Michio Yamaguchi

Reactive Oxygen Species in Reoxygenation Injury of Rat Brain Capillary Endothelial Cells

OBJECTIVE To clarify the mechanism of anoxia/reoxygenation (A/R) injury of rat brain capillary endothelial cells (BCEC). METHODS BCEC isolated from Sprague-Dawley rats by enzymatic treatment and centrifugation were subjected to anoxia (95% N2, 5% CO2) for 20 minutes and then to reoxygenation (95% air, 5% CO2) for 3 hours. Enzyme inhibitors, including oxypurinol, indomethacin, and N(G)-nitro-L-arginine methyl ester, or specific free-radical scavengers, such as superoxide dismutase, catalase, and the ferric iron chelator deferoxamine, were added before A/R injury. The BCEC were incubated in a range of Ca2+ concentrations from 1 to 0.01 mmol/L during A/R injury. Cytotoxicity was assayed by release of intracellular lactate dehydrogenase (LDH). RESULTS With A/R injury, LDH release from the control group (no protective agents) significantly increased (44.8 +/- 3.3%), compared with a small increase in a normoxic group. BCEC treated with oxypurinol, indomethacin, or N(G)-nitro-L-arginine methyl ester showed suppression of LDH release. LDH release was almost totally suppressed by superoxide dismutase and partially by catalase or deferoxamine. The LDH release was partly dependent on calcium concentration. CONCLUSION BCEC subjected to A/R become potent generators of free radicals, especially superoxide anion. Free radical production depends on both xanthine oxidase and cyclooxygenase pathways. Peroxynitrite and extracellular Ca2+ both contribute importantly to reoxygenation injury of BCEC.

The Role of Calcium Ion in Anoxia/Reoxygenation Damage of Cultured Brain Capillary Endothelial Cells

Capillary endothelial cells are critical targets in both ischemia and reperfusion of the brain. Arachidonic acids and oxygen free radicals have been shown to cause disruption of blood-brain barrier (BBB) by destruction of capillary endothelial cell membrane. However, the exact mechanism of BBB breakdown by cerebral ischemia/reperfusion remains undetermined. The aim of the present study is to clarify the mechanism of intracellular calcium ion ([Ca2+]i) change in brain capillary endothelial cells under anoxia/reoxygenation. Brains capillary endothelial cells were isolated from ten male Sprague-Dawley rats by a two step enzymatic process. [Ca2+]i was measured by means of a confocal laser scanning microscope using Indo 1-A/M as a calcium indicator. The endothelial cells were subjected to anoxia and reoxygenization under different conditions. [Ca2+]i increased gradually during anoxia and slightly decreased after reoxygenation. Indomethacin and SOD suppressed the elevation of [Ca2+]i during anoxia. NG-nitro-L-arginine methyl ester and catalase moderately suppressed the elevation, however nifedipine did not suppress it at all. In this model, rapid [Ca2+]i change was not observed during the reoxygenation phase. The results indicate that the anoxia induced elevation of [Ca2+]i in the brain capillary endothelial cells depends on superoxide and peroxynitrite generation.

The Mechanism of Reversible Osmotic Opening of the Blood-Brain Barrier: Role of Intracellular Calcium Ion in Capillary Endothelial Cells

Despite clinical and experimental interest in the osmotic opening of the blood-brain barrier (BBB), the mechanism underlying the phenomenon remain undetermined. The aim of this study is to investigate the mechanism of intracellular Ca2+ change in brain microvascular endothelial cells subjected to hyperosmotic stress. Cultured rat brain capillary endothelial cells were obtained by two-step enzymatic purification. Intracellular Ca2+ was measured by a confocal laser scanning microscope. After exposing the endothelial cells to 1.4 M mannitol for 30 seconds, the change of intracellular Ca2+ concentration was monitored. Intracellular Ca2+ concentration increased rapidly and reached its peak value within 10 seconds after the application of mannitol. The Ca2+ concentration returned to the basal level within 200 seconds. A calcium channel blocker nifedipine (100 microM, 10 microM) did not block the increase. A specific blocker (KB-R7943) of Na+/Ca2+ exchange did not affect the rapid elevation of intracellular Ca2+. However, it blocked the return phase almost completely. The results indicated that the Na+/Ca2+ exchanger pumped out the increased intracellular Ca2+ during the return phase. Reversible osmotic disruption and reconstruction of the BBB is not due to simple mechanical shrinkage of the endothelial cells but is due to the intracellular Ca(2+)-activated complex mechanism. The manipulation of the reconstruction phase, which depends on Na+/Ca2+ exchanger, may have clinical implications.

