Visceral fat accumulation is associated with cerebral small vessel disease.

BACKGROUND AND PURPOSE: Obesity is associated with the risk of coronary artery disease and stroke. Visceral fat plays a significant role in the atherogenic effects of obesity. Whether visceral fat accumulation, as measured by computed tomography (CT), is an independent risk factor for the presence of cerebral small vessel disease (SVD) was investigated. METHODS: This study comprised 506 Japanese subjects 35-74 years of age (mean 55.3 years) without a history of symptomatic cerebrovascular disease who underwent health screening tests, including brain magnetic resonance imaging, carotid echography and measurements of the visceral fat area (VFA) and subcutaneous fat area (SFA) on abdominal CT. Visceral fat accumulation was defined as VFA ≥ 100 cm(2) . Logistic regression analysis was performed to examine the associations between visceral fat accumulation and cerebral SVD such as white matter lesions (WMLs) and silent lacunar infarction (SLI). RESULTS: The prevalence of WMLs and SLI but not carotid plaque were significantly higher in subjects with VFA ≥ 100 cm(2) than those with VFA < 100 cm(2) . A VFA ≥ 100 cm(2) was associated with WMLs and SLI independent of age, cardiovascular risk factors and other measurements of obesity, such as waist circumference and body mass index. A large waist circumference was independently associated with SLI. SFA, the combination of VFA and SFA, and body mass index were not associated with WMLs or SLI. CONCLUSIONS: Visceral fat accumulation was independently associated with the presence of cerebral SVD in subjects without a history of symptomatic cerebrovascular disease.

Activation of tyrosine hydroxylase prevents pneumonia in a rat chronic cerebral hypoperfusion model.

Pneumonia is a common complication with the highest attributable proportion of deaths in patients with stroke. Cilostazol is a potent type III phosphodiesterase inhibitor, approved as an anti-platelet aggregation agent. The present study was designed to determine the protective mechanism of cilostazol against post-stroke pneumonia using a rat chronic cerebral hypoperfusion model. Rats were subjected to bilateral common carotid artery ligation (LBCCA) and divided randomly into the vehicle group (n=72) and cilostazol group (n=72). Rats of each group were sacrificed at baseline and at days 14, 28 and 42 after LBCCA. Cilostazol significantly improved the swallowing reflex by shortening the latency to elicited swallowing and increasing the numbers of swallows (P<0.05) at 14 days of hypoperfusion. It also decreased the numbers of bacterial colonies grown in cultures from homogenized lungs. Cilostazol markedly upregulated cyclic AMP responsive element binding protein (CREB) phosphorylation, increased tyrosine hydroxylase (TH) expression in the substantial nigra, and maintained dopamine (84.7+/-2.3 vs. 79.2+/-4.1% control; P=0.0512) and substance P levels (86.6+/-7.9 vs. 73.9+/-6.5% control; P<0.05) in the striatum, compared with the vehicle group. Our results indicate that cilostazol improves the swallowing reflex by enhancing the expression of TH through the CREB phosphorylation signaling pathway, and suggest that cilostazol could be useful in preventing pneumonia in the chronic stage of stroke.

The effect of magmatic activity on hydrothermal venting along the superfast-spreading East pacific rise.

A survey of hydrothermal activity along the superfast-spreading (approximately 150 millimeters per year) East Pacific Rise shows that hydrothermal plumes overlay approximately 60 percent of the ridge crest between 13 degrees 50' and 18 degrees 40'S, a plume abundance nearly twice that known from any other rige portion of comparable length. Plumes were most abundant where the axial cross section is inflated and an axial magma chamber is present. Plumes with high ratios of volatile ((3)He, CH(4), and H(2)S) to nonvolatile (Mn and Fe) species marked where hydrothermal circulation has been perturbed by recent magmatic activity. The high proportion of volatile-rich plumes observed implies that such episodes are more frequent here than on slower spreading ridges.

Focal cerebral ischemia/reperfusion injury in mice induces hematopoietic prostaglandin D synthase in microglia and macrophages.

