Ryo Ohtani

White Matter Lesions and Glial Activation in a Novel Mouse Model of Chronic Cerebral Hypoperfusion

Background and Purpose— Cerebrovascular white matter (WM) lesions are closely associated with cognitive impairment and gait disorders in the elderly. We have successfully established a mouse model of chronic cerebral hypoperfusion that may provide new strategies for the molecular analysis of cerebrovascular WM lesions. Methods— Adult C57Bl/6 male mice were subjected to bilateral common carotid artery stenosis (BCAS) using external microcoils with varying inner diameters from 0.16 to 0.22 mm. Cerebral blood flow (CBF) in the frontal cortices was measured by laser-Doppler flowmetry at 2 hours and at 1, 3, 7, 14, and 30 days after BCAS. The brains were then removed and examined at 30 days with histological stains and immunohistochemistry for markers of microglia and astroglia. Results— At 2 hours, the CBF values (ratio to the preoperative value) did not change in the 0.22 mm group but decreased significantly to 77.3±13.4% in the 0.20 mm group, 67.3±18.5% in the 0.18 mm group, and 51.4±11.5% in the 0.16 mm group. At day 1, the CBF began to recover in all groups but remained significantly lower until 14 days in comparison to the control group. In the 0.20 mm and 0.18 mm groups, WM lesions occurred after 14 days without any gray matter involvement. These lesions were the most intense in the corpus callosum adjacent to the lateral ventricle but were mild in the anterior commissure and optic tract. In contrast, 4 of 5 mice developed some gray matter changes in the 0.16 mm group. The proliferation of activated microglia and astroglia was observed in the WM beyond 3 days after BCAS. Conclusions— WM lesions were successfully induced after chronic cerebral hypoperfusion with relative preservation of the visual pathway. These features in this mouse model are appropriate for cognitive assessment and genetic analysis, and it may provide a powerful tool to understand the pathophysiology of WM lesions.

Synthesis of Prussian Blue Nanoparticles with a Hollow Interior by Controlled Chemical Etching

Hollow particles, an important class of materials with large internal cavities and thin shells, present a wide range of potential applications, such as energy storage, chemical catalysis, photonics, and biomedical carriers. The properties of hollow particles are strongly affected by the compositions and exquisite nanostructures of the shell regions. Many efforts have been made to control these parameters. Recently, hollow particles with nanoporous shells have attracted great interest because the large pore volumes and high surface areas provided by the nanoporous shells show large storage capacity and allow guest species to pass easily into the internal cavity. For instance, metal oxide hollow particles present a superior lithium storage capacity and good cycle performance, 8] which could improve gas sensitivity and catalytic performance 10] in other applications. Inspired by the superiority of nanoporous shells, the creation of microporous crystal shells with outstanding properties is also expected. Porous coordination polymers (PCPs) (or metal–organic frameworks, MOFs) and zeolites are representative microporous crystals. Having adjustable porosity and properties, microporous crystals show great potential in applications such as separation, catalysis, adsorption, and gas storage. 13] To date, however, there have been no reports on the successful synthesis of uniformly nanosized hollow particles with microporous crystalline shells. 15] Very few reports on microporous zeolite and PCP hollow particles have been published, and several problems have been encountered. A major problem is that the synthesized hollow particles are very large and have a broad size distribution. Considering their practical use in various catalysis reaction processes, uniformly sized hollow particles are ideal because they can densely fill reactors or columns. Another problem is that the microporous crystallinity in the shells decreases seriously during the formation process (that is, amorphous regions are formed in the shells), causing a loss of thermal stability, acidic sites, and surface area. Therefore, the development of a general method of synthesizing uniformly nanosized hollow particles with highly crystalline microporous shells is in demand. Herein, we present a facile route to the fabrication of uniform-sized Prussian blue (PB) hollow particles by utilizing a controlled self-etching reaction in the presence of PVP. PB crystals consisting of metal ions coordinated by CN bridges are very typical coordination polymers with a high surface area, showing excellent properties in many applications such as catalysis, sensors, molecular magnets, gas storage, and bioimaging. The critical point in our procedure is the selection of PB mesocrystals as a starting material. PB mesocrystals are formed by the aggregation of PB nanocrystals in an oriented way, thereby showing single-crystal-like behavior. Through small pores (or defects) in the aggregated PB nanocrystals, the etching solution can be diffused into the core of the mesocrystals. We succeeded in the formation of an interior hollow cavity with the retention of the original PB crystallinity. Although a few attempts to prepare PB hollow structures have been reported, the obtained shells were amorphous or poorly crystalline, with large organic impurities. Such hollow particles cannot show high surface areas and good magnetic responses. Two types of PB mesocrystals with different particle sizes were used as starting materials (details regarding the synthetic conditions are given in the Experimental Section). SEM images indicated that the particle-size distributions of the original PB mesocrystals were very narrow and their average diameters were around 110 nm (Figure 1a) and 190 nm (Supporting Information, Figure S1a). From highly magnified SEM images (insets of Figure 1 a; Supporting Information, Figure S1a), very rough surfaces were observed at the edges and corners of the cubes, suggesting that the cube shapes were created by the aggregation of small PB nano[*] Dr. M. Hu, Dr. Y. Nemoto, Prof. Dr. Y. Yamauchi World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS) 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan) E-mail: yamauchi.yusuke@nims.go.jp