Congenital frontal bone defect with intact overlying scalp

An unusual case of a congenital frontal bone defect with intact overlying scalp and intact underlying dura mater is reported. Although spontaneous healing by the intact underlying dura mater by regeneration was expected, it did not occur. Cranioplasty was done for protective and cosmetic purposes. A review of the literature of congenital skull defects shows that spontaneous regeneration does not occur in this rare anomaly.

The Mechanism of Free Radical Generation in Brain Capillary Endothelial Cells After Anoxia and Reoxygenation

We studied the mechanism of reoxygenation injury of cerebral microvessels in cultured rat brain capillary endothelial cells (BCECs). BCECs were isolated from rat cerebral cortices by a two step enzymatic treatment. The monolayers of BCECs were subjected to anoxia for 20 minutes followed by reoxygenation for 3 hours. Cell damage was assessed by measuring the leakage of intracellular lactic dehydrogenase (LDH). The control group was anoxia/reoxygenated BCECs without any protective reagents. To study the protective effect of free radical scavengers and antioxidants, superoxide dismutase, catalase, deferoxamine, oxypurinol, indomethacin, or NG-nitro-L-arginine methyl ester (L-NAME) was applied during anoxia/reoxygenation. Thus 7 experimental conditions were established. Lactic dehydrogenase (LDH) leaked from reoxygenated BCECs due to cell membrane damage. This leakage was almost totally suppressed by superoxide dismutase, indicating that reoxygenation injury of BCECs is mediated by superoxide generation. The other scavengers and antioxidants partially suppressed LDH leakage. Reduction of Ca2+ in the culture medium from 1.6 mM to 0.016 mM also suppressed LDH leakage. These results indicate that BCECs subjected to anoxia/reoxygenation become potent generators of superoxide anion, which is thought to be responsible for reoxygenation injury. The superoxide generation partially depends on the xanthine oxidase and cyclooxygenase pathways. As L-NAME partially suppressed LDH leakage peroxynitrite may contribute to reoxygenation injury of BCECs. The extracellular Ca2+ concentration also plays a critical role in the reoxygenation injury of BCECs.

Cerebral ischemia alters glucose transporter kinetics across rat brain microvascular endothelium. Quantitative analysis by an in situ brain perfusion method.

The purpose of this study was to quantify the changes of blood-brain barrier glucose transporter kinetics following cerebral ischemia using an in situ brain perfusion technique. Sixty-four adult male Sprague-Dawley rats were divided into control and ischemia groups, and a four-vessel occlusion model was used to provide an ischemic insult. To obtain regional capillary permeability area products of glucose and regional perfusion fluid flow rates, the perfusion fluid was dually labeled with 2-deoxy[14C]glucose and [3H]diazepam, and the brain was perfused at a constant rate via the external carotid artery. After sampling tissues from the brain, dual scintillation counting was performed and both regional perfusion fluid flow rates and regional capillary permeability area products were calculated. We determined kinetic parameters, including Vmax, Km and Kd as described in the Michaelis-Menten equation, by the non-linear least squares method. In the ischemia group, a decrease in Vmax and an increase in Km were recognized, which mean decreases in the affinity and the number of functioning glucose transporters. These results suggest that cerebral ischemia downregulates the blood-brain barrier glucose transporters.

