Vascular density of superficial esophageal squamous cell carcinoma determined by direct observation of resected specimen using narrow band imaging with magnifying endoscopy.

Observation of the microvasculature using narrow band imaging (NBI) with magnifying endoscopy is useful for diagnosing superficial squamous cell carcinoma. Increased vascular density is indicative of cancer, but not many studies have reported differences between cancerous and noncancerous areas based on an objective comparison. We observed specimens of endoscopic submucosal dissection (ESD) using NBI magnification, and determined the vascular density of cancerous and noncancerous areas. A total of 25 lesions of esophageal squamous cell carcinoma that were dissected en bloc by ESD between July 2013 and December 2013 were subjected to NBI magnification. We constructed a device that holds an endoscope and precisely controls the movement along the vertical axis in order to observe submerged specimens by NBI magnification. NBI image files of both cancerous (pathologically determined invasion depth, m1/2) and surrounding noncancerous areas were created and subjected to vascular density assessment by two endoscopists who were blinded to clinical information. The invasion depth was m1/2 in 20, m3/sm1 in four and sm2 in one esophageal cancer lesion. Mean vascular density was significantly increased in cancerous areas (37.6 ± 16.3 vessels/mm2) compared with noncancerous areas (17.6 ± 10.0 vessels/mm2) (P < 0.05). The correlation coefficients between vascular density determined by two endoscopists were 0.86 and 0.81 in cancerous and noncancerous areas, respectively. Receiver operating curve (ROC) analysis revealed that the area under the curve (AUC) of vascular density was 0.895 (95% CI, 0.804-0.986). For this ROC curve, sensitivity was 78.3% and specificity was 87.0% when the cutoff value of vascular density was 26 vessels/mm2. NBI magnification confirmed significant increases in vascular density in cancerous areas compared with noncancerous areas in esophageal squamous cell carcinoma. The rates of agreement between vascular density values determined by two independent operators were high.

Hypertension induced by a nonpressor dose of angiotensin II in kininogen-deficient rats.

Brown Norway Katholiek rats with very low levels of plasma kininogens excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats (132 +/- 2 mmHg, n = 7) was not different from that of normal rats. Angiotensin II (Ang II) (20 micrograms/d SC) from 7 weeks of age for 2 weeks with a micro-osmotic pump caused significant increases in blood pressure (181 +/- 5 mm Hg, n = 7, 9 weeks old) in the deficient rats, although the same treatment induced no blood pressure increase in the normal rats. Also during this period, the deficient rats had significantly higher heart rates, tended to excrete less urinary sodium, and showed significantly higher sodium levels in serum, erythrocytes, and cerebrospinal fluid compared with the normal rats. Ang II increased urinary excretion of aldosterone in both deficient and normal rats (P < .05). Spironolactone treatment (50 mg/kg per day) for 7 days in deficient rats restored blood pressure and heart rate to normal levels and significantly reduced sodium levels in erythrocytes and cerebrospinal fluid. Subcutaneous infusion of bovine low-molecular-weight kininogen with an osmotic pump in Ang II-treated deficient rats induced significant reductions in blood pressure, heart rate, and erythrocyte sodium levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 in Ang II-treated normal rats induced a hypertensive response in parallel with significant increases in heart rate and erythrocyte sodium level. These results suggest that the lack of kinin generation observed in the kininogen-deficient rats may cause the hypertensive response during the administration of a nonpressor dose of Ang II mainly through sodium retention probably caused by aldosterone release.

High sensitivity to salt in kininogen-deficient brown Norway Katholiek rats.

Brown Norway Katholiek rats, which have very low levels of plasma kininogens, excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats fed low (0.3%) NaCl diets (131 +/- 4 mm Hg, n = 12) was not different from that in normal rats. Two percent NaCl diets given from 7 weeks of age for 4 weeks caused rapid increases in blood pressure (167 +/- 4 mm Hg, n = 12, 9 weeks old) in deficient rats, although the same diets induced no blood pressure increase in normal rats. Urinary excretion of active kallikrein and prokallikrein remained constant in both rat groups throughout NaCl loading. During this period, the deficient rats secreted less urine (9 weeks old, P < .05) and less urinary sodium (11 weeks old, P < .05). Serum levels of sodium in deficient rats were higher (P < .05) than in normal rats at 9 weeks of age. Intracellular concentrations of sodium in the erythrocytes of deficient rats were higher (P < .05) than in normal rats throughout NaCl loading. Subcutaneous infusion of bovine low molecular weight kininogen with an osmotic pump in NaCl-loaded deficient rats induced a reduction (P < .01) in blood pressure and increases (P < .05) in urine volume and urinary sodium and kinin levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 or of aprotinin in NaCl-loaded normal rats induced a hypertensive response. This antagonist treatment reduced urine volume and urinary sodium. These results indicate that the lack of kinin generation observed in the kininogen-deficient rats was related through sodium retention to the hypertensive response to NaCl loading.

