Keisuke Tsutsumi

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke‐prone spontaneously hypertensive rats subjected to a 10‐min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of 125I‐ET‐1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ETB receptor. The de novo 125I‐ET‐1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET‐1‐ and ET‐3‐like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ETB receptor are activated to participate in the pathophysiology of ischemia‐related neural tissue damage by astrocytic ET‐1 and ET‐3 produced in response to transient forebrain ischemia would have to be considered.

Estrogens regulate angiotensin-converting enzyme and angiotensin receptors in female rat anterior pituitary

We studied the effects of the estrous cycle, ovariectomy and estrogen replacement on angiotensin-converting enzyme (ACE) (kininase II, EC 3.4.15.1) and angiotensin II (AT) receptors in the pituitary gland of the female rat. Quantitative autoradiography, with the use of consecutive pituitary sections, allowed for simultaneous determination of changes in binding and in the potential AT synthetic ability of individual pituitaries, and for a correlation between these two phenomena. In the anterior pituitary, ACE activity and binding of the ACE inhibitor [125I]-351A were not changed during the estrous cycle. Ovariectomy produced a significant increase in ACE activity and binding, and both of these parameters returned to normal after estrogen replacement. There were no changes in ACE activity or binding in the posterior pituitary during the estrous cycle or after ovariectomy or hormone replacement. AT receptors were characterized as of the AT1 type, since they were displaced by the selective AT1 antagonist DuP 753 and not by the AT2 competitor PD 123177. There were marked changes in the concentration of AT1 receptors during the estrous cycle, with highest numbers in metestrus, lower in estrus and diestrus, and lowest during proestrus. Estrogen replacement in ovariectomized rats decreased AT1 receptor number in the anterior pituitary. Our results indicate a dual effect of estrogen on anterior pituitary AT, physiologically on AT receptor expression and pharmacologically on ACE activity.

Adrenomedullin Improves the Blood–Brain Barrier Function Through the Expression of Claudin-5

Summary1. Aims: Brain vascular endothelial cells secret Adrenomedullin (AM) has multifunctional biological properties. AM affects cerebral blood flow and blood–brain barrier (BBB) function. We studied the role of AM on the permeability and tight junction proteins of brain microvascular endothelial cells (BMEC).2. Methods: BMEC were isolated from rats and a BBB in vitro model was generated. The barrier functions were studied by measuring the transendothelial electrical resistance (TEER) and the permeability of sodium fluorescein and Evans’ blue albumin. The expressions of tight junction proteins were analyzed using immunocytochemistry and immunoblotting.3. Results: AM increased TEER of BMEC monolayer dose-dependently. Immunocytochemistry revealed that AM enhanced the claudin-5 expression at a cell–cell contact site in a dose-dependent manner. Immunoblotting also showed an overexpression of claudin-5 in AM exposure.4.Conclusions: AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.

Angiotensin II receptor subtypes and phosphoinositide hydrolysis in rat adrenal medulla.

Angiotensin II (ANG) receptor subtypes were characterized by quantitative autoradiography after incubation with the ANG agonist [124I]Sar1-ANG in rat adrenal medulla. ANG receptors are highly localized in adrenal medulla. Specific binding was displaced by 4% and by 95% with the AT, receptor blocker losartan and the AT2 receptor competitor CGP 42112A, respectively. Analysis of competition curves indicated relative binding potencies for the AT2 population of CGP 42112A>PD 123319> PD 123177. ANG stimulated +-nositol phosphate formation in a dose-dependent manner in rat adrenal medulla. Losartan at concentrations of 10(-9) to 10(-5) M antagonized the effect of ANG, whereas PD 123177 or PD 123319 had no antagonistic action. However, at a higher concentration (10(-5) M) PD 123177 or PD 123319 potentiated the effect of ANG on InsP1-accumulation. In the presence of PD 123319 (10(-5) M) ANG dose-response curve was shifted to the left with no change in the maximal effect. This affect was blocked by the addition of losartan (10(-5) M). On the contrary, the addition of CGP 42112A (10(-6) M) inhibited ANG-induced increase in InsP1-accumulation. On the other hand, ANG and CGP 42112A reduced basal cyclic GMP formation, this effect was partially reverted by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. Our results further demonstrate the presence of two ANG receptor subtypes in adrenal medulla: ANG binding to AT, receptor stimulates inositol phospholipid metabolism, whereas ANG binding to AT2 receptors decreases both inositol phosphate production and cGMP formation.