Selective Impairment of Working Memory in a Mouse Model of Chronic Cerebral Hypoperfusion

Background and Purpose— We recently designed a mouse model of chronic cerebral hypoperfusion, in which the cerebral white matter is damaged without significant gray matter lesions. The behavioral characteristics of these mice were studied using a test battery for neurological and cognitive functions. Methods— Adult C57Bl/6 male mice were subjected to either sham-operation or bilateral common carotid artery stenosis (BCAS) using microcoils with an internal diameter of 0.18 mm. At 30 days after BCAS, 70 animals were divided into 3 groups and subjected to behavioral test batteries. The first group underwent comprehensive behavioral test, including the neurological screen, prepulse inhibition, hot plate, open field, light/dark transition, Porsolt forced swim and contextual and cued fear conditioning (BCAS n=13; sham-operated n=11). The second group was for the working memory task of the 8-arm radial maze test (BCAS n=12; sham-operated n=10), and the third for the reference memory task of the 8-arm radial maze test (BCAS n=13; sham-operated n=11). Another batch of animals were examined for histological changes (BCAS n=11; sham-operated n=12). Results— The white matter including the corpus callosum was consistently found to be rarefied without clear ischemic lesions in the hippocampus. No apparent differences were observed in the comprehensive test batteries between the control and BCAS mice. However, in the working memory tasks tested with the 8-arm radial maze, the BCAS mice made significantly more errors than the control mice (P<0.0001). Again, there were no detectable differences in the reference memory tasks between the groups. Conclusions— At 30 days after BCAS, working memory deficits as well as white matter changes were apparent in the mice. Working memory deficit was attributable to damage of the frontal-subcortical circuits, suggesting the BCAS model is useful to evaluate the substrates of subcortical vascular dementia.

Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis.

Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimers disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimers disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.

Precise control and consecutive modulation of spin transition temperature using chemical migration in porous coordination polymers.

Precise control of spin transition temperature (T(c)) is one of the most important challenges in molecular magnetism. A Hofmann-type porous coordination polymer {Fe(pz)[Pt(II)(CN)(4)]} (1; pz = pyrazine) exhibited cooperative spin transition near room temperature (T(c)(up) = 304 K and T(c)(down) = 284 K) and its iodine adduct {Fe(pz)[Pt(II/IV)(CN)(4)(I)]} (1-I), prepared by oxidative addition of iodine to the open metal sites of Pt(II), raised the T(c) by 100 K. DSC and microscopic Raman spectra of a solid mixture of 1-I and 1 revealed that iodine migrated from 1-I to 1 through the grain boundary after heating above 398 K. We have succeeded in precisely controlling the iodine content of {Fe(pz)[Pt(CN)(4)(I)(n)]} (1-In; n = 0.0-1.0), which resulted in consecutive modulation of T(c) in the range 300-400 K while maintaining the hysteresis width. Furthermore, it was demonstrated that iodine migration in the solid mixture was triggered by the spin transition of 1-I. The magnetically bistable porous framework decorating guest interactive open-metal-site in the pore surface makes it possible to modulate T(c) ad arbitrium through unique postsynthetic method using iodine migration.

Enhanced bistability by guest inclusion in Fe(ii) spin crossover porous coordination polymers

Inclusion of thiourea guest molecules in the tridimensional spin crossover porous coordination polymers {[Fe(pyrazine)[M(CN)(4)]} (M = Pd, Pt) leads to novel clathrates exhibiting unprecedented large thermal hysteresis loops of ca. 60 K wide centered near room temperature.

A Switchable Molecular Rotator: Neutron Spectroscopy Study on a Polymeric Spin-Crossover Compound

A quasielastic neutron scattering and solid-state (2)H NMR spectroscopy study of the polymeric spin-crossover compound {Fe(pyrazine)[Pt(CN)(4)]} shows that the switching of the rotation of a molecular fragment--the pyrazine ligand--occurs in association with the change of spin state. The rotation switching was examined on a wide time scale (10(-13)-10(-3) s) by both techniques, which clearly demonstrated the combination between molecular rotation and spin-crossover transition under external stimuli (temperature and chemical). The pyrazine rings are seen to perform a 4-fold jump motion about the coordinating nitrogen axis in the high-spin state. In the low-spin state, however, the motion is suppressed, while when the system incorporates benzene guest molecules, the movements of the system are even more restricted.

