Masