Hideyuki Ishihara

Molecular and cellular permeability control at the blood-brain barrier

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells. These cells have at least three properties which distinguish them from their peripheral counterparts: (1) tight junctions (TJs) of extremely low permeability; (2) low rates of fluid-phase endocytosis; (3) specific transport and carrier molecules. In combination, these features restrict the nonspecific flux of ions, proteins, and other substances into the central nervous system (CNS) environment. The restriction protects neurons from harmful compositional fluctuations occurring in the blood and allows uptake of essential molecules. Breakdown of the BBB is associated with a variety of CNS disorders and results in aggravation of the condition. Restoration of the BBB is thus one strategy during therapy of CNS diseases. Its success depends on a precise knowledge of the structural and functional principles underlying BBB functionality. In this review we have tried to summarise the current knowledge of TJs, including information gained from non-neuronal systems, and describe selected mechanisms involved in permeability regulation.

Distribution and functional activity of P-glycoprotein and multidrug resistance-associated proteins in human brain microvascular endothelial cells in hippocampal sclerosis.

Multidrug resistance protein, also referred as P-glycoprotein (P-gp, MDR1; ABCB1) and multidrug resistance-associated protein (MRP) 1 (ABCC1) and 2 (ABCC2) are, thus far, candidates to cause antiepileptic drug (AED) resistance epilepsy. In this study, we investigated P-gp, MRP1 and MRP2 expression, localization and functional activity on cryosections and isolated human brain-derived microvascular endothelial cells (HBMEC) from epileptic patients (HBMEC-EPI) with hippocampal sclerosis (HS), as compared with HBMEC isolated from normal brain cortex (HBMEC-CTR). We examined the expression and distribution of three transporters, P-gp, MRP1 and MRP2 on two major parts of the resected tissue, the hippocampus and the parahippocampal gyrus (Gph). P-gp showed diffuse expression not only in endothelium but also by parenchymal cells in both the hippocampus and the Gph. MRP1 labeling was observed in parenchymal cells in the Gph. By contrast, MRP2 was mainly found in endothelium of the hippocampus. P-gp and MRP1 expression in the Gph was relatively high in the patient with long-term seizure history. Quantitative RT-PCR analysis of HBMEC revealed that MDR1, MRP1 as well as MRP5 (ABCC5) and MRP6 (ABCC6) were overexpressed in HBMEC-EPI at the mRNA level. HBMEC from both normal and epilepsy groups displayed protein expression of P-gp, whereas MRP1 and MRP2 were seen only in HBMEC-EPI. Accordingly, it is of particular interest that MRP functional activities were observed in HBMEC-EPI, but not in HBMEC-CTR. Our results suggest that complex MDR expression changes not only in the hippocampus but in the Gph may play a role in AED pharmacoresistance in intractable epilepsy patients with mesial temporal lobe epilepsy (MTLE) by altering the permeability of AEDs across the blood-brain barrier (BBB).

Down‐regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type‐1 receptor and nuclear factor‐κB activation

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin‐1, ZO‐1 and ZO‐2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO‐1 expression in astrocytes. TNF decreased occludin, but not ZO‐1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down‐regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor‐κB (NF‐κB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type‐1 receptor (TNFR1), TNF type‐2 receptor (TNFR2), or both, respectively, revealed that down‐regulation was mediated entirely by TNFR1. ZO‐1, which can interact with occludin, was found to co‐precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down‐regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF‐κB is involved as a negative regulator.

Endothelial cell barrier impairment induced by glioblastomas and transforming growth factor beta2 involves matrix metalloproteinases and tight junction proteins

Abstract Gliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor &bgr;2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor &bgr;2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

Preoperative Prediction of Outcome in 283 Poor-Grade Patients with Subarachnoid Hemorrhage: A Project of the Chugoku-Shikoku Division of the Japan Neurosurgical Society

Background: The management of patients with poor-grade subarachnoid hemorrhage (SAH) continues to be controversial. The objective of this study was to examine predictors of outcome of poor-grade SAH after surgical obliteration of the aneurysm. Methods: The study was performed as a retrospective review of 283 patients with poor-grade SAH who underwent surgical obliteration of the aneurysm at multiple centers in Chugoku and Shikoku, Japan. Results: A favorable outcome at discharge was achieved in 97 of the 283 patients (34.3%). Age (p < 0.001), World Federation of Neurosurgical Societies (WFNS) grade V at admission (p = 0.002), improvement in WFNS grade after admission (p = 0.002), Fisher grade (p = 0.039) and a low-density area (LDA) associated with vasospasm on computed tomography (CT; p < 0.001) showed a significant association with outcome. Further analysis of WFNS grades indicated that most patients who only improved to preoperative grade IV from grade V at admission did not have a favorable outcome. Multivariate analysis identified age (especially of ≧65 years; p < 0.001), WFNS grade V (p < 0.001) and LDA associated with vasospasm on CT (p < 0.001) as predictors of a poor outcome, and improvement in WFNS grade (p = 0.001) as a predictor of a favorable outcome after surgical obliteration of the aneurysm. Conclusions: Advanced age, WFNS grade V, improvement in WFNS grade, and LDA associated with vasospasm on CT were found to be independent predictors of clinical outcome, whereas rebleeding, early aneurysm surgery and treatment modality (surgical clipping or Guglielmi detachable coil embolization) were not independently associated with outcome in patients with poor-grade aneurysm.

