Immunoblot analysis and immunohistochemical staining were performed to investigate the participation levels of RhoA, RhoB, RhoC, ROCK1, and ROCK2. The E(max) values induced by carbachol and norepinephrine were similar in the rat prostate. Fasudil significantly inhibited carbachol- or norepinephrine-induced prostatic contractions in a dose-dependent manner. Fasudil 10(-5) M reduced the initial prostatic contraction (without fasudil) to 56.7 ± 5.9% for carbachol and to 45.7 ± 12.3% for norepinephrine. Amounts of RhoA, RhoB, RhoC, ROCK1, and ROCK2 were detected by immunoblot analysis in the prostate. Immunohistochemical study revealed that RhoA, RhoB, RhoC, ROCK1, and ROCK2 were all positive in the prostatic smooth muscle, while there were some differences of distributions of Immunoreactivities between these enzymes in the prostatic glandula. Our data indicated that rat prostate contains RhoA, RhoB, RhoC, ROCK1, and ROCK2, which play an important role in the autonomic nerve-mediated contractile responses in the prostate.

Protective effect of sivelestat, a neutrophil elastase inhibitor, on ipsilateral and contralateral testes after unilateral testicular ischaemia-reperfusion injury in rats.

OBJECTIVE: To investigate the effect of a neutrophil elastase inhibitor, sivelestat sodium hydrate, on testicular ischaemia-reperfusion (IR)-injury. MATERIAL AND METHODS: Eight-week-old male Sprague-Dawley rats were divided into four groups: sham-operated control rats; IR rats (group IR); and IR rats that received intra-abdominal administration of 15 mg/kg or 60 mg/kg sivelestat (group IR15 and group IR60, respectively). Right testicular vessels were clamped for 90 min in groups IR, IR15 and IR60. Sivelestat had been administered 45 min after the induction of the ischaemia in groups IR15 and IR60. In subpopulations of IR, IR15 and IR60 rats, reperfusion was performed after ischaemia for 2 h (groups IR-A, IR15-A and IR60-A, respectively) or 48 h (groups IR-B, IR15-B and IR60-B, respectively). At the end of the reperfusion period, blood samples were aspirated from both spermatic veins of each rat and testosterone was evaluated. Then both testes from all rats were collected and tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), and heat-shock protein-70(HSP-70) were evaluated. Testicular tissue samples were also processed for histological evaluation and TUNEL staining. RESULTS: MDA, MPO and HSP-70 levels in the ischemic testis were significantly higher in the IR group compared with the control group. MDA and HSP-70 in the contralateral testis were significantly higher in the IR group compared with the control group. Bilateral testosterone levels were lower in all rat groups in comparison with the control group. Bilateral testicular samples in group IR showed extensive histopathologic degenerative alterations and increased percentage of apoptotic cells. Sivelestat treatment lowered the MDA concentration and the percentage of apoptotic cells bilaterally and ameliorated the testicular histological pattern bilaterally. CONCLUSIONS: Unilateral testicular ischaemia causes significant contralateral testicular damage. Sivelestat may be a novel adjunct tool for reducing oxidative stress and partially preventing bilateral testicular damage.

Edaravone ameliorates diabetes-induced dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the rat.

INTRODUCTION: Diabetes mellitus (DM) represents a major risk factor for erectile dysfunction (ED). Although the etiology of diabetes-induced ED is multifactorial and still unknown, reactive oxygen species are thought to be one of the key factors. AIM: The aim of this article is to investigate whether administration of edaravone, a free radical scavenger, could prevent type 1 diabetes-induced dysfunction of nitric oxide (NO)-induced relaxation in corpus cavernosum smooth muscle in the rat. METHODS: Six-week-old male Wistar rats were randomly divided into three groups. One group was treated with citrate-phosphate buffer plus normal saline (group Cont), whereas in the other two groups, diabetes was induced by streptozotocin (50 mg/kg intraperitoneally [i.p.]). Subsequently, the diabetic rats were treated for 4 weeks either with edaravone (10 mg/kg/day, i.p.; group DM + E) or with normal saline (group DM). MAIN OUTCOME MEASURES: Serum glucose and malondialdehyde levels as well as penile cyclic guanosine monophosphate (cGMP) concentrations were determined, and penile function was estimated by organ bath studies with norepinephrine-mediated contractions and acetylcholine-mediated relaxations. The participation mRNA levels of muscarinic M(3) receptors, neuronal nitrous oxide synthase (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), and participation protein levels of nNOS, eNOS, phosphorylated nNOS, and phosphorylated eNOS were investigated by quantitative real-time polymerase chain reaction (PCR) and immunoblot analysis, respectively. RESULTS: Treatment with edaravone prevented partially but significantly the decreased body and penile weight induced by diabetes. Treatment with edaravone significantly improved the increased diabetes-induced malondialdehyde levels, the decreased penile cGMP concentrations, the increased diabetes-induced norepinephrine-mediated contractions, and the decreased acetylcholine-mediated relaxation. Although there were no significant differences in expression levels of mRNAs in nNOS, diabetes-induced upregulation of muscarinic M(3) receptor and iNOS mRNAs as well as diabetes-induced downregulations of eNOS, phosphorylated nNOS, and phosphorylated eNOS were significantly prevented by edaravone. CONCLUSIONS: Edaravone decreases the oxidative insult in the penile corpus cavernosum by ameliorating the NO-NOS system and thus preventing partially the developing ED in DM in the rat.

Muscarinic receptors and their mRNAs in type 2 Goto-Kakizaki diabetic rat prostate.

BACKGROUND: As increasing evidence is pointing towards the relationship between diabetes and benign prostatic hyperplasia/lower urinary tract symptoms, we investigated the pharmacological properties and gene expressions of the muscarinic receptors in type 2 diabetes rat prostate. METHODS: Twelve- and 70-week-old male Goto-Kakizaki (GK) rats and age-matched male Wistar rats were used in this study. The densities of muscarinic receptors (B(max) values) were determined by saturation studies with [(3)H]NMS ([N-methyl-(3)H] scopolamine methyl chloride) in the prostatic membrane particulates. The participation levels of M(1), M(2), and M(3) receptor protein and mRNA levels in the prostate were investigated by immunoblot analysis and real-time polymerase chain reaction (PCR), respectively. RESULTS: The B(max) values in 12-week-old Wistar and GK, and in 70-week-old Wistar and GK rat prostates were 36.0 +/- 2.8, 49.4 +/- 11.4, 22.0 +/- 2.2, and 47.0 +/- 4.1 fmol/mg protein, respectively. However, there were no significant differences in the affinity constants between any groups. Immunoblot analysis showed the existence of significant amounts of M(1), M(2), and M(3) receptor subtypes in each rat prostate. According to real-time PCR studies the rank order of expression levels of muscarinic receptors mRNA subtypes in the prostate were M(3) > M(2) > M(1). In each receptor subtype in each group, diabetes induced up-regulation of mRNAs while the advanced age of the rats was related with down-regulation of mRNAs. CONCLUSIONS: Our data indicated that type 2 diabetes induced up-regulation and age-related down-regulation of the expressions of muscarinic receptors and their mRNAs in the rat prostate.

Bladder dysfunction after acute urinary retention in the rats: a novel over active bladder model.

As there is increasing evidence that benign prostatic hyperplasia and its related acute urinary retention (AUR) induce over active bladder (OAB) syndrome, we investigated the effects of AUR on bladder function over a 4-week period in a rat model. Ten-week-old female Sprague-Dawley rats were used in this study. AUR was induced by clamping the distal urethra of each rat with a small clip, and then infusing 3 ml (0.6 ml/min) of saline with an infusion pump through a transurethral catheter (22G). The obstruction was sustained for 60 min and the clip was removed and then the bladder was allowed to drain through the catheter. The bladder function was estimated by voiding behavior studies (at 3 days, 1, 2, 3, and 4 weeks), cystometric studies (at 2 and 4 weeks) and organ bath studies using KCl and carbachol (at 2 and 4 weeks). Furthermore, we evaluated histological changes in the rat bladder 2 and 4 weeks after the induction of AUR. The same parameters were also measured in non-AUR rats (control group). The rat bladder weight in the AUR group at 2 weeks was significantly larger than that of the controls, and returned to the control level 4 weeks after the AUR episode. The voiding behavior studies showed significant increase in micturition frequency per day and decrease in single voiding volume 3 days after the induction of AUR, and this voiding behavior was continued for more than 2 weeks. The cystometric studies showed a significant decrease in single-voided volume at 2 weeks rat. However, no significant changes of the other parameters were observed in the rats. The histological studies showed significant infiltration of neutrophils and lymphocytes, as well as increase in turnover of epithelium in AUR rats at 2 weeks, while significant increases in fibrosis in submucosal layer were observed in AUR rats at 4 weeks. This study demonstrated that bladder dysfunction in the rat model caused by AUR needs more than 2 weeks of recovery period. The AUR-associated alterations in the bladder may represent a key clue to understand the underlying pathophysiological mechanisms, which take place in OAB syndrome.

Protective effect of edaravone, a free-radical scavenger, on ischaemia-reperfusion injury in the rat testis.

OBJECTIVE: To investigate the effect of edaravone, a radical scavenger, on ischaemia-reperfusion (I-R) injury in the testes. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were allocated to one of four groups: a no-drug group subjected to induction of 30-min of ischaemia and 60-min reperfusion; two drug groups administered edaravone at 1 or 10 mg/kg intraperitoneal and then subjected to 30-min ischaemia and 60-min reperfusion; and a sham-operated control group administered edaravone at 10 mg/kg intraperitoneal. To induce testicular I-R, the right testis was exposed outside of the body and the testicular artery was clamped with a small clip for 30 min. Blood flow and nitric oxide (NO) release were monitored in real time simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. After death the tissue levels of NO(2)-NO(3) (a marker of NO production), malondialdehyde (a marker of lipid peroxidation), 8-hydroxydeoxyguanosine (a marker of oxidative DNA damage), myeloperoxidase (a marker of neutrophil infiltration), and heat-shock protein 70 (HSP 70) and its mRNA were measured. The testicular tissue was also analysed histologically. RESULTS: Clamping the testicular artery resulted in a decrease of blood flow to 0-5% of the basal level measured before clamping. NO release was increased during clamping and gradually recovered to the basal level on removing the clip. Interestingly, the peak of NO release in rats of the no-drug group occurred at the start of reperfusion, while that in the high-dose drug group occurred several minutes later. The levels of NO(2)-NO(3), malondialdehyde, 8-hydroxydeoxyguanosine, myeloperoxidase and HSP 70 and its mRNA, and histological variables, were significantly greater in the no-drug I-R group than in the control, and these variables were ameliorated by treatment with edaravone. CONCLUSION: These results indicate that edaravone reduces the oxidative stress and prevents the testicular damage induced by I-R.

Ischemic preconditioning and post-conditioning to decrease testicular torsion-detorsion injury.

PURPOSE: The main pathophysiology of torsion-detorsion is associated with ischemia-reperfusion injury in the testis caused by the twisted spermatic cord and its release. It is most likely mediated by oxygen free radicals. We investigated the effects of ischemic preconditioning and post-conditioning on rat testicular ischemia-reperfusion injury. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats (SLC, Shizuoka, Japan) were randomly divided into 4 age matched groups, including 1-control sham operation, 2-60-minute ischemia/120-minute reperfusion, 3-3 cycles of 5-minute ischemia/5-minute reperfusion and then 60-minute ischemia/120-minute reperfusion (ischemic preconditioning) and 4-60-minute ischemia, 5 cycles of 10-second reperfusion/10-second ischemia and then 120-minute reperfusion (ischemic post-conditioning). After sacrifice the levels of malondialdehyde, 8-hydroxydeoxyguanosine, myeloperoxidase, superoxide dismutase, catalase, heat shock protein 70 protein and mRNA, and DNA fragmentation were measured in the rat testes. Testicular tissue was also histologically analyzed. RESULTS: The levels of malondialdehyde, 8-hydroxydeoxyguanosine, myeloperoxidase, heat shock protein 70 mRNA, superoxide dismutase, catalase, DNA fragmentation and apoptosis cells were significantly higher in the ischemia-reperfusion group than in controls. Ischemic preconditioning decreased histological parameters, including vacuolation and necrosis, and decreased malondialdehyde, 8-hydroxydeoxyguanosine, myeloperoxidase, heat shock protein 70 mRNA but not protein, superoxide dismutase, catalase, DNA fragmentation and apoptosis compared to the ischemia-reperfusion group. Ischemic post-conditioning ameliorated 8-hydroxydeoxyguanosine, superoxide dismutase, heat shock protein 70 mRNA, DNA fragmentation and apoptosis compared to the ischemia-reperfusion group. CONCLUSIONS: Our data indicate that ischemic preconditioning and post-conditioning ameliorated the testicular damage induced by ischemia-reperfusion injury.

Endothelial dysfunction in the early- and late-stage type-2 diabetic Goto-Kakizaki rat aorta.

As there are increasing evidences that human diabetes induces cardiovascular dysfunction, we investigated the type-2 diabetes-induced endothelial dysfunction in the early and late-stage Goto-Kakizaki (GK) rat aorta. We performed organ bath studies, and examined the changes in expression levels of muscarinic M(3) receptor, endothelial, inducible, and neuronal nitric oxide synthase (eNOS, iNOS, and nNOS, respectively) mRNAs in the rat aorta utilizing real-time polymerase chain reaction in 12-week-old and 70-week-old GK rats as well as in age-matched Wistar rats. In the 12-week-old GK rat aorta, a significant increase in norepinephrine-induced contraction and a significant decrease in acetylcholine-induced relaxation as well as significant increases in expression levels of muscarinic M(3) receptor and eNOS and a significant decrease in nNOS mRNAs were observed compared to age-matched controls. In the older GK rat aorta, significant decreases in acetylcholine- and nitroglycerine-induced relaxations as well as significant decreases in the expression levels of muscarinic M(3) receptor, eNOS, iNOS, and nNOS mRNAs were observed compared to those in the younger GK rats. In contrast, although significant decreases in acetylcholine and nitroglycerine-induced relaxations were observed, the expression levels of muscarinic M(3) receptor, eNOS, iNOS, and nNOS mRNAs in the older Wistar rats aorta were unchanged, increased, increased and decreased, respectively, compared to the younger Wistar rat aorta. These results indicate that endothelial dysfunction in the rat aorta progresses with age and development of diabetes condition, and that decreased relaxations in the late-stage rat aorta may be due to these alterations.

Acute urinary retention and subsequent catheterization cause lipid peroxidation and oxidative DNA damage in the bladder: preventive effect of edaravone, a free-radical scavenger.

OBJECTIVE: To investigate the effect of a free-radical scavenger, edaravone, on the changes occurring with acute urinary retention (AUR) and subsequent catheterization in the rat bladder. MATERIALS AND METHODS: Eight-week-old male Sprague Dawley rats were allocated to one of four groups; an AUR group that had urinary retention induced, with subsequent catheterization; two edaravone groups, given edaravone at 1 or 10 mg/kg body weight for 60 min and then the same urinary retention and subsequent catheterization; and a sham-operated control group given edaravone 10 mg/kg. Urinary retention was induced by the clamping the rat penile urethra with a small clip, making a cystostomy, and then infusing 3 mL (0.6 mL/min) of saline with an infusion pump. The obstruction was sustained for 30 min and then the bladder was allowed to drain with a catheter in place for 60 min as the studies continued. After killing the rats the function of the bladder was assessed, with carbachol and 100 mM KCl, and the levels of malondialdehyde (MDA, a marker of lipid peroxidation), 8-hydroxydeoxyguanosine (8-OHdG; a marker of oxidative DNA damage), heat-shock protein 70 (HSP 70) and its mRNA were measured. RESULTS: AUR increased the intravesical pressure and decreased blood flow, and subsequent catheterization decreased the intravesical pressure and increased blood flow. Edaravone induced a decrease in blood flow in the bladder during the urinary retention and subsequent catheterization compared to the blood flow in the AUR group. Edaravone resulted in protection of the contractile responses to both carbachol and KCl in a dose-dependent manner. The MDA concentration, 8-OHdG content and expressions of HSP-70 and its mRNA in the AUR group were significantly larger than those of the control group. Edaravone markedly suppressed the accumulations of MDA and 8-OHdG in the bladder, and reduced the expressions of HSP 70 and its mRNA. CONCLUSION: These results indicate that edaravone reduces the oxidative stress and prevents the bladder dysfunction caused by AUR and subsequent catheterization.

Effect of the rho-kinase inhibitor hydroxyfasudil on bladder overactivity: an experimental rat model.

OBJECTIVES: To investigate the effects of the rho-kinase inhibitor hydroxyfasudil on bladder overactivity in cyclophosphamide (CYP)-induced cystitis. METHODS: Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (200 mg/kg). Four days later, bladder function was evaluated by: (i) monitoring micturition behavior in metabolic cages between hydroxyfasudil- and vehicle-treated animals; (ii) measuring changes in continuous cystometrograms in response to intravenous hydroxyfasudil under anesthesia; and (iii) conducting a functional study examining the effect of hydroxyfasudil on the concentration-response curves to carbachol in bladder tissue strips. RESULTS: Intraperitoneal injection of hydroxyfasudil (10 mg/kg) significantly increased both the average and maximal voided volumes. Hydroxyfasudil significantly decreased the maximal detrusor pressure, whereas the intercontraction interval was not significantly affected. After administration of 0.1, 0.3, 1, and 3 microM hydroxyfasudil, the maximal contraction of the concentration-response curves to carbachol was significantly reduced to 74.5 +/- 4.2%, 55.2 +/- 5.6%, 29.4 +/- 5.6%, and 21.6 +/- 8.2% of the control values, respectively. CONCLUSIONS: The present findings indicate that hydroxyfasudil might be a new treatment option for CYP-induced detrusor overactivity.

Pharmacological properties, functional alterations and gene expression of muscarinic receptors in young and old type 2 Goto-Kakizaki diabetic rat bladders.

PURPOSE: We investigated pharmacological properties, functional alterations and gene expression of the muscarinic receptor system in young and old Goto-Kakizaki rat bladders. MATERIALS AND METHODS: Male 12 and 70-week-old Goto-Kakizaki rats and age matched male Wistar rats were used in this study. Bladder function was estimated by voiding behavior, cystometric and functional studies using KCl, carbachol and various concentrations of subtype selective muscarinic antagonists, ie pirenzepine, methoctramine, 4-DAMP (Sigma) and atropine (Wako Pure Chemical Industries, Osaka, Japan). The participation levels of M(2) and M(3) receptor mRNA in the bladder were investigated by real-time polymerase chain reaction. RESULTS: In voiding behavior studies there were no significant differences in urine output, although an age related decrease in micturition frequency and an age related increase in single voided volume were observed in Goto-Kakizaki and Wistar rats. In cystometric studies there were no significant differences in maximum detrusor pressure or bladder capacity, although residual urine volume was significantly increased in 70-week-old Goto-Kakizaki rats. In functional studies carbachol induced detrusor contractility was significantly increased in Goto-Kakizaki rats in each age group. Estimated pA(2) values for atropine, pirenzepine, methoctramine and 4-DAMP (Sigma) indicated that the carbachol induced contractile response was mediated through the M(3) receptor subtype in all groups. Furthermore, muscarinic M(2) and M(3) receptor mRNA was significantly up regulated in 70-week-old Goto-Kakizaki rat bladders. CONCLUSIONS: Our data indicate that noninsulin dependent diabetes induces alterations in the muscarinic receptor system, which may contribute to the development of diabetic cystopathy.

Cyclohexenonic long-chain fatty alcohol has therapeutic effects on diabetes-induced angiopathy in the rat aorta.

We studied the effects of cyclohexenonic long-chain fatty alcohol (N-hexacosanol) on diabetes-induced angiopathy in the rat aorta. Male Sprague-Dawley rats were divided into 4 groups, a control group and 3 other groups in which diabetes was induced by streptozotocin (50 mg/kg i.p.). Four weeks after the induction of diabetes, the 3 groups received treatment with either vehicle or N-hexacosanol (2 or 8 mg/kg, i.p. every day) for another 4 weeks. To determine the mechanisms of diabetic vascular dysfunction and the effects of N-hexacosanol, we conducted organ bath studies and real-time polymerase chain reaction on muscarinic M(3) receptor, and endothelial and inducible nitric oxide synthase (eNOS and iNOS) mRNAs in the rat aorta. Treatment with N-hexacosanol did not alter the diabetic status, but improved the diabetes-induced hypercontraction produced by norepinephrine and the damaged endothelium-dependent relaxation of the rat aorta induced by acetylcholine. Furthermore, in the diabetic rats, both muscarinic M(3) receptor and iNOS mRNAs were significantly increased, and N-hexacosanol reversed these upregulations. However, the expression of eNOS mRNA showed no change in all groups. These results indicate that N-hexacosanol has beneficial effects on functional dysfunction and reverses the upregulation of muscarinic M(3) receptor and iNOS mRNAs in the diabetic rat aorta.

Beneficial effect of preconditioning on ischemia-reperfusion injury in the rat bladder in vivo.

We investigated the effect of preconditioning on ischemia-reperfusion injury in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder. Twelve-week-old male SD rats were divided into three groups; sham-operated control (Cont), 30 min ischemia-60 min reperfusion (IR) and three times of 5 min ischemia and then 30 min ischemia-60 min reperfusion (PC) groups. The bladder functions were estimated by cystometric and functional studies. Contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists, i.e. atropine (non-selective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective). We also measured tissue levels of malonaldehyde (MDA) and examined possible histological changes in these rats' bladders. Preconditioning partially prevented the reduction of bladder dysfunction induced by ischemia-reperfusion. Estimation of the pA2 values for atropine, pirenzepine, methoctramine, and 4-DAMP indicates that the carbachol-induced contractile response in bladder dome is mediated through the M3 receptor subtype in all groups. The MDA concentration in the IR group was significantly larger than that of the control group, and preconditioning significantly reduced MDA production in the bladder. In histological studies, the ischemia-reperfusion with or without preconditioning caused infiltration of leukocytes and rupture of microcirculation in the regions of submucosa and smooth muscle without a corresponding sloughing of mucosal cells. Our data indicate that preconditioning has a beneficial effect on ischemia-reperfusion injury in the rat bladder.