Cerebroprotective properties of SM-20220, a potent Na(+)/H(+) exchange inhibitor, in transient cerebral ischemia in rats.

The aim of this study was to investigate the contribution of the Na(+)/H(+) exchanger to cerebral ischemia using SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate), a newly synthesized compound. In in vitro experiments, we evaluated the inhibitory effect of SM-20220 on the Na(+)/H(+) exchanger in cultured neurons and glial cells. The IC(50) of SM-20220 in neurons and glial cells was 5 nM and 20 nM, respectively. To examine the in vivo effects of SM-20220 on brain injury, we used a transient middle cerebral artery occlusion model in rats. SM-20220 given intravenously 1 h after occlusion significantly reduced the extent of cerebral edema, Na(+) content and infarcted area in a dose-dependent manner. The results of the present study suggest that the Na(+)/H(+) exchanger is involved in the aggravation of brain edema and infarction, and its inhibitor may exert protective effects on post-ischemic brain damage.

Ebelactone B, an inhibitor of urinary carboxypeptidase Y-like kininase, prevents the development of deoxycorticosterone acetate-salt hypertension in rats.

Kininogen-deficient Brown Norway Katholiek rats (BN-Ka) excrete little urinary kinin, compared with normal rats of the same strain (BN Kitasato rats (BN-Ki)). Deoxycorticosterone acetate-salt treatment increased systolic blood pressure in both rats, but much faster in BN-Ka than in BN-Ki. Daily subcutaneous administration of ebelactone B (15 and 5 mg/kg/day), a rat urinary carboxypeptidase Y-like kininase inhibitor, significantly reduced systolic blood pressure in BN-Ki, but not in BN-Ka. This treatment significantly increased urinary Na+ excretion and reduced Na+ concentration in the erythrocytes in BN-Ki, but not in BN-Ka. An angiotensin-converting enzyme inhibitor, lisinopril (5 mg/kg/day s.c.), did not reduce the systolic blood pressure in either BN-Ki or BN-Ka. These results suggested that ebelactone B has promise as a preventive agent for the development of hypertension acting through the inhibition of urinary kinin degradation.

Poststatin, a novel inhibitor of bradykinin-degrading enzymes in rat urine.

Incubation of bradykinin with rat urine resulted in the successive degradation of bradykinin to bradykinin-(1-8), bradykinin-(1-7) and bradykinin-(1-6). In contrast, in rat plasma, bradykinin was degraded via either bradykinin-(1-8) or bradykinin-(1-7) to bradykinin-(1-5). Phosphoramidon (1 mM) partially inhibited the degradation of bradykinin by rat urine, as well as the conversion of bradykinin-(1-7) to bradykinin-(1-6). D,L-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid (1 mM) and captopril (1 mM) did not have a significant effect on any of the degradation steps in rat urine. In contrast, all of the degradation steps in urine, namely, from bradykinin to bradykinin-(1-8), from bradykinin-(1-8) to bradykinin-(1-7) and from bradykinin-(1-7) to bradykinin-(1-6), were markedly inhibited by poststatin (1 mM), even though this compound was reported originally to be a novel inhibitor of post-proline cleaving enzyme. Poststatin (1 mM) did not inhibit the degradation of bradykinin in rat plasma. These results indicate that poststatin is an effective inhibitor of kinin-degrading enzyme in rat urine.

Effect of catalase and thioredoxin addition to sperm incubation medium before in vitro fertilization on sperm capacity to support embryo development.