Evaluation of brain toxicity following near infrared light exposure after indocyanine green dye injection

Indocyanine green (ICG) has excellent safety records and is widely used in medical diagnosis. Recently, a new method has been developed to estimate cerebral blood flow (CBF) using ICG in combination with near-infrared spectroscopy (NIRS). The new technique may be of wide clinical interest, as it is noninvasive and easy to perform at the bedside in stroke patients. Additionally, ICG with the use of specific wavelength lasers is documented to be effective in photodynamic therapy (PDT). Under normal conditions ICG does not cross the intact blood brain barrier (BBB). However, in patients with brain injuries where the BBB may be disturbed, ICG could accumulate in brain parenchyma and in combination with NIR-light exposure, phototoxicity could occur. The aim of the present study was to examine the possible toxicity of ICG in combination with NIRS in a specific setting for CBF measurements. In five rats with mannitol induced BBB breakdown no traces of ICG were found during spectrophotometric analysis of the brain cell suspensions. In ten rats with disrupted BBB there were no significant increases of brain temperature or histological signs of brain damage following 1 h NIR-light exposure after ICG injection. The existing literature concerning the application of ICG in combination with NIR light is reviewed.

Multicenter Prospective Cohort Study on Volume Management After Subarachnoid Hemorrhage Hemodynamic Changes According to Severity of Subarachnoid Hemorrhage and Cerebral Vasospasm

Background and Purpose— Systemic circulation management has not been established for patients with poor grade aneurysmal subarachnoid hemorrhage (SAH) or delayed cerebral ischemia (DCI) after SAH. The aims of the study were to examine hemodynamic variables in these patients and to establish treatment strategies. Methods— A multicenter prospective cohort study of hemodynamic variables from days 1 to 14 was performed using a transpulmonary thermodilution system (PiCCO Plus). Parameters were analyzed by Mann–Whitney test. Multivariate analysis was performed to identify parameters involved in onset of DCI. Results— The subjects were 204 patients, including 138 with poor grade SAH (World Federation of Neurological Surgeons grades IV and V) and 52 who developed DCI. The extravascular lung water index, pulmonary vascular permeability index, and systemic vascular resistance index were significantly greater in patients with poor grade SAH compared with those with good grade SAH (World Federation of Neurological Surgeons I–III) on day 2 (P=0.049, P=0.039, and P=0.038). Cardiac index was significantly lower in patients with poor grade SAH on days 1 and 2 (P=0.027 and P=0.011). In patients with DCI, the global end-diastolic volume index was significantly lower than in those without DCI on days 3 to 5 (P=0.0053; P=0.048; and P=0.048). In multivariate analysis, median global end-diastolic volume index, cardiac index, and systemic vascular resistance index at an early stage of SAH (days 3–6) were independently related to onset of DCI (P=0.023, P=0.013, and P=0.003). Conclusions— Patients with poor grade SAH developed heart failure–like afterload mismatch at an early stage, and those with DCI had decreased global end-diastolic volume index (hypovolemia) in the early stage of SAH. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: UMIN000003794.

Continuous hypertension and tachycardia after resection of a hemangioblastoma behind the dorsal medulla oblongata: relationship to sympathetic overactivity at the neurogenic vasomotor center

A very rare case of continuous hypertension and tachycardia after excision of a cerebellar hemangioblastoma at the dorsal medulla oblongata is presented. This 21-year-old man was admitted to the authors hospital with a headache and dizziness. Radiological examination revealed a tumor located behind the dorsal medulla oblongata and compressing it substantially. The tumor was completely resected, but after the surgery the patient experienced prolonged hypertension and tachycardia. Postoperative MR imaging showed a small injury at the dorsocaudal medulla that was located at the caudal site of the nucleus of the tractus solitarius (NTS). Because the NTS has been reported to play a central role in cardiovascular regulation along with the rostral ventrolateral medulla, the authors considered it possible that the NTS injury was the cause of the prolonged elevation of sympathetic tone.

Seasonal Variation in the Incidence of Aneurysmal Subarachnoid Hemorrhage Associated with Age and Gender: 20-Year Results from the Yamaguchi Cerebral Aneurysm Registry

Background: This study was a cerebral aneurysm registry study conducted in a region with few climatic differences. Based on data collected for over 20 years, seasonal variations and characteristics of subarachnoid hemorrhage (SAH) due to ruptured aneurysms were analyzed. Methods: This study included 5,007 patients in the Yamaguchi Prefecture with aneurysmal SAH between 1986 and 2005. Incidence rates by month, sex, age, severity, and aneurysm site were analyzed. Results: In women, seasonal variation was observed, in particular among those aged ≥50 years. Among those aged 50-69 years, the highest incidence was in October, and the nadir was in June (peak-to-trough ratio = 1.72). At age ≥70 years, this was slightly different, with the highest incidence in December and the nadir in July (peak-to-trough ratio = 1.48). However, there was no seasonal variation in men overall; it was limited to elderly men at age ≥70 years, with the highest incidence in January and the nadir in July (peak-to-trough ratio = 2.9). Aneurysm site and severity showed no relationship with seasonal variation. Conclusion: The present study shows seasonal variations in the onset of SAH. Seasonal variations in SAH differed depending on age and sex.