Itaru Satoh

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.

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 M3 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 M3 receptor and eNOS and a significant decease 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 M3 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 M3 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

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.

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.

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.

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

To investigate the effect of edaravone, a radical scavenger, on ischaemia‐reperfusion (I‐R) injury in the testes.

Protective effect of ischaemic post-conditioning on ipsilateral and contralateral testes after unilateral testicular ischaemia-reperfusion injury

The aim of this study was to investigate the effect of ischaemic post-conditioning (IPostC) against ischaemia-reperfusion (IR) injury on bilateral testes after unilateral testicular ischaemia in the rat. Eight-week-old male Sprague-Dawley rats were divided into control group; IR group (60 min ischaemia-24 h reperfusion); IPostC1 × 10 group (60 min ischaemia followed by one cycle of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion); IPostC3 × 10 group (three cycles of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion); IPostC5 × 10 group (five cycles of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion) and IPostC3 × 30 group (three cycles of 30 sec reperfusion-30 sec ischaemia; then 24 h reperfusion). In the IR and IPostC groups, the right testicular vessels were clamped using a special vascular clip. Malondialdehyde (MDA) and myeloperoxidase (MPO) levels were measured in testicular tissue samples bilaterally. Additionally, bilateral testicular tissue samples were processed for histological evaluation including haematoxylin-eosin, 4-hydroxy-2-nonenal (4-HNE) and TdT-mediated dUTP Nick End Labelling (TUNEL) staining. The levels of MDA and MPO as well as the positive cells per seminiferous tubule in TUNEL and 4-HNE stain in bilateral testes from the IR group were significantly higher compared with the control group. IPostC3 × 30 protocol significantly ameliorated the aforesaid parameters in both testes compared with the IR group. For the first time, we have demonstrated that IPostC protects both testes after unilateral testicular ischaemia-reperfusion. IPostC3 × 30 protocol offered the most effective protection.

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

What’s known on the subject? and What does the study add?

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

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).

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.