A Case of Paroxysmal Cold Hemoglobinuria Possessing Moderate Paroxysmal Nocturnal Hemoglobinuria-Type Erythrocytes.

BACKGROUND Paroxysmal cold hemoglobinuria (PCH) is an autoimmune hemolytic disease caused by the Donath-Landsteiner (DL) antibody. Paroxysmal nocturnal hemoglobinuria (PNH) is a non-autoimmune hemolytic disease that is caused by a dysfunction in the synthesis of the glycosyl phosphatidylinositol anchor protein, resulting in the deregulation of the complement cascade and hypersensitivity for a hemolytic attack against erythrocytes. The mechanisms of these 2 hemolytic diseases are distinct. If PCH and PNH coexist in a patient, it is difficult to perform a differential diagnosis. We introduce a case of PCH that had DL antibodies and large PNH-type clones. CASE REPORT An 82-year-old female patient was referred to our hospital because of anemia. Initial workup revealed a negative antiglobulin test and a positive DL test. For the differential diagnosis, we surveyed the population of cells that had PNH-type clones, which revealed erythrocyte PNH clones (19.6%) and granulocyte PNH clones (73.3%). During the patient's clinical course, mild hemolysis persisted without any attack. The percentage of the PNH-type erythrocytes was not obviously changed, and the DL antibody was detected 8 months after the initial admission. We determined that the persistent mild anemia was caused by concomitant diseases of PCH and PNH, although determining which of the 2 hemolytic systems was primarily responsible for the anemia was difficult. CONCLUSIONS When considering the differential diagnosis for hemolytic diseases, an adequate combination of laboratory tests for hemolysis is required.

Apoptosis inhibitor of macrophage ameliorates fungus-induced peritoneal injury model in mice.

Fungal peritonitis in a patient on peritoneal dialysis (PD) is a refractory injury accompanied by severe inflammation, predisposing patients to a poor prognosis. Defective clearance of necrotic tissue interferes with amelioration of tissue injury and induces abnormal tissue remodeling. In the recent reports, apoptosis inhibitor of macrophage (AIM, also called CD5L) prevents obesity, hepatocellular carcinoma and acute kidney injury. Here, we investigated potential roles of AIM in prevention of progression of fungal peritonitis models. AIM -/- mice subjected to zymosan-induced peritonitis exhibited progressive inflammation and sustained peritoneal necrosis tissue on day 28 after the disease induction, whereas there was an improvement in AIM +/+ mice. This appeared to be caused by deposition of AIM at the necrotic peritoneum in AIM +/+ mice. In vitro, AIM enhanced the engulfment of necrotic debris by macrophages derived from zymosan-induced peritonitis, M1- and M2a-like bone marrow derived macrophages, as well as by mesothelial cells. In addition, administration of recombinant AIM dramatically ameliorated severe inflammation associated with necrosis in zymosan-induced peritonitis of AIM -/- mice. Our observations suggest that AIM appears to be involved in the repair process of zymosan-induced peritonitis, and thus, could be the basis of development of new therapeutic strategies for PD-related fungal peritonitis.

Sodium chloride promotes tissue inflammation via osmotic stimuli in subtotal-nephrectomized mice.

Chronic inflammation, which is often associated with high all-cause and cardiovascular mortality, is prevalent in patients with renal failure; however, the precise mechanisms remain unclear. High-salt intake was reported to induce lymphangiogenesis and autoimmune diseases via osmotic stimuli with accumulation of sodium or chloride. In addition, sodium was recently reported to be stored in the extremities of dialysis patients. We studied the effects and mechanisms of high salt loading on tissue and systemic inflammation in subtotal-nephrectomized mice (5/6Nx) and in cultured cells. Macrophage infiltration in the peritoneal wall (P<0.001), heart (P<0.05) and para-aortic tissues (P<0.001) was significantly higher in 5/6Nx with salt loading (5/6Nx/NaCl) than in 5/6Nx without salt loading (5/6Nx/Water); however, there were no significant differences in blood pressure and renal function between the groups. Tissue interleukin-6, monocyte chemotactic protein-1 (MCP-1), serum- and glucocorticoid-inducible kinase 1 (Sgk1) and tonicity-responsive enhancer binding protein (TonEBP) mRNA were significantly elevated in the peritoneal wall and heart with 5/6Nx/NaCl when compared with 5/6Nx/Water. Sodium was stored in the abdominal wall, exerting high-osmotic conditions. Reversal of salt loading reduced macrophage infiltration associated with decreased TonEBP in 5/6Nx/NaCl. Macrophage infiltration associated with fibrosis induced by salt loading was decreased in the 5/6Nx/NaCl/CC chemokine receptor 2 (CCR2, receptor of MCP-1)-deficient mice when compared with 5/6Nx/NaCl/Wild mice, suggesting that CCR2 is required for macrophage infiltration in 5/6Nx with NaCl loading. In cultured mesothelial cells and cardiomyocytes, culture media with high NaCl concentration induced MCP-1, Sgk1 and TonEBP mRNA, all of which were suppressed by TonEBP siRNA, indicating that both MCP-1 and Sgk1 are downstream of TonEBP. Our study indicates that high NaCl intake induces MCP-1 expression leading to macrophage infiltration via the TonEBP-MCP-1 pathway in 5/6Nx/NaCl mice, and that TonEBP has a central role in inflammation in patients with renal failure taking high salt.