Hematopoietic prostaglandin D synthase is a key enzyme in synthesis of prostaglandin D. Hematopoietic prostaglandin D synthase is expressed in microglia of the developing mouse brain. This study determined the serial changes and cellular localization of hematopoietic prostaglandin D synthase, and its role in cerebral ischemia/reperfusion injury using C57BL/6 mice (n=84) and bone marrow chimera mice (n=16). The latter mice were selected based on their expression of enhanced green fluorescent protein in bone marrow/blood-derived monocytes/macrophages. The middle cerebral artery was occluded for 60 min, followed by reperfusion. Hematopoietic prostaglandin D synthase expression was examined by immunohistochemistry and Western blotting. Hematopoietic prostaglandin D synthase-positive cells were mainly expressed in the peri-ischemic area at 12 h (P<0.05) and 24 h (P<0.001) after reperfusion, while they were mostly found in the transition area at 48-72 h postreperfusion (P<0.001). There was a significant increase in staining intensity as well as number of hematopoietic prostaglandin D synthase-positive cells in the ischemic core at 5-7 (P<0.001) days postreperfusion. Hematopoietic prostaglandin D synthase-positive cells also co-expressed ionized calcium-binding adapter molecule 1, a marker of microglia/macrophages, and cyclooxygenase-2, but not markers of neurons, oligodendrocytes and astrocytes. Until 72 h postreperfusion, many enhanced green fluorescent protein-positive cells were negative for hematopoietic prostaglandin D synthase, but the number of hematopoietic prostaglandin D synthase-enhanced green fluorescent protein coexpressing cells increased significantly at 5-7 days after reperfusion. Our results indicate that hematopoietic prostaglandin D synthase is mainly produced by endogenous microglia until 72 h after reperfusion, but at 7 days after reperfusion, it is also produced by migrating bone marrow/blood-derived macrophages in the ischemic brain tissue. We speculate that hematopoietic prostaglandin D synthase in the brain has different functions during early and late phases of ischemia.

Induction and selective accumulation of mutant ubiquitin in CA1 pyramidal neurons after transient global ischemia.

Accumulation of mutant ubiquitin-B (UBB(+1)) in neurons is considered the hallmark of proteasomal dysfunction in neurodegenerative disorders, however no such evidence in ischemic brain has been reported. We investigated the contribution of UBB(+1) in delayed neuronal death after transient global ischemia. Transient global ischemia was achieved by occlusion of bilateral common carotid arteries for 5 min and reperfusion in male Mongolian gerbils (n=6 per each time point). In the CA1 region, UBB(+1) immunoreactivity appeared in the cytoplasm of pyramidal cells at 30 min post-ischemia, and the density of these neurons increased at day 2 (P<0.001) and further increased at day 4 post-ischemia. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive (apoptotic) cells appeared selectively in the CA1 region at day 3 and their density increased further at day 4 post-ischemia (P<0.001). In contrast, UBB(+1) immunoreactivity was only transiently detected from 30 min to 1 day post-ischemia in CA3, dentate gyrus, and frontal cortex, but disappeared at day 2 post-ischemia. No TUNEL-positive cells were observed in these three regions. UBB(+1) mRNA was detected by reverse transcription-polymerase chain reaction in every region of the hippocampus and frontal cortex of ischemic gerbils and even in the non-ischemic control animals, and its expression level was independent of brain region and time after ischemia. Our results indicate induction and selective accumulation of UBB(+1) protein in dying neurons of the CA1 region and suggest that UBB(+1) expression may be induced by proteasomal dysfunction after transient global ischemia.

Carrier-mediated transport system for cephalexin in human placental brush-border membrane vesicles.

The uptake of cephalosporin antibiotics, cephalexin, was studied with brush-border microvillous plasma membrane vesicles prepared and purified from human full-term placental syncytiotrophoblasts. The uptake of cephalexin by the membrane vesicles was not stimulated in the presence of an Na+ gradient from the outside to the inside of the vesicles, whereas alpha-(methylamino)isobutyrate uptake into the vesicles of the same preparation was stimulated by an Na+ gradient. The equilibrium level of cephalexin uptake decreased with increasing osmolarity of the medium, which indicates that cephalexin is transported into the membrane vesicles. When cephalexin concentrations were varied, the initial rate of uptake obeyed Michaelis-Menten kinetics with Km and Vmax values of 2.29 mM and 2.98 nmol/mg of protein per 60 s, respectively. The uptake of cephalexin was inhibited by structural analogues and sulfhydryl modifying reagents. These results indicate the existence of a carrier-mediated transport system for cephalexin in the human placental brush-border membranes.

Type 2 diabetes reduces the proliferation and survival of oligodendrocyte progenitor cells in ishchemic white matter lesions.