Ibudilast, a phosphodiesterase inhibitor, protects against white matter damage under chronic cerebral hypoperfusion in the rat.

Cerebrovascular white matter (WM) lesions, which are frequently observed in vascular cognitive impairment and vascular dementia, can be produced in rats by clipping the common carotid arteries bilaterally. Since TNF-alpha is known to cause the degeneration of myelin, we examined whether these lesions can be ameliorated by ibudilast, a cyclic AMP phosphodiesterase (PDE) inhibitor that suppresses tumor necrosis factor (TNF)-alpha production. After the ligation of both common carotid arteries in 29 rats, 21 rats received a daily oral administration of 10, 30 or 60 mg/kg ibudilast and 8 rats received vehicle for 14 days. The pathological changes in the white matter were quantified in terms of white matter lesions and the emergence of activated microglia immunoreactive for major histocompatibility complex (MHC) antigen. In the vehicle-treated animals, white matter lesions and microglial activation occurred in the optic tract, internal capsule and corpus callosum. A low dose (10 mg/kg) of ibudilast failed to suppress the white matter lesions and microglial activation, whereas a dose of either 30 or 60 mg/kg ibudilast ameliorated these lesions (p<0.001). Without an alterations in laboratory blood data, 60 mg/kg ibudilast exhibited percent reduction of the white matter lesions ranging between 50% and 70%, which was more effective than 30 mg/kg ibudilast (p<0.05). The TNF-alpha immunoreactive glia decreased in number in the 60 mg/kg ibudilast-treated group as compared to the vehicle-treated group (p<0.001). These results indicate a dose-dependent protective effect of ibudilast against cerebrovascular white matter lesions and suggest a potential use for ibudilast in the treatment of vascular dementia.

Upregulation of ceramide and its regulating mechanism in a rat model of chronic cerebral ischemia.

Ceramide is a key mediator of apoptosis, and is involved in the cellular stress response. We examined the alterations in the ceramide levels and their synthetic/degradative pathway in a rat model of chronic cerebral ischemia, in which ischemic white matter (WM) lesions occur in association with oligodendroglial cell apoptosis. Chronic cerebral ischemia was induced by clipping both common carotid arteries in male Wistar rats. After predetermined periods of 1, 3, 7 and 14 days, the animals were subjected to immunohistochemical and biochemical investigations for ceramide in the region containing the frontal cortex and corpus callosum (region 1), and the region containing the internal capsule and globus pallidus (region 2). After 14 days, the myelin was degraded in the corpus callosum, internal capsule and the optic tract in Klüver-Barrera staining. There was a significant increase in the ceramide level and the activity of its synthetic enzyme, acidic sphingomyelinase (SMase), whereas its degrading enzyme, glucosylceramide synthase (GCS), was downregulated in both regions 1 and 2 as compared to the sham-operated rats. Simultaneously, ceramide immunoreactive glia increased in number in the corpus callosum and the internal capsule after 3, 7 and 14 days. Double labeling for ceramide with glial fibrillary acidic protein but not with leukocyte common antigen indicated the astroglial nature of these glia. These findings indicate that chronic cerebral ischemia induces an increased ceramide level in astroglia as a result of downregulation of GCS and an upregulation of ASMase activity.

Loss of Cholinergic Pathways in Vascular Dementia of the Binswanger Type

We sought to determine the changes in the cholinergic pathways, which project from the nucleus basalis of Meynert (nBM) and travel in the subinsular region, in vascular dementia of the Binswanger type (VDBT) and Alzheimer’s disease (AD). The subinsular regions were examined in 6 autopsied brains with VDBT, 5 brains with AD and 4 control brains without any neurologic diseases. The cholinergic pathway was labeled either by histochemistry for acetylcholine esterase (AChE), a degradatory enzyme of ACh, or by immunohistochemistry for choline acetyltransferase, its synthetic enzyme. The numerical density of nBM neurons did not differ significantly between these groups (163 ± 49 in the VDBT, 105 ± 82 in the AD and 198 ± 76 in the control groups), but with a tendency towards a decrease in the AD group. The subinsular cholinergic fibers were impaired, with relative preservation of the nBM neurons in VDBT, whereas both the subinsular cholinergic fibers and the nBM neurons were degraded in AD. These results indicate that the cholinergic pathway is damaged not only in AD, but also in VDBT, and may further provide a pharmacological basis for treatment with AChE inhibitors in VDBT.