The Effect of Leukotriene C4 on the Permeability of Brain Capillary Endothelial Cell Monolayer

The role of leukotrienes as mediator of brain edema is still controversial. Recently, the ability of gamma-GTP to act as enzymatic barrier and to inactivate leukotrienes in normal brain capillaries was pointed out. A hypothesis tested in our experiments was that Leukotriene C4 (LTC4) increases permeability of a cerebral capillary endothelial monolayer which lacks gamma-GTP activity. Brain capillary endothelial cells were obtained of 10 rats from cerebral cortex by an enzymatic isolation procedure. The cells have an intact function, however, lack gamma-GTP activity. The endothelial cells were cultured on an optically clear collagen membrane mounted on a plastic frame. Effects of bradykinin (1 x 10(-5) M) and LTC4 (1 x 10(-7) M, 1 x 10(-6) M, 5 x 10(-6) M, 1 x 10(-5) M) were tested on permeability of the endothelial cell monolayer by measuring leakage of 14C-sucrose. The effect of LTC4 and bradykinin on intracellular calcium was studied by laser scanning confocal microscopy. LTC4 did not increase permeability of the brain capillary endothelial cell monolayer which lacked gamma-GTP activity. LTC4 did neither increase the concentration of intracellular calcium. Differences of LTC4 receptor function in normal brain capillaries and tumor capillaries remain to be studied.

Reoxygenation Injury of Human Brain Capillary Endothelial Cells

Abstract1. Many studies have demonstrated that endothelial cells from several species can generate oxygen free radicals when subjected to anoxia and reoxygenation. However, due to the heterogeneity of the endothelium within different organs and species, the effects of superoxide dismutase (SOD), catalase, and allopurinol on reoxygenated cultured cells remain quite controversial.2. This review outlines the possible sources of oxygen free radicals within brain endothelial cells.3. We examine the aspects of the effects of SOD catalase and allopurinol on cultured human brain capillary endothelial cells upon reoxygenation.4. Also, we introduce briefly a method of culturing human brain capillary endothelial cells and present our experimental results on the effects of SOD, catalase, and allopurinol in these cultured cells following anoxia and reoxygenation.

Cerebral Blood Flow of Rats with Water-Intoxicated Brain Edema

Water intoxication brain edema was produced in rats by intraperitoneal loading of excessive amounts of distilled water (DW). In 10% and 20% groups, DW in amounts of 10 or 20% of body weight was injected, respectively. Water content of brain tissue increased proportionally to the amount of injected water, as follows: 79.8% of wet weight in control, 80.5 and 82.4 in 10% and 20% DW groups, respectively. Since cerebral blood flow (CBF) values measured by laser Doppler (LD) flowmetry were found to give a good correlation with those by hydrogen clearance method in a preliminary experiment, CBF measurement were carried out using LD flowmetry thereafter. Before the injection, CBF values were around 50 ml/min/100 g. Two hours after the water loading, CBF values in 10% and 20% DW groups were 25.6 and 20.3 ml/100 g/min, respectively. CBF values under these edematous condition decreased significantly (p < 0.001 by paired t-test) in proportion to the severity of the brain edema.

Impaired Learning of Active Avoidance in Water-Intoxicated Rats

Brain edema is an important clinical condition. Pathophysiological findings on behavioral changes may be helpful for a comprehensive understanding of brain edema. However, only few reports on behavioral studies of brain edema have so far appeared. Experiments using psychological techniques on animals are rather time-consuming and may not be suitable for the study of transient conditions, as brain edema caused by trauma, vascular accidents, or others. We have developed a method for avoidance learning of rats using a running wheel apparatus with computer assistance. This model was employed in studies on brain edema from water introxication in rats. As a result, avoidance learning was significantly impaired by water intoxication. Either direct overhydration of the brain or indirect effects, as a decrease in cerebral blood flow, or both, are suggested as mechanisms underlying the impairment of behavior.