Diabetes mellitus (DM) is a major risk factor for stroke and it exacerbates tissue damage after ischemic insult. Diabetes is one of the important causes of the progression of white matter lesion, however, the pathological mechanisms remain unclear. The present study evaluated the influences of type 2 DM on ischemic subcortical white matter injury and the recruitment of oligodendrocyte progenitor cells (OPCs) under chronic cerebral hypoperfusion using type 2 diabetic (db/db) mice. After bilateral common carotid artery stenosis (BCAS), the rarefaction in the white matter was more severe in db/db mice than in db/+ mice, and the number of glutathione S-transferase-pi (GST-pi)-positive mature oligodendrocytes (OLG) was lower in db/db mice than in db/+ mice at 4 and 8 weeks after ischemia. There were no significant differences in the number of single-stranded DNA (ssDNA)-positive apoptotic cells in the deep white matter between the db/db and db/+ mice. We found a transient increase in the platelet-derived growth factor receptor-α (PDGFRα)-positive OPCs in white matter lesions after ischemia. However, significantly fewer PDGFRα-positive OPCs were detected in db/db than db/+ mice from 4weeks after BCAS. The number of Ki67-positive proliferating cells in the deep white matter was significantly lower in db/db mice than in db/+ mice from 4 to 8weeks after BCAS. Most of the Ki67-positive cells were PDGFRα-positive OPCs. Finally, we assessed the survival of 5-bromo-2'-deoxyuridine (BrdU)-positive proliferating cells in ischemic white matter, and found significantly poorer survival of BrdU/PDGFRα-positive OPCs or BrdU/GST-pi-positive OLGs in the db/db mice compared to the db/+ mice in the white matter after BCAS. Our findings suggest that the type 2 DM mice exhibited more severe white matter injury 8 weeks after chronic ischemia. Decreased proliferation and survival of OPCs may play an important role in the progression of white matter lesions after ischemia in diabetics.

Polymorphism of the lipoprotein lipase gene and risk of atherothrombotic cerebral infarction in the Japanese.

BACKGROUND AND PURPOSE: Lipid and lipoprotein abnormalities have been implicated in the pathogenesis of ischemic cerebrovascular disease and atherosclerosis. Lipoprotein lipase (LPL) plays an important role in plasma lipoprotein metabolism. Several studies have recently reported the presence of a relationship between Ser447Stop mutation of LPL and coronary artery disease. Other polymorphisms (HindIII and PvuII) of the LPL gene have already been shown to correlate significantly with dyslipidemia. We investigated whether these polymorphisms are associated with increased risk of ischemic cerebrovascular disease (CVD). METHODS: We recruited 177 CVD patients (atherothrombotic infarction, n=71; cardioembolic infarction, n=30; lacunar infarction, n=76) and 177 healthy control subjects. Subjects were genotyped for the Ser447Stop mutation and for HindIII/PvuII restriction fragment length polymorphisms of the LPL gene, and the findings were investigated for associations with the clinical subtypes of CVD and with lipid levels. RESULTS: The Ser447Stop mutation correlated significantly with CVD (0.107 versus 0.158; P=0.035). For the CG+GG versus CC genotype, the odds ratio between control subjects and CVD patients with atherothrombotic infarction was 0.42 (95% CI, 0.18 to 0.99) (P=0.046). Serum HDL cholesterol and triglyceride levels did not correlate significantly with the Ser447Stop genotype. HindIII polymorphism correlated significantly with CVD (0.234 versus 0.169; P=0.031), but the frequency of PvuII polymorphism was not significantly different between groups. CONCLUSIONS: Our results suggest that the Ser447Stop mutation of the LPL gene is a novel genetic marker for low risk of atherothrombotic cerebral infarction.

Accumulation of 4-hydroxynonenal-modified proteins in hippocampal CA1 pyramidal neurons precedes delayed neuronal damage in the gerbil brain.

It has been proposed that reactive oxygen species and lipid peroxidation have a role in the delayed neuronal death of pyramidal cells in the CA1 region. To explore the in situ localization and serial changes of 4-hydroxy-2-nonenal-modified proteins, which are major products of membrane peroxidation, we used immunohistochemistry of the gerbil hippocampus after transient forebrain ischemia with or without preconditioning ischemia. The normal gerbil hippocampus showed weak immunoreactivity for 4-hydroxy-2-nonenal-modified proteins in the cytoplasm of CA1 pyramidal cells. 4-hydroxy-2-nonenal immunoreactivity showed no marked changes after preconditioning ischemia. In the early period after ischemia and reperfusion, there was a transient increase of nuclear 4-hydroxy-2-nonenal immunoreactivity in CA1 pyramidal neurons. In contrast, cytoplasmic immunoreactivity transiently disappeared during same period and then increased markedly from 8h to seven days. One week after ischemia, 4-hydroxy-2-nonenal immunoreactivity was observed within reactive astrocytes in the CA1 region. Early nuclear accumulation of 4-hydroxy-2-nonenal in CA1 neurons may indicate a possible role in signal transduction between the nucleus and cytoplasm/mitochondria, while delayed accumulation of 4-hydroxy-2-nonenal-modified proteins in the cytoplasm may be related to mitochondrial damage. We conclude that 4-hydroxy-2-nonenal may be a key mediator of the oxidative stress-induced neuronal signaling pathway and may have an important role in modifying delayed neuronal death.

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia.

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48-72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.

Effects of delayed nerve repair on regeneration of rat sciatic nerve.

The optimal time for nerve suture in transected rat sciatic nerve was investigated. The sciatic nerve on both side nerves were transected and repaired by an epineurial suture technique 0-28 days after nerve transection. The regeneration distance was measured by the sensory pinch reflex test 2-6 days after nerve repair. The initial delay period and the regeneration rate were calculated by using a new mathematical model. One day delayed repair was sufficient to reduce the initial delay period and the largest reduction on the initial delay period was observed in the three day delayed repair group. The initial delay period was reduced to 0.87 days as compared to 2.31 days for nerves repaired immediately (controls). The seven day and ten day delayed repair had similar effects but they also showed a decrease of the regeneration rate. The regeneration rate was not affected except for these two time points. The results suggest that the repair delay period can influence the initial delay as well as the regeneration rate. In our model an optimal effect was achieved on both these measures with a one or three day delay between transection and repair.

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.

Postischemic accumulation of lipid peroxidation products in the rat brain: immunohistochemical detection of 4-hydroxy-2-nonenal modified proteins.

We report an immunohistochemical study on the distribution and alterations of 4-hydroxy-2-nonenal (HNE)-modified proteins, an indicator of lipid peroxidation, in the rat brain after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. HNE immunoreactivity was not observed in intact neurons, but it appeared in some shrunken neurons within the infarcted zone at 3 h after reperfusion. The number of HNE-positive neurons increased with the spread of the infarcted area. The pyramidal neurons in the third layer of the frontoparietal cortex were HNE-positive and the intensity of their HNE immunoreactivity was highest at 24 h after reperfusion. At 48 h, HNE-positive neurons were observed in the medial part of the striatum, the lateral side of the frontoparietal cortex, and at the boundary between the infarcted and noninfarcted zones. In addition, strong HNE immunoreactivity was seen in microglia (identified by OX-42 immunostaining). This method seems to be useful to follow the progress of lipid peroxidation at the cellular level after ischemic injury.

Glucose metabolism in cholinoceptive cortical rat brain regions after basal forebrain cholinergic lesion.

To address the question whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease are interrelated with, or consequences of, basal forebrain cholinergic cell loss, an experimental approach was employed to produce cortical cholinergic dysfunction in rat brain by administration of the cholinergic immunotoxin 192IgG-saporin. [14C]D-glucose utilization in brain homogenates, D-glucose-displaceable [3H]cytochalasin B binding to glucose transporters (GLUT). Northern and Western analyses, as well as in vivo [14C]2-deoxyglucose autoradiography were used to quantify the regional glucose metabolism. Basal forebrain cholinergic lesion resulted in transient increases in glucose transporter binding in cortical regions displaying reduced acetylcholinesterase activity, already detectable seven days after lesion with peak values around 30 days post lesion. Western analysis revealed that the changes in total glucose transporter binding are mainly due to changes in the GLUT3 subtype only, while the levels of GLUT1 and GLUT3 mRNA (Northern analysis) were not affected by cholinergic lesion. Both immunocytochemistry and in situ hybridization demonstrated preferential localizations of GLUT1 on brain capillaries and GLUT3 on neurons, respectively. A lesion-induced transient decrease in [14C]D-glucose utilization seven days post lesion was detected in the lesion site, whereas cholinoceptive cortical regions were not affected. In vivo [14C]deoxyglucose uptake was transiently increased in cholinoceptive cortical regions and in the lesion site being highest between three to seven days after lesion. The cholinergic lesion-induced transient up-regulation of cortical glucose transporters and deoxyglucose uptake reflects an increased glucose demand in regions depleted by acetylcholine suggesting functional links between cortical cholinergic activity and glucose metabolism in cholinoceptive target regions.