Vascular endothelial growth factor receptor-3 is a novel target to improve net ultrafiltration in methylglyoxal-induced peritoneal injury.

Appropriate fluid balance is important for good clinical outcomes and survival in patients on peritoneal dialysis. We recently reported that lymphangiogenesis associated with fibrosis developed in the peritoneal cavity via the transforming growth factor-ß1-vascular endothelial growth factor-C (VEGF-C) pathway. We investigated whether VEGF receptor-3 (VEGFR-3), the receptor for VEGF-C and -D, might be a new target to improve net ultrafiltration by using adenovirus-expressing soluble VEGFR-3 (Adeno-sVEGFR-3) in rodent models of peritoneal injury induced by methylglyoxal (MGO). We demonstrated that lymphangiogenesis developed in these MGO models, especially in the diaphragm, indicating that lymphangiogenesis is a common feature in the peritoneal cavity with inflammation and fibrosis. In MGO models, VEGF-D was significantly increased in the diaphragm; however, VEGF-C was not significantly upregulated. Adeno-sVEGFR-3, which was detected on day 50 after administration via tail vein injections, successfully suppressed lymphangiogenesis in the diaphragm and parietal peritoneum in mouse MGO models without significant effects on fibrosis, inflammation, or neoangiogenesis. Drained volume in the peritoneal equilibration test using a 7.5% icodextrin peritoneal dialysis solution (the 7.5% icodextrin peritoneal equilibration test) was improved by Adeno-sVEGFR-3 on day 22 (P<0.05) and day 50 after reduction of inflammation (P<0.01), indicating that the 7.5% icodextrin peritoneal equilibration test identifies changes in lymphangiogenesis. The solute transport rate was not affected by suppression of lymphangiogenesis. In human peritoneal dialysis patients, the dialysate to plasma ratio of creatinine positively correlated with the dialysate VEGF-D concentration (P<0.001). VEGF-D mRNA was significantly higher in the peritoneal membranes of patients with ultrafiltration failure, indicating that VEGF-D is involved in the development of lymphangiogenesis in peritoneal dialysis patients. These results indicate that VEGFR-3 is a new target to improve net ultrafiltration by suppressing lymphatic absorption and that the 7.5% icodextrin peritoneal equilibration test is useful for estimation of lymphatic absorption.

Estrogen-dependent activation of neutral cholesterol ester hydrolase underlying gender difference of atherogenesis in apoE-/- mice.

OBJECTIVE: Mechanisms underlying gender difference of atherogenesis were investigated focusing on direct effects of estrogen on the artery. METHODS: First, male and female apoE(-/-) mice were fed an atherogenic diet for 16 weeks from 10 weeks of age. Second, female apoE(-/-) mice were ovariectomized (ovx) or sham operated at 8 weeks of age, and 2-weeks afterwards, one-third of each ovx-group received conjugated equine estrogens (CEE) (0, 2.5 or 5.0 µg/day) for 16 weeks. Atherosclerotic lesions were examined after experimental periods. To clarify anti-atherogenic effect of 17ß-estradiol (E2) on artery, neutral cholesteryl ester hydrolase (N-CEase) activity in aorta and peritoneal macrophages, and E2-treated J774A.1 cells were measured. RESULTS: First, atherosclerotic lesion in female mice was significantly less than male mice without any changes in serum lipids and lipoprotein profile. N-CEase activity in aorta and peritoneal macrophages in female mice was significantly higher than male mice. Second, atherosclerotic lesion in ovx-group was significantly greater than sham-group. CEE-replacement to ovx-group decreased atherosclerotic lesion in a dose-dependent manner. N-CEase activity in aorta and peritoneal macrophages was decreased in ovx-group compared to sham-group, and restored by CEE-replacement in macrophages. To study detailed mechanisms, J774A.1 cells were treated with E2. E2 significantly increased N-CEase activity, and cAMP-dependent protein kinase (A-kinase) type II activity and the protein in cytosol fraction without any changes of total protein of A-kinase type II. CONCLUSION: These results suggest that gender difference of atherogenesis is partly accounted for activation of N-CEase through estrogen-dependent translocation of A-kinase type II in macrophages.

