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.

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.

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.

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.

N-hexacosanol prevents diabetes-induced rat ileal dysfunction without qualitative alteration of the muscarinic receptor system.

We evaluated the effects of N-hexacosanol, a cyclohexenonic long-chain fatty alcohol, on muscarinic receptors in diabetic rat ileal dysfunction. Eight-week-old male SD rats were divided into four groups. After induction of diabetes (streptozotocin 50 mg/kg, i.p.), three groups were maintained for eight weeks with treatment by N-hexacosanol (0, 2 or 8 mg/kg, s.c. every day). Ileum function was investigated by organ bath studies using carbachol and KCl, and the expression levels of muscarinic M(2) and M(3) receptors were investigated by real-time polymerase chain reaction. Various concentrations of subtype-selective muscarinic antagonists, i.e., atropine (non-selective), pirenzepine (M(1) selective), methoctramine (M(2) selective), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M(1)/M(3) selective), were used in this study. In the presence and absence of these antagonists, contractile response curves to increasing concentrations of carbachol were investigated. Treatment with N-hexacosanol did not alter the diabetic status of the rats, but did significantly prevent the carbachol-induced hypercontractility in diabetic rat ileum. Estimation of the pA(2) values for atropine, pirenzepine, methoctramine, and 4-DAMP indicated that the carbacholinduced contractile response in the ileum is mainly mediated through the muscarinic M(3) receptor subtype in all groups. Furthermore, N-hexacosanol significantly prevented the diabetes-induced up-regulation of intestinal muscarinic M(2) and M(3) receptor mRNAs in streptozotocin-diabetic rats. Our data indicated that N-hexacosanol exerts preventive effects with respect to carbachol-induced hypercontractility in the diabetic rat ileum without qualitative alteration of the muscarinic receptor system.

N-hexacosanol ameliorates streptozotocin-induced diabetic rat nephropathy.

In this study we investigated the effects of N-hexacosanol on streptozotocin-induced rat diabetic nephropathy. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an intraperitoneal injection of streptozotocin (50 mg/kg). The rats were divided into four groups and maintained for 8 weeks: control rats, diabetic rats without treatment with N-hexacosanol, and diabetic rats treated with N-hexacosanol (2 mg/kg and 8 mg/kg i.p. every day). Although N-hexacosanol failed to modify the diabetic status, increases in serum creatinine as well as in kidney weight were significantly reduced. The malonaldehyde and transforming growth factor beta-1 (TGF-beta1) concentrations as well as the protein kinase C (PKC) activities in the diabetic kidney were significantly higher than those of the control, which were decreased by treatment with N-hexacosanol. Histological examinations revealed that N-hexacosanol significantly ameliorated diabetic-induced tubulointerstitial pathological changes. Our data suggest that N-hexacosanol could prevent increases in the malonaldehyde and TGF-beta1 concentrations and PKC activities in the kidney, and ameliorate diabetic-induced nephropathy.

N-hexacosanol reverses diabetic induced muscarinic hypercontractility of ileum in the rat.

Diabetic neuropathy, a major complication of diabetes mellitus, is associated with development of gastrointestinal motility dysfunction and autonomic neuropathy. N-hexacosanol has neurotrophic effects and exhibits a wide variety of biological actions. In this study, we investigated the effects of cyclohexenonic long-chain fatty alcohol (N-hexacosanol) on streptozotocin-diabetic hypercontractility in the rat ileum longitudinal muscles. Treatment with N-hexacosanol did not alter the diabetic status of the animals, i.e., body weight, serum glucose, and serum insulin levels, but significantly restored the thickness of intestine wall and ameliorated diabetes-induced hypercontractility of the rat ileum in a dose-dependent manner. Furthermore, N-hexacosanol reversed the diabetes-induced upregulation of intestinal muscarinic M(2) and M(3) receptors mRNAs in the streptozotocin-diabetic rats. These results indicate that N-hexacosanol has therapeutic effects on hypercontractility in the diabetic ileum by ameliorating overexpression of muscarinic M(2) and M(3) receptors mRNAs.

General administration of cyclohexenonic long-chain fatty alcohol ameliorates hyperreactivity of STZ-induced diabetic rat aorta.

Diabetic neuropathy, a major complication of diabetes mellitus, is associated with the development of vascular dysfunction and autonomic neuropathy. We studied the effects of cyclohexenonic long-chain fatty alcohol (FA) on streptozotocin-diabetic hyperreactivity in the rat aorta smooth muscle. The rats were divided randomly into four groups and were maintained for 4 weeks: age-matched control rats, diabetic rats without treatment with FA, and diabetic rats treated with FA (2 and 8 mg/kg, i.p. everyday). The serum glucose and insulin levels were determined, and the contractile responses of the aorta induced by a thromboxane A2 agonist, U46619 and KCl were investigated. Treatment with FA did not alter rats' diabetic status, i.e., body weight, thickness of the aorta, serum glucose levels, and serum insulin levels, but significantly improved the diabetic-induced hyperreactivity of the rat aorta in a dose-dependent manner. Removal of endothelium did not change contractile force between groups. In histological examinations, thinning of smooth muscle bundle in the wall of aorta was observed in the diabetic rat, which was not significantly improved by treatment with FA. Our data indicate that FA can prevent hyperreactivity in the diabetic aorta.

Effects of cyclohexenonic long-chain fatty alcohol on diabetic rat trachea.

In order to investigate the diabetes-associated neuropathy and prevent effects of cyclohexenonic long-chain fatty alcohol, a neurotrophic substance, in trachea, we studied its effect on streptozotocin-diabetic hyper-reactivity in the rat trachea. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an intraperitoneal injection of streptozotocin (50 mg/kg). The rats were divided randomly into four groups and were maintained for four weeks: age-matched control rats, diabetic rats without treatment with cyclohexenonic long-chain fatty alcohol, and diabetic rats treated with cyclohexenonic long-chain fatty alcohol (2 and 8 mg/kg, i.p. every day). The serum glucose and insulin levels were determined, and the contractile responses of the trachea induced by carbachol and KCl were investigated. Treatment with cyclohexenonic long-chain fatty alcohol did not alter the rats' diabetic status, i.e., body weight, thickness of the trachea, serum glucose levels, and serum insulin levels, but significantly improved the diabetic-induced hyper-reactivity of the rat trachea in a dose-dependent manner. There was no significant difference in either the carbachol- or KCl-induced contractile forces between groups with or without mucosa in the functional studies. In histological examinations, thinning of cricoid cartilage, thickness of basal membrane, and degeneration, fragmentation of elastic fibers in the submucosal layer, and hypertrophy of smooth muscle bundle in the membranous wall of trachea were observed in the diabetic rat trachea, which were improved by treatment with cyclohexenonic long-chain fatty alcohol. Our data indicate that this drug can prevent hyper-reactivity in the diabetic trachea.

Effects of cyclohexenonic long-chain fatty alcohol in type 2 diabetic rat nephropathy.

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (CHLFA) on the alterations of type 2 diabetes-induced nephropathy. Forty-week-old male Goto-Kakizaki (GK) and Wistar rats were divided into four groups of 6 to 8 animals. Group A consisted of eight Wistar rats and served as an age-matched control group. Group B (7 GK rats) received no treatment and served as a diabetic group. Group C (6 GK rats) was treated daily with low-dose CHLFA (2 mg/ kg/body weight, subcutaneously) for 30 weeks, and Group D (6 GK rats) with high-dose CHLFA (8 mg/kg/body weight) for 30 weeks. At the end of the treatment period, urinary protein excretion, blood chemistry, renal histological, and immunohistological analyses were conducted. Although CHLFA administration did not influence serum glucose or insulin levels, it reversed diabetes-induced increases in urinary protein excretion and serum creatinine. Light microscopically, CHLFA treatment ameliorated the otherwise elevated glomerular sclerotic scores in the diabetic group.Immunohistochemically, increased expression of desmin and decreased expression of rat endothelial cell antigen-1 in the group with untreated diabetes both showed a reversal to control levels in the high-dose CHLFA treatment group. In conclusion, CHLFA may ameliorate type 2 diabetes-induced nephropathy.

The role of ATP-sensitive potassium channel on acute urinary retention and subsequent catheterization in the rat.

We investigated the role of K(ATP) channel on acute urinary retention (AUR) induced bladder dysfunction. Eight-week-old female Sprague-Dawley rats were divided into seven groups: a sham-operated control group, an AUR group, and five AUR groups treated with: two different K(ATP) channel openers namely nicorandil (3 or 10mg/kg), or cromakalim (100 or 300microg/kg), or one K(ATP) channel inhibitor namely glibenclamide (5mg/kg). The drugs were administered 30min before induction of AUR. After the urethra was obstructed with a clip, AUR was induced by intravesical infusion of 2.5ml of saline via cystostomy. Following a 30min obstruction the bladder was allowed to drain with a catheter in place for 60min with real-time monitoring of intravesical pressure and blood flow. After the experimental period, the bladder function was assessed, using organ bath techniques (carbachol and 100mM KCl). AUR increased the intravesical pressure and decreased the blood flow. The subsequent catheterization decreased the intravesical pressure and increased the blood flow. AUR group reduced significantly the contractile responses to both carbachol and KCl compared with the control group. Nicorandil and cromakalim but not glibenclamide prevented the bladder dysfunction after AUR suggesting that K(ATP) channel openers may prevent the bladder dysfunction caused by AUR and subsequent catheterization.

Characterization of the ileal muscarinic receptor system in 70-week-old type II Goto-Kakizaki diabetic rats; effects of cyclohexenonic long-chain fatty alcohol.

As gastrointestinal motility disorders are frequently reported in patients with diabetes, we attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol in type 2 Goto-Kakizaki (GK) diabetic enteropathy. At 40 weeks of age male GK rats divided into three groups (treated with 0, 2 or 8 mg/kg of cyclohexenonic long-chain fatty alcohol; started at the age of 40 weeks). Age-matched male Wistar rats were used in this study. At 70 weeks of age the ileal functions were estimated by organ bath studies using 100 mM KCl and carbachol. The expression levels of muscarinic M(2) and M(3) receptor mRNAs in the ileum were investigated by real-time polymerase chain reaction (PCR). Treatment with cyclohexenonic long-chain fatty alcohol did not alter the diabetic status of the GK rats, i.e., body weight, serum glucose, and serum insulin levels, but significantly ameliorated diabetes-induced hypercontractility of the rat ileum caused by carbachol in a dose-dependent manner. Although there were no significant differences in the expression levels of muscarinic M(3) receptor mRNAs in any of the groups, cyclohexenonic long-chain fatty alcohol reversed the diabetes-induced up-regulation of intestinal muscarinic M(2) receptor mRNAs in treatment groups. These results indicate that cyclohexenonic long-chain fatty alcohol exerts its therapeutic effects on hypercontractility in the ileum of 70-week-old GK type 2 diabetic rats by ameliorating overexpression of muscarinic M(2) receptors.

Ability of cyclohexenonic long-chain fatty alcohol to reverse diabetes-induced cystopathy in the rat.

OBJECTIVES: We investigated the ability of 3-(15 hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen 1-one (N-hexacosanol), a neurotrophic substance, to reverse diabetes-induced cystopathy in the rat. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were divided randomly into four age-matched groups. In three of these groups, diabetes was induced by streptozotocin (STZ; 50mg/kg intraperitoneal [IP]). Four weeks after the induction of diabetes, the three groups received another 4 weeks of treatment by vehicle or N-hexacosanol (2 or 8 mg/kg IP every day). The serum glucose and serum insulin levels were determined, and the bladder functions were estimated by voiding behavior studies, cystometric studies, and functional studies using carbachol and KCl. The participation levels of M(2) and M(3) receptors were investigated by real-time polymerase chain reaction and immunohistochemical staining. Typical hematoxylin-eosin staining was also performed. RESULTS: Treatment with N-hexacosanol did not alter the rats' diabetic status, but did significantly improve the diabetes-induced dysfunction of the detrusor in a dose-dependent manner. Furthermore, N-hexacosanol significantly reversed the upregulation of muscarinic M(2) and M(3) receptor messenger RNAs (mRNAs) in STZ-diabetic rats. Muscarinic M(2) and M(3) receptors were localized in detrusor and urothelium, and there was no difference between any of the groups in the distribution of muscarinic M(2) and M(3) receptors. CONCLUSIONS: These results indicate that N-hexacosanol has a beneficial effect on hyperreactivity in the diabetic detrusor by ameliorating overexpression of muscarinic M(2) and M(3) receptor mRNAs.

Real-time monitoring of nitric oxide and blood flow during ischemia-reperfusion in the rat testis.

In the present study, we attempted to clarify the role of nitric oxide (NO) and its release during the ischemia-reperfusion rat testis. Eight-week-old male Sprague-Dawley rats were divided into seven groups: age-matched control rats, ischemia (30 minutes)-reperfusion (30 minutes) rats without NG-nitro-L-arginine methyl ester (L-NAME) and L-arginine (L-Arg) treatment, ischemia (30 minutes)-reperfusion (30 minutes) rats treated with L-NAME (10, 30, and 100 mg/kg), ischemia-reperfusion rats treated with L-Arg (10 and 30 mg/kg). Sixty minutes prior to induction of ischemia, L-NAME or L-Arg was administrated intraperitoneally. Real-time monitoring of blood flow and NO release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. NO2-NO3 and malonaldehyde (MDA) concentrations were measured in the experimental testes. Furthermore, we investigated possible morphological changes in the testis. Clamping of the testicular artery decreased blood flow to 5-20% of the basal level measured before clamping. Immediately following clipping of the artery, NO release rapidly increased. After removing the clip, NO release gradually returned to the basal level. This phenomenon was enhanced by treatment with L-Arg and inhibited by treatment with L-NAME. NO2-NO3 concentrations were increased by treatment with L-Arg and decreased by treatment with L-NAME, while MDA concentrations were increased by treatment with L-NAME and were decreased by treatment with L-Arg. In histological studies, the ischemia-reperfusion caused infiltration of leukocytes and a rupture of microvessels in the testis. Our data suggest that NO has cytoprotective effects on ischemia-reperfusion injury in the rat testis.