Colocalization of Bcl-2 and 4-hydroxynonenal modified proteins in microglial cells and neurons of rat brain following transient focal ischemia.

Bcl-2 has a role in suppressing the production of reactive oxygen species and lipid peroxidation. To explore the in situ localization of 4-hydroxy-2-nonenal (HNE)-modified proteins and the Bcl-2 oncoprotein, we used double immunofluorescence labeling and confocal imaging in the rat brain after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. Immunoreactivity for HNE or Bcl-2 was not detected at 1 h, but appeared in some intact neurons in the boundary between the infarcted and non-infarcted zones at 12 h. At 48 h, HNE-positive microglia were colocalized with Bcl-2 in the infarcted area and the boundary zone. Bcl-2 may play an important role in the antioxidant system promoting survival of the neurons and activated microglia following reperfusion injury.

Differentiation of embolic and thrombotic middle cerebral artery occlusion using ultrasonic carotid flow velocity analysis.

The purpose of this study was to determine the value of Duplex ultrasound of the carotid arteries in the differentiation of embolic from thrombotic middle cerebral artery (MCA) occlusion. We report here the results of carotid Duplex ultrasound study from 164 patients with acute ischemic stroke. Flow velocity and diameter were measured in bilateral common carotid arteries (CCA). The end-diastolic flow velocity (Ved) and the pulsatility index (PI) were calculated from Doppler waves. The PI is an index of peripheral vascular resistance. We compared the relationship between percent carotid stenosis and percent decrease in Ved. The patients studied could be classified into three groups using ultrasound parameters. Group I was characterized by > 30% decrease in Ved and < 80% carotid stenosis, group II by < 30% decrease in Ved and < 80% carotid stenosis, and group III by > 30% decrease in Ved and > 80% carotid stenosis. All 23 patients in group I had embolic MCA stem occlusion. 28 out of 115 patients in group II had thrombotic MCA stem occlusions. All 26 patients in group III had internal carotid artery occlusion or severe stenosis. Ved was markedly reduced in group I and group III compared to group II (p < 0.01). PI in the affected artery was increased in groups I and III (p < 0.01). Embolic occlusion was characterized by > 30% decrease in Ved in the absence of > 80% carotid stenosis, and an increase in PI. The results indicate that these two conditions can be differentiated using Duplex ultrasound in carotid arteries.

Interferon-alpha modulates resistance to cisplatin in three human hepatoma cell lines.

We investigated the expression of the drug resistance-related genes, multidrug resistance gene 1 (MDR1), multidrug resistance associated protein gene (MRP), and the DNA topoisomerase IIalpha, DNA topoisomerase IIbeta, and glutathione-S-transferase pi gene (GST-pi) in three human hepatoma cell lines (HepG 2, HuH 7, SK-Hep-1) with or without drug treatment with interferon-alpha (IFN-alpha) and cisplatin (CDDP), by a reverse transcription-polymerase chain reaction (RT-PCR) method and a competitive PCR method. The signals of the MDR1, MRP, topoisomerase IIalpha, and topoisomerase IIbeta genes in HepG2 were weakened when IFN-alpha was added to CDDP. In SK-Hep-1, the administration of CDDP alone increased the signals of MDR1 while the addition of IFN-alpha decreased the signals, and the signals of GST-pi were decreased by IFN-alpha plus CDDP. In summary, our results concerning the expression of drug resistance-related genes in three human hepatoma cell lines demonstrate that IFN-alpha may modulate the mechanism of resistance to CDDP in liver cancer.

Distant metastasis of hepatocellular carcinoma after successful treatment of the primary lesion.

Recent advances in both diagnosis and treatment of hepatocellular carcinoma (HCC) have improved the prognosis and changed the clinical significance of the subsequently increasing distant metastases. Of 163 patients with HCC, 76 (47%) were treated successfully. The cumulative recurrence rate in these patients was 65% after 3 years, and 82% after 5 years. Six patients with HCC in whom distant metastases were detected after successful treatment of the primary lesion, are reported. Two patients underwent curative surgical resection, and four cases were treated medically, resulting in a 31.9 to 94.3% reduction in tumor size in the CT scan image. Distant metastases without intrahepatic recurrence were diagnosed 10 to 46 months after the treatment of the primary lesions. The sites of the metastases included bone 3; lung 2; and adrenal gland 1. Distant metastases found after successful treatment of the primary lesions are of great clinical significance for the treatment of HCC.