Ascorbic acid levels and neutrophil superoxide production in blood of pre-, early and late hypertensive stroke-prone spontaneously hypertensive rats.

We compared ascorbic acid (AA) levels in the blood and TPA- and fMLP-stimulated superoxide (O(2)(•-)) production in neutrophils of pre-, early, and late hypertensive stroke-prone spontaneously hypertensive rats (SHRSP) with those of age-matched Wistar Kyoto rats (WKY), or two other normotensive strains of rats. Plasma and lymphocyte AA levels were about two-fold higher in SHRSP as early as 4 weeks old compared to WKY, and also higher than those of Wistar and Sprague-Dawley (SD) rats. Levels of AA were high in the liver and adrenal glands of SHRSP, indicating congenitally high AA levels. The production of O(2)(•-) in neutrophils was about two-fold higher in SHRSP than in WKY even at 4 weeks of age, and increased with age in both strains. Among SHRSP, AA levels in lymphocytes decreased at the late hypertensive stages with a decrease in hepatic l-gulono-γ-lactone oxidase (GLO) activities. These data suggest that bi-phasic AA levels in the blood of SHRSP comprise congenitally high levels and a decrease after persistent hypertension due to enhanced O(2)(•-) production and a decrease in de novo AA synthesis through GLO.

Antiatherosclerotic effect of the edible mushrooms Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji) in apolipoprotein E-deficient mice.

In the present study, we examined the antiatherosclerotic effects of 3 edible mushrooms, Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji), in atherosclerosis-susceptible C57BL/6J, apolipoprotein E-deficient (apoE(-/-)) mice. Male apoE(-/-) mice (6 weeks of age) were fed a normal diet (cholesterol concentration <66 mg/100 g) or a normal diet containing 3% dried Eringi, Maitake, or Bunashimeji mushroom powder for 10 weeks. Food intake, body weight, serum total cholesterol (TC), and serum triacylglycerols (TG) were measured periodically during the experimental period. At the end of the experiment (at 16 weeks of age), the atherosclerotic lesion area was measured in cross-sections of the aortic root. Serum TC concentrations in the Bunashimeji group were significantly lower than that in the control group at 8, 10, 12, 14, and 16 weeks of age. Serum TC concentrations in the Eringi, and Maitake groups were significantly lower than that in the control group only at 12 weeks of age. There was no significant difference in the serum TG concentrations in all groups during the experimental period. The atherosclerotic lesions were significantly decreased in the Eringi, Maitake, and Bunashimeji groups than that in the control group at the end of the experiment. Dietary supplementation with the Bunashimeji mushroom powder had the strongest antiatherosclerotic effect among 3 mushrooms. In conclusion, supplementation of the 3 edible mushrooms prevents the development of atherosclerosis, even normal diet. Antiatherosclerotic effect is partly via lowering of serum TC concentrations; further mechanisms should be investigated.

[Anesthetic management of cesarean section in a patient with pulmonary embolism due to protein S deficiency diagnosed postoperatively].

A 27-year-old pregnant woman with pulmonary embolism was scheduled for cesarean section. She received anticoagulant therapy with continuous infusion of heparin and prophylactic placement of an inferior vena cava (IVC) filter. Heparin was discontinued 7 hours before operation. Spinal anesthesia using hyperbaric bupivacaine 12 mg was performed and the operation was completed uneventfully. She was given a diagnosis of protein S deficiency after discharge. Preoperative anticoagulant therapy and placement of IVC filter may be effective in preventing new pulmonary embolism.

Preventive effects of green tea catechins on spontaneous stroke in rats.

