Naoki Kitagawa

Effects of hypoxia on endothelial/pericytic co-culture model of the blood–brain barrier

The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytic foot processes. Most research for the in vitro BBB is performed endothelial cells with or without astrocytes. Hypoxia damage to the BBB induces vasogenic brain edema. We have generated a new model of the BBB with brain endothelial cells and pericytes and have examined the effects of hypoxia using this model. Brain microvascular endothelial cells and pericytes were isolated from three-week-old male Wister rats using enzyme and mechanical homogenization. Three models (A: only endothelial monolayer, B: endothelial monolayer with pericytes non-contact condition, and C: contact condition) were made by culturing these cells using Transwell co-culture system and were exposed to hypoxic condition. We evaluated barrier function with transendothelial electrical resistance (TEER) and permeability of Evans blue-albumin and sodium fluorescein. The tightest barrier was observed in the endothelial/pericytic contact model. Despite hypoxia-induced disruption of the barrier in endothelial monolayer and non-contact co-culture models, a minimum of dysfunction was seen in the contact co-culture model. Therefore, it is considered that pericytes effect on the endothelia by secreting factors or through a gap junction. In short, pericytes induce endothelial maturation and a tighter barrier function, which supports the function against the hypoxic injury. Intercellular communication might be important to keep the BBB functional and stabilize in hypoxia.

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.

Efficacy of DynaCT Digital Angiography in the Detection of the Fistulous Point of Dural Arteriovenous Fistulas

BACKGROUND AND PURPOSE: Identifying the precise hemodynamic features, including the fistulous point, is essential for treatments of dural arteriovenous fistulas (DAVFs). This study illustrates the efficacy of DynaCT digital angiograms obtained from a 3D C-arm CT to directly visualize the location of the fistulous points in DAVFs. MATERIALS AND METHODS: This retrospective study observed 14 consecutive patients with DAVFs, which included 7 cavernous sinuses, 4 transverse-sigmoid sinuses, 2 convexity-superior sagittal sinuses, and 1 tentorial sinus. In the assessment of the practical applicability for the diagnosis of DAVFs, images obtained from 2D digital subtraction angiography (DSA) and DynaCT were comparatively evaluated. RESULTS: In all patients, DynaCT digital angiography could clearly demonstrate the feeding arteries, the fistulous points, and the draining veins. Significant anatomic landmarks for the fistulous points with relationships to osseous structures were also provided. Compared with 2D DSA, DynaCT digital angiograms demonstrated 12 additional findings in 8 patients (57%), including the detection of the fistulous points (n = 7), the feeders (n = 1), the retrograde leptomeningeal drainage (n = 1), the draining veins (n = 1), and the venous anomaly (n = 2). CONCLUSIONS: In comparison with 2D DSA, DynaCT may provide more detailed information to evaluate DAVFs. DynaCT digital angiograms have a high contrast and isotropic spatial resolution, allowing a reliable visualization of small vessels and fine osseous structures. Such detailed information, especially for the location of the fistulous points, could be very useful for either the endovascular or the surgical treatments of DAVFs.

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.

N-acetylcysteine inhibited nuclear factor-κB expression and the intimal hyperplasia in rat carotid arterial injury

Abstract Neointima formation associated with vascular restenosis is a complex local inflammatory process actively involving the vascular smooth muscle cell (SMC) proliferation. Nuclear factor-κB (NF-κB) is a transactivator of a diverse group of genes whose activation has been strongly associated with the cellular response to inflammation. Since anti-oxidant N-acetylcysteine (NAC) inhibit NF-κB activity in vascular SMC in vitro , we examined the in vivo effect of the NAC on balloon-induced neointimal formation in the carotid artery of rats. Sprague-Dawley rats underwent balloon dilatation injury of the left carotid artery to induce neointimal formation. One group of these rats (n = 9) were treated with daily intraperitoneal injection of NAC (200 mg kg-1) for 14 consecutive days, whereas the control group (n = 9) was treated with saline. Fourteen days after the injury, the left carotid arteries were removed and analyzed under microscope. Several rats underwent the same treatment as above and were sacrificed three days after injury for immunohistochemistry and Western blot studies. A morphometric analysis revealed that there were significant differences in intima/media ratio between the two groups. Immunohistochemical and Western blotting studies demonstrated that NAC suppressed the injury-induced NF-κB activity in the medial SMC layer. Treatment with NAC suppresses vascular NF-κB activation and this inhibition reduced the pathological thickening of the arterial wall. The NF-κB pathway, therefore, represents an attractive therapeutic target for strategies to prevent vascular restenosis. [Neurol Res 2001; 23: 731-738]

Endovascular treatment for cervical carotid artery stenosis presenting with progressing stroke: three case reports

BACKGROUND Progressing stroke is said to occur when symptoms and signs worsen in cases of ischemic stroke. Although conservative methods using volume expansion with antithrombotic or anticoagulative agents are widely used for progressing stroke, in some hospitals, emergency carotid endarterectomy (CEA) has been performed for carotid stenosis, with mixed results. Here we report three cases with progressing ischemic stroke that were managed by endovascular surgical intervention. CASE DESCRIPTION We performed endovascular surgery in three patients with cervical carotid artery stenosis presenting with progressing stroke or crescendo transient ischemic attacks. Endovascular treatment was less invasive and feasible for acute phase treatment. While local thrombolysis alone was found to be less effective, stent placement induced complete resolution of stenosis, but may result in hyperperfusion syndrome or hemorrhagic infarction. CONCLUSIONS In an emergency, percutaneous transluminal angioplasty with proper dilatation is preferred, and then CEA or stenting should be considered after the patients condition stabilizes.