OBJECTIVE: To determine whether addition of catalase and thioredoxin to sperm incubation medium before IVF improves sperm potential to support embryo development. DESIGN: CD-1 mouse spermatozoa were preincubated without or with catalase or thioredoxin for 1 hour before IVF, and sperm motility parameters, fertilization rate, and embryo development were determined. SETTING: A conventional laboratory setting. INTERVENTION(S): Mice were superovulated with pregnant mare serum gonadotropin and hCG. Eggs in cumulus oophorus were collected and used for fertilization with epididymal spermatozoa. MAIN OUTCOME MEASURE(S): Sperm motility parameters, fertilization rate, and embryo development. RESULT(S): Sperm motility parameters and fertilization rates were not affected by catalase treatment. However, the rates of blastocyst and hatching blastocyst formation when catalase-treated (16 micrograms/mL) spermatozoa were used were significantly higher than those observed with nontreated spermatozoa (44% versus 24%, 31% versus 2%, respectively). Addition of thioredoxin to preincubation media did not affect the percentage of motility and the fertilization rate but increased the rate of blastocyst formation to an extent similar to that triggered by catalase (2.7- and 3.3-fold, respectively). CONCLUSION(S): These results suggest that H2O2 produced in sperm suspensions before IVF reduces their potential to promote embryo development, that these toxic effects of H2O2 are latent, appearing mainly 3 to 5 days after IVF, and that catalase and thioredoxin are efficient agents to protect sperm potential to support embryo development.

Effect of sperm lipid peroxidation on fertilization.

This study was aimed at determining, with homologous mouse gametes, whether a level of membrane lipid peroxidation insufficient to affect sperm motility parameters can alter sperm fertilizing potential. The addition of ferrous ions and ascorbic acid (Fe2+/ Asc) to mouse sperm suspensions increases the formation of thiobarbituric acid-reactive substances (TBARS), an indicator of lipid peroxide breakdown products, without significantly affecting the level of sulfhydryl groups. In the presence of Fe2+/Asc concentrations above > 0.4/2.0 mM, spermatozoa become immotile. However, at concentrations < or = 0.4/2.0 mM of Fe2+/Asc, i.e., conditions in which the TBARS formation is increased by < or = 4.6-fold over that of controls, motility remains unaffected for up to 3 hours. In the presence of 0.4/ 2.0 mM Fe2+/Asc, treated spermatozoa increase their fertilizing potential by 50%, as measured by the formation of two-cell embryos. This increase is not caused by improvements in sperm motility parameters (percentage, linearity, velocity, hyperactivation) or sperm capacitation. On the other hand, there is a significant increase in the capacity of mouse spermatozoa to bind to homologous zona pellucida. In conclusion, mild peroxidative conditions, that increase lipid peroxide formation 4.6-fold without significantly modifying free sulfhydryl groups and sperm motility parameters, improve the fertilizing potential of spermatozoa by increasing their binding capacity to zona pellucida.

Activation of urinary inactive kallikrein by an extract from the rat kidney cortex.

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodoacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.

Noradrenaline concentrations in human preovulatory follicular fluid exceed those in peripheral plasma.

The concentrations of noradrenaline in preovulatory follicular fluid obtained from women undergoing in vitro fertilization were determined by high-performance liquid chromatography with fluorometric detection and compared with those in peripheral plasma obtained concurrently. All of the follicular fluid samples contained noradrenaline at concentrations substantially higher than those in the corresponding plasma samples. A positive correlation was found between noradrenaline levels in follicular fluid and plasma in each woman (n=11; r=0.952; p<0.001). However, there was no significant difference between noradrenaline concentrations in follicular fluid aspirated from follicles with or without an oocyte [mean+/-SEM, 0.207+/-0.002 ng/microl for follicular fluid samples with an oocyte (n=44), and 0.221+/-0.003 ng/microl for follicular fluid samples without an oocyte (n=13)]. The data indicate that noradrenaline accumulates in follicular fluid, supporting the physiological significance of noradrenaline in the local regulation of human ovarian functions.

SM-20220, a potent Na+/H+ exchange inhibitor, improves consciousness recovery and neurological outcome following transient cerebral ischaemia in gerbils.