Retrospective analysis of neurological outcome after intra-arterial thrombolysis in basilar artery occlusion

BACKGROUND Basilar artery occlusion usually has a very poor outcome and is associated with a high mortality rate. Local intra-arterial thrombolysis may improve the clinical outcome and reduce mortality in the treatment of acute basilar artery occlusion. We evaluated the possible variables affecting recanalization and clinical outcome in patients with basilar artery occlusions undergoing thrombolytic therapy. METHODS We analyzed retrospectively the clinical course and outcome of a series of 26 patients between 1998 and 2001. All patients who were examined within 24 hours after onset of symptoms underwent emergency cerebral angiography and subsequent intra-arterial thrombolysis. Three patients additionally received percutaneous transluminal angioplasty of underlying stenosis at the site of thrombosis. RESULTS Outcome was good in 9 patients (34.6%) and poor in 17 (65.4%). Recanalization could be achieved in 24 patients (92.3%) and was not affected by age, sex, site of occlusion, etiology, thrombolytic drugs, or time interval. Good outcome was associated with younger age, good initial clinical condition, and no evidence of brain stem infarction. There was no association between the interval (greater or less than 6 hours) from the onset of symptoms until the end of thrombolysis and survival. CONCLUSIONS We confirm that intra-arterial thrombolysis reduces mortality in basilar artery occlusion. Young patients (<75 years) without any infarct in brain stem before the start of treatment seem to be the ideal candidates for thrombolysis. Basilar artery thrombosis could and should be reopened, even late (after 6 hours) after symptom onset.

Role of Diffusion-Weighted Magnetic Resonance Imaging in Diffuse Axonal Injury

Purpose: To determine whether the signal changes on magnetic resonance imaging (MRI), including fluid attenuated inversion recovery (FLAIR), T2*-weighted gradient echo (GE) imaging, and diffusion-weighted imaging (DWI) in diffuse axonal injury (DAI) patients correlate with the clinical outcome. Material and Methods: We diagnosed patients with DAI based on the following criteria: 1) a loss of consciousness from the time of injury that persisted beyond 6 h; 2) no apparent hemorrhagic contusion on computed tomography (CT); 3) the presence of white matter injury on MRI. Twenty-one DAI patients were analyzed (19 M, 2 F, mean age 34 years) with MRI (FLAIR, T2*-weighted GE imaging, and DWI). Results: 325 abnormalities were detected by MRI within a week after injury. The T2*-weighted GE imaging was significantly more sensitive than FLAIR and DWI in diagnosing DAI. DWI detected only 32% of all lesions, but could depict additional shearing injuries not visible on either T2*-weighted GE imaging or FLAIR. The mean number of lesions in brainstem detected by DWI in the favorable group (good recovery/moderately disabled) was significantly smaller than in the unfavorable group (severely disabled/vegetative survival/death). This trend was not observed on the T2*-weighted GE imaging and FLAIR findings. Conclusion: DWI cannot detect all DAI-related lesions, but is a potentially useful imaging modality for both diagnosing and assessing patients with DAI.

High-resolution magnetic resonance imaging for detection of carotid plaques.

OBJECTIVE: We report our experience using high-resolution magnetic resonance imaging (MRI) to identify carotid plaques and also discuss these MRI findings while comparing them with carotid endarterectomy specimens. METHODS: Eighteen carotid plaques from 17 different patients were observed using plaque MRI. The patients included 14 men and 3 women, aged 53 to 75 years (mean, 68.6 yr). Eight patients experienced a stroke and four patients experienced transient ischemic attack. The remaining five patients did not experience any neurological symptoms. Two-dimensional time-of-flight (TOF) MR angiography; T1-weighted imaging; fat-suppressed, cardiac gated, black-blood proton density imaging; and T2-weighted imaging were obtained with a 1.5-T MRI. RESULTS: Symptomatic plaques showed either vast or partially dotted high signals for each contrast. The high signal intensity on time-of-flight and T2-weighted imaging predicted the instability of the plaques (100% sensitivity and specificity). In particular, time-of-flight imaging predicted intraplaque hemorrhaging with 100% sensitivity and 80% specificity. MRI revealed that three of four asymptomatic lesions were unstable plaques. CONCLUSION: High-resolution MRI was able to detect various signal patterns related to the plaque components, and it was thus considered to be very useful for evaluating plaque instability. The application of plaque MRI therefore may positively affect the decision-making process when selecting optimal therapeutic strategies to treat with carotid plaques.