Lowered glucose suppressed the proliferation and increased the differentiation of murine neural stem cells in vitro

Cerebral ischemia is known to activate endogenous neural stem cells (NSCs), but its mechanisms remain unknown. Since lowered glucose supply seems to mediate ischemic actions, we examined the effect of low glucose on NSC activities in vitro. Low glucose applied during the proliferation period diminished EGF‐induced proliferation of NSCs without affecting subsequent differentiation, but low glucose directly exposed during the differentiation period facilitated the differentiation of NSCs into neurons and astrocytes. These findings suggest that low glucose facilitated NSC differentiation, but it diminished NSC proliferation. Moreover, the effect of low glucose may be dependent on the timing of application.

Positional vertebral artery compression and vertebrobasilar insufficiency due to a herniated cervical disc

The authors report a case of vertebrobasilar insufficiency caused by vertebral artery (VA) compression due to a herniated cervical disc, which was surgically treated with the aid of intraoperative angiography. This 78-year-old man visited the hospital because of syncope following head rotation. Admission CT scans revealed a calcified mass adjacent to the right lateral process of the C-4 spine. Cervical angiography demonstrated an obstruction of the right VA at this region on rotation of the head to the right. The operation revealed a cervical disc protruding toward the right VA. The disc was surgically removed, and then the decompression of the right VA was confirmed on intraoperative angiography studies. A histopathological examination showed fibrohyaline cartilage, indicating an ossified intervertebral disc. The postoperative course was uneventful, and he has not experienced any syncope since treatment. A cervical disc herniation could be a cause of vertebrobasilar insufficiency by exerting positional compression of the VA. Intraoperative angiography could be quite useful to confirm this condition during decompression surgery for a cervical VA.

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized 125I‐endothelin‐1 (125I‐ET‐1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High‐affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (KD = 4.7 ± 1.0 × 10−10 and 1.6 ± 0.3 × 10−10M, respectively; Bmax = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ‐123, a selective antagonist for the ETA receptor, potently competed for 125I‐ET‐1 binding to sections of the microvessels with IC50 values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10−6M BQ‐123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET‐3 showed two components (IC50 = 5.7 ± 2.5 × 10−10 and 1.4 ± 0.2 × 10−6M for tumor microvessels, 1.8 ± 0.6 × 10−10 and 1.1 ± 0.3 × 10−6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high‐affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ETA subtype.

A selective endothelin ETA antagonist, BQ-123, inhibits125I-endothelin-1 (125I-ET-1) binding to human meningiomas and antagonizes ET-1-induced proliferation of meningioma cells

Summary1. We studied the effects of BQ-123, a selective ETA receptor antagonist, on125I-endothelin-1 (125I-ET-1) binding to cell surface receptors in surgically excised human meningiomas and on ET-1-induced DNA synthesis in cultured human meningioma cellsin vitro, using a quantitative receptor autoradiographic technique with radioluminography and3H-thymidine incorporation, respectively.2. All of the human meningiomas expressed high-affinity binding sites for125I-ET-1, regardless of differences in histological subtypes (Kd=2.6±0.2 nM,Bmax=374±93 fmol/mg; mean ± SE;n=9).3. BQ-123 competed for125I-ET-1 binding to sections of meningiomas with IC50s of 3.2±0.9×10−7M, and 10−4M BQ-123 displaced 80% of the binding.4. ET-1 significantly stimulated DNA synthesis in cultured human meningioma cells, up to 170% of the basal level in the presence of 10−9M ET-1. BQ-123 inhibited ET-1 (10−9M)-induced DNA synthesis in meningioma cells, in a dose-dependent manner, and 10−5M BQ-123 reduced it to 120% of the basal level.5. The number of meningioma cells determined after 4 days in culture was dose dependently increased in the presence of ET-1 (10−9 and 10−7M). The growth rate of meningioma cells, incubated with 10−9M ET-1, was reduced by 50% in the presence of 10−7M BQ-123.6. Our data suggest that (a) human meningioma cells express a large number of ETA endothelin receptors, with a small proportion of non-ETA receptors linked to proliferation of the cells, and (b) ET receptor antagonists, including BQ-123, might prove to be effective treatment for patients with meningioma.