BACKGROUND: Green tea catechins possess potent antioxidative properties and protect against various oxidative diseases. Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) develop severe hypertension and spontaneous stroke at early ages. We previously reported that ingestion of green tea catechins prevents cerebral ischemic damage in a middle cerebral artery occlusion and reperfusion rat stroke model, in association with increased plasma epigallocatechin-gallate (EGCG) concentrations. In this study, we examined whether tea catechin intake decreases the incidence of spontaneous stroke in M-SHRSP. MATERIAL/METHODS: Male M-SHRSP ingested 0.5% green tea catechin extract (Polyphenon E) in their drinking water beginning at 5 weeks of age, and blood pressure, heart rate, and locomotor activity were continuously monitored from 8 weeks using a telemetry system. Stroke onset was assessed by the appearance of neurologic symptoms, body weight loss, and circadian rhythm disturbances in heart rate, blood pressure, and locomotor activity. RESULTS: Tea catechin ingestion significantly delayed stroke onset by 10 days compared to the control group. Although there was no difference in blood pressure at 10 weeks, the rate of in blood pressure increase in the tea catechin group was significantly smaller than that in the control group. Plasma NO2- and NO3- concentrations increased after stroke in both groups without significant difference between the two groups. Plasma EGCG concentration significantly decreased at post-stroke compared with that of pre-stroke. CONCLUSIONS: Continuous ingestion of green tea catechins from an early age prevented the development of spontaneous stroke in M-SHRSP, probably by inhibiting the further development of high blood pressure at later ages.

[Anesthetic management of a patient with mitochondrial encephalomyopathy under total intravenous anesthesia].

This report describes a patient with mitochondrial encephalomyopathy who underwent tracheostomy under total intravenous anesthesia. This 15-year-old girl had been suffering from aspiration pneumonia repeatedly. Anesthesia was induced with propofol (30 mg) and fentanyl (50 microg), and the trachea was intubated without a muscle relaxant. The patient was mechanically ventilated also without a relaxant, and anesthesia was maintained with a continuous infusion of propofol 4-10 mg x kg(-1) x hr(-1) and a bolus injection of fentanyl 25 microg. Bispectral index (BIS) was monitored and maintained at 15-65. The patient showed smooth recovery from anesthesia, and the BIS value returned to the pre-anesthetic level 15 minutes after completion of the anesthesia. Her postoperative course was uneventful. We conclude that total intravenous anesthesia by propofol and fentanyl is a preferable method for the management of the patient with mitochondrial encephalomyopathy.

Repeated in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure affects male gonads in offspring, leading to sex ratio changes in F2 progeny.

The effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the reproductive system of male rat offspring (F1) and the sex ratio of the subsequent generation (F2) were examined. Female Holtzman rats were gavaged with an initial loading dose of 400 ng/kg TCDD prior to mating, followed by weekly maintenance doses of 80 ng/kg during mating, pregnancy, and the lactation period. Maternal exposure to TCDD had no significant effects on fetus/pup (F1) mortality, litter size, or sex ratio on gestation day (GD) 20 or postnatal day (PND) 2. The TCDD concentration in maternal livers and adipose tissue on GD20 was 1.21 and 1.81 ng/kg, respectively, and decreased at weaning to 0.72 in the liver and 0.84 in the adipose tissue. In contrast, the TCDD concentration in pup livers was 1.32 ng/kg on PND2 and increased to 1.80 ng/kg at weaning. Ventral prostate weight of male offspring was significantly decreased by TCDD exposure on PND28 and 120 compared with that of controls. Weight of the testes, cauda epididymides, and seminal vesicle, and sperm number in the cauda epididymis were not changed by TCDD exposure at PND120. TCDD- or vehicle-exposed male offspring were mated with unexposed females. The sex ratio (percentage of male pups) of F2 offspring was significantly reduced in the TCDD-exposed group compared with controls. These results suggest that in utero and lactational TCDD exposures affect the development of male gonads in offspring (F1), leading to changes in the sex ratio of the subsequent generation (F2).

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats disrupts brain sexual differentiation.