EFFECT OF TUMOR NECROSIS FACTOR-ALPHA ON THE PERMEABILITY OF BOVINE BRAIN MICROVESSEL ENDOTHELIAL CELL MONOLAYERS

The administration of chemotherapy to patients with tumors of the central nervous system is often blocked by the blood-brain barrier. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that promotes vascular permeability in addition to its pro-inflammatory effects. However, no direct evidence exists as to whether TNF-alpha may increase permeability of the BBB. We evaluated the effect of TNF-alpha on the transport of cisplatin (CDDP) or high molecular weight dextran labeled with fluorescein isothiocyanate (FITC-dextran) across bovine brain microvessel endothelial cell (BMEC) monolayers that was conducted on side-by-side diffusion chambers in vitro. The permeability coefficient for the transport of CDDP across the untreated monolayer was 3.80 x 10(-5) cm sec-1 at 30 minutes. After treating the BMEC monolayer with TNF-alpha (50 U ml-1 and 500 U ml-1) for 36 hours, the PC of CDDP increased significantly to 8.94 x 10(-5), and 14.43 x 10(-5) cm sec-1 respectively (p < 0.01). TNF-alpha had no effect on the transport of FITC-dextran across the BMEC monolayers. Electron microscopy showed that the tight junctions between the BMECs persisted even after treatment with TNF-alpha, whereas they had been partially disrupted following exposure to mannitol, 1600 mOsm kg-1. TNF-alpha selectively promoted the in vitro permeability of the blood-brain barrier to CDDP without disrupting tight junctions. This system could be used as a model for experimental studies of chemotherapy. Findings suggested that the combined administration of TNF-alpha and CDDP may be clinically useful.

Kidney angiotensin II receptors and converting enzyme in neonatal and adult Wistar-Kyoto and spontaneously hypertensive rats

The aim of the present study was to correlate the development of the renin angiotensin system (RAS) in the kidney of the rat with the development of genetic hypertension. Immature (1-week-old) and adult (12-week-old) normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive kidney rats (SHR) were used for quantification of angiotensin II (ANG II) receptors and angiotensin converting enzyme (ACE) binding sites using quantitative autoradiography. In both neonatal and adult animals of either strain, ANG II receptors were of AT1 subtype. In all kidney areas of 1-week-old rats. ANG II receptor density was higher in SHR than WKY. Binding density increased with age in WKY rats; thus, in the glomeruli and the outer stripe of the outer medulla of 12-week-old WKY, binding was significantly higher than that present in age-matched SHR. [125I]351A binding to ACE was highest in the outer medulla and not detectable in glomeruli. In 1-week-old rats, binding to ACE was higher in WKY than in SHR strain. No differences in ACE binding were found between adult SHR and WKY rats, with the exception of the inner stripe of the outer medulla, where no binding was detected in SHR. Our results support the hypothesis that the RAS in kidney is developmentally regulated and is involved in the development and maintenance of genetic hypertension in SHR.

Angiotensin II AT1 receptors in rat superior cervical ganglia : characterization and stimulation of phosphoinositide hydrolysis

Angiotensin II receptor number was higher in superior cervical ganglia of 2-week-old when compared to 8-week-old rats. In both young and adult rats, specific binding of [125I][Sar1]angiotensin II was displaced competitively by the AT1-receptor antagonist DuP 753 but not by the AT2-receptor competitor PD 123177. In ganglia from adult rats, DuP 753 competed with an IC50 of 113 nM. The stable guanine nucleotide GTP gamma S inhibited binding of [125I][Sar1]angiotensin II in young and adult rats by approximately 50% with IC50 values of 105 and 120 nM, respectively, suggesting that the angiotensin receptor is G-protein linked. Angiotensin II at a dose of 1 microM stimulated inositol phosphate formation 58% over control values in superior cervical ganglia from 8-week-old rats. This effect was totally blocked by 10 microM DuP 753 but not by 10 microM PD 123177. Our findings demonstrate that rat superior cervical ganglia contain AT1-type angiotensin receptors that are probably G-protein linked, and their stimulation results in increased inositol phospholipid metabolism.