We studied the cerebroprotective effect of SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulphonate), a newly synthesized Na+/H+ exchanger (NHE) inhibitor, in Mongolian gerbil global ischaemia. Transient cerebral ischaemia was induced by clipping both common carotid arteries for 30 min followed by 24h reperfusion. Intravenous administration of SM-20220 (0.3 or 1.0 mg kg(-1)) immediately after reperfusion significantly shortened the consciousness recovery time (P < 0.01). SM-20220 also improved the neurological outcome (McGraw's scale) after reperfusion. At the dose of 1.0 mg kg(-1), the mortality rate was significantly reduced at 24 h after reperfusion (P < 0.01). This study shows that NHE is involved in the aggravation of cerebral function, represented by consciousness recovery, and neurological outcome following transient forebrain ischaemia, and that its inhibitor may exert protective effects on post-ischaemic brain damage.

Lectin histochemistry in rat liver fibrosis induced by heterologous serum sensitization.

The localization of carbohydrates in rat livers with fibrosis induced by heterologous serum was examined by lectin histochemical and biochemical techniques. Twenty-four lectins were used to visualize the different carbohydrates in paraffin sections of normal and fibrotic liver tissues. No differences in staining patterns of these lectins were observed between normal and fibrotic livers in hepatocyte cell membranes including bile canaliculi, sinusoidal endothelial, or bile ductal cells. Kupffer cells strongly stained with Vicia villosa agglutinin (VVA) were seen only in the periportal zone of the normal liver, but they were observed in the periportal zone and scattered throughout the pseudolobular zone in the fibrotic liver. The cytoplasm of some hepatocytes was strongly stained by Bandeiraea simplicifolia lectin-I (BSL-I). BSL-I positive hepatocytes in normal liver were localized in the periportal zone, but those in the fibrotic liver were scattered in the periportal and perifibrous zones. After polyacrylamide gel electrophoresis of liver glycoproteins, differences in molecular sizes of BSL-I positive glycoproteins (79 and 81 kD) were detected by lectin blotting. Cell density of perifibrous BSL-I positive hepatocytes may be useful as a diagnostic parameter for liver fibrosis and/or cirrhosis. Two distinct staining patterns with twelve lectins were observed in fibrotic septa of the fibrotic liver. The fibrotic septa were stained with six characteristic lectins, and the centrilobular septa were stained with all these twelve of lectins. Histopathological assessment of the centrilobular fibrotic septa stained with these characteristic lectins may contribute to the diagnosis and prognosis of hepatic fibrosis.

Inhibitory effects of a phosphate diester of alpha-tocopherol and ascorbic acid (EPC-K1) on myocardial infarction in rats.

The inhibitory effect of a phosphate diester of alpha-tocopherol and ascorbic acid (EPC-K1) was examined in myocardial infarction induced in rats, in comparison with a selective 5-lipoxygenase inhibitor, AA-861. EPC-K1 significantly reduced the infarct size at 24 and 48 h after ligation, whereas AA-861 reduced it only at 48 h after ligation. In in-vitro experiments, EPC-K1 inhibited not only superoxide anion generation (IC50 = 4.2 x 10(-5) M), but also acid phosphatase activity (IC50 = 2.4 x 10(-5) M) in rat polymorphonuclear leukocytes in a concentration-dependent manner, while AA-861 showed marginal effects on both actions. These results indicated that EPC-K1 induced cardioprotective effects by affecting neutrophil functions by inhibition of generation of superoxide-anion generation and acid-phosphatase activity. The mechanism of the reduction of the infarct size by EPC-K1 differed from that of AA-861, which latter inhibited 5-lipoxygenase and the formation of leukotriene B4.

Delayed treatment of Na+/H+ exchange inhibitor SM-20220 reduces infarct size in both transient and permanent middle cerebral artery occlusion in rats.

SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate) is a Na+/H+ exchanger (NHE) inhibitor which has been shown to attenuate cerebral edema in the rat transient focal ischemia model. However, to date, the effect of SM-20220 on cerebral infarction has not been examined. The present experiments were designed to investigate these effects, using both transient and permanent middle cerebral artery (MCA) occlusion models in rats. A dose of 1 mg/kg given intravenously 30 min after the onset of transient MCA occlusion reduced the infarcted area. In the permanent MCA occlusion model, SM-20220 reduced the infarcted area when treatment was delayed for 5, 30 or 60 min after the onset of ischemia. The present results show that NHE has a crucial role in the pathogenesis of ischemic brain damage. This NHE inhibitor may be useful for treating stroke because of its effectiveness with both forms of ischemia and because of its postischemic administration.