The effects of in utero and lactational exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on brain sexual differentiation were investigated. TCDD was orally administered to pregnant Holtzman rats on gestation day (GD) 15, and the activity of brain aromatase, a key enzyme for sexual differentiation, was measured in offspring on postnatal day (PND) 2. Changes in sexual dimorphisms of saccharin preference and the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were examined in adult offspring. In controls, litter means of brain aromatase activity were higher in males than in females. In utero exposure to 200 ng/kg TCDD significantly decreased the sex ratio of aromatase activity (male/female) on PND 2. Offspring were weaned on PND28 and the saccharin test was started on PND84. In controls, saccharin (0.25%) intake (g/kg body weight) was significantly higher in female offspring than in males. In utero exposure to 200 ng/kg TCDD significantly increased saccharin intake in male offspring compared with control males, whereas 800 ng/kg TCDD had no effect. Neither dose of TCDD influenced saccharin intake of female offspring. In controls, SDN-POA volume was significantly greater in males than in females at 14 weeks of age. Exposure to 200 ng/kg TCDD significantly decreased SDN-POA volume in males, whereas 800 ng/kg TCDD had no effect. Neither doses of TCDD influenced the SDN-POA volume in female offspring. These results suggest that in utero and lactational TCDD exposure dose-dependently induces demasculinization in male offspring by inhibiting brain aromatase activity in the hypothalamus-preoptic area during central nervous system development.

Nitric oxide and effect of a radical scavenger N-tert-butyl-alpha-phenylnitrone on stroke in a rat model.

To characterize the role of nitric oxide (NO) in stroke, NO was measured using an in vivo microdialysis technique and electron spin resonance spectrometry in malignant stroke-prone spontaneously hypertensive rats (M-SHRSP), stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The brain dialysate NO level was higher in SHRSP than in WKY. NO was not detected in M-SHRSP hippocampus microdialysate after stroke except after the administration of N-tert-butyl-alpha-phenylnitrone (PBN). In addition, very little NO was generated in M-SHRSP brain tissue with hemorrhage. These data demonstrate an association between NO and stroke in M-SHRSP. Further, PBN administration results in maintenance of NO levels after stroke in M-SHRSP.

Protective effects of green tea catechins on cerebral ischemic damage.

BACKGROUND: Green tea catechins possess potent antioxidative properties, and the preventive effects against various oxidative diseases have been reported. The purpose of this study is to examine preventive and protective effects of green tea catechins on various deteriorative processes following stroke. MATERIAL/METHODS: Male Wistar rats were given ad libitum water with or without 0.25 and 0.5% tea catechin extract for 5 days prior to the operation and during the experiment. Right middle cerebral artery was occluded for 2 h, then reperfused for 22 h. Brain slices were stained with triphenyltetrazolim chloride to assess infarct area. Concentrations of plasma EGCg, and serum NOx were analyzed by HPLC. Detection of iNOS expression, neutrophil infiltration and peroxynitrite formation in the penumbra was performed by immunostain. Neurologic deficit was scored by posture reflex. RESULTS: Tea catechins dose-dependently reduced the brain infarct area and volume. Infarct volume was inversely correlated with plasma EGCg concentration. Dark staining for iNOS, neutrophils and peroxynitrite were observed in vessel wall of small arteries in control ischemic hemisphere, while in catechins (0.5%)-treated rats iNOS was detected slightly, and staining for neutrophils and peroxynitrite was not seen. Catechin ingestion blocked a 3-fold increase in serum NOx concentration in the jugular vein, and also reduced by 35% a 2-fold increase of plasma lipid peroxide level seen in control rats after reperfusion. Neurologic deficits were significantly alleviated by 0.5% catechin ingestion. CONCLUSIONS: Daily intake of green tea catechins efficiently protects the penumbra from irreversible damage due to cerebral ischemia, and consequent neurologic deficits.

Sterol-mediated regulation of hormone-sensitive lipase in 3T3-L1 adipocytes.

We previously reported that intracellular free cholesterol at physiological concentrations regulates the activity of neutral cholesterol esterase (N-CEase) in macrophages. The objective of the present study is to investigate whether the regulation of N-CEase by cholesterol is generally observed in other types of cells such as adipocytes with high activity of hormone-sensitive lipase (HSL), the same gene product as N-CEase. 3T3-L1 adipocytes were cultured with and without cholesterol (1-30 microg/mL) or 25-hydroxycholesterol (0.1-10 microg/mL), and changes in the N-CEase activity, expression of HSL mRNA, and protein were examined. Incubation (24 h) of cells with cholesterol did not change N-CEase activity, but incubation with 25-hydroxycholesterol decreased the activity in a concentration-dependent manner by 24 (24 h) and 54% (36 h). Quantitative reverse transcription-PCR indicated that 25-hydroxycholesterol (10 microg/mL) did not influence expression of HSL mRNA. However, Western blot analysis showed that this sterol reduced HSL protein by 72 (24 h) and by 93% (36 h), respectively. It was concluded that sterol-mediated regulation of HSL/N-CEase occurs not only in macrophages but also in adipocytes, and regulation appears to occur not at a transcriptional level but by a post-transcriptional process. Sterol-mediated proteolysis may be involved in the loss of HSL protein.

Concurrent formation of peroxynitrite with the expression of inducible nitric oxide synthase in the brain during middle cerebral artery occlusion and reperfusion in rats.

Peroxynitrite is assumed to play a crucial role in brain damage associated with the overproduction of nitric oxide (NO). The purpose of this study is to examine time-dependent changes of nitrite and nitrate (NOx) concentration in the circulation, and peroxynitrite formation as well as the expression of inducible nitric oxide synthase (iNOS) in the penumbra of rat brains during transient middle cerebral artery occlusion (MCAO) of Wistar rat for 2 h and reperfusion for 4-70 h. NOx concentration in the circulation was continuously monitored at the right jugular vein by microdialysis. The expression of iNOS was detected at 22-70 h after reperfusion in vascular walls and the cortex. Nitrotyrosine, a marker of peroxynitrite, appeared 4 h after reperfusion in the cortex, increasing substantially at 22-46 h in vascular walls. NOx level in dialysate increased immediately after MCAO. After a gradual decrease, the level increased again 4 h after reperfusion, reaching a maximum at 46 h. Brain myeloperoxidase activity, a marker of neutrophil infiltration, was not detected 4 h after reperfusion, but greatly increased at 22 h and then decreased. These results suggest that a marked increase of NOx level in the circulation might reflect the expression of iNOS, while neuronal NOS may contribute to peroxynitrite formation in the cortex observed at an earlier phase of reperfusion. This study indicates that monitoring NOx level in the circulation serves to assess the progress of stroke, and to determine appropriate therapeutic measures.

Fluctuation of serum NO(x) concentration at stroke onset in a rat spontaneous stroke model (M-SHRSP). Peroxynitrite formation in brain lesions.

Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) develop hypertension and stroke at earlier ages than do nonmalignant SHRSP. Our previous findings suggested that reactive oxygen species were involved in the development of stroke in this strain. Nitric oxide (NO) which is more released at ischemia, might play a crucial role in stroke development by producing peroxynitrite, a neurotoxic substance. This study investigated whether the development of cerebrovascular lesion in M-SHRSP could be assessed by the fluctuation of serum NO(x) concentration, and whether peroxynitrite is associated with brain damage. Serum NO(x) levels were examined using an automated NO detector. Stroke-onset was temporally assessed according to a known method: changes in body weight, water intake, and neurologic symptoms. Cerebral lesions were confirmed by magnetic resonance imaging (MRI), and Evans blue extravasation at autopsy. MRI taken just after estimated stroke onset disclosed brain lesions. The baseline serum NO(x) level remained at 15-18 micromol/l, but the level gradually increased prior to stroke, and significantly at stroke onset. A marked rise in serum NO(x) occurred subsequently at poststroke. Immunohistochemical staining of nitrotyrosine, a peroxynitrite marker, was detected around vessels, neuronal cells and parenchyma in cerebral lesions. Stroke occurred in 50% of male M-SHRSP at 80 days of age. In conclusion, this study provides the first evidence for fluctuation of serum NO(x) at the onset of spontaneous stroke accompanying the appearance of peroxynitrite in brain lesions. Monitoring serum NO(x) would serve to assess the development of brain lesions at least in spontaneous stroke model.

Changes in fetal brain aromatase activity following in utero 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in rats.

Aromatase catalyzes intraneuronal conversion of androgens to estrogens in the hypothalamus-preoptic area at a specific time during development. This local estrogen formation has a central role in sexual differentiation. To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sexual differentiation, TCDD (800 and 1600 ng/kg) was orally administered to pregnant Holtzman rats on gestation day (GD) 15, and the change in brain aromatase activity of fetuses (GD20) and pups (postnatal day 2) was examined. Litter means of brain aromatase activity in the hypo-preoptic area of control fetuses were higher in males than in females (female/male ratio was 0.7). This ratio of aromatase activity increased in a TCDD-concentration dependent manner, reaching 1 at the higher dose. There was a similar change in aromatase activity in brains of pups (PND 2). These results suggest that in utero TCDD exposure induces demasculinization in male offspring by inhibiting the aromatase activity in the brain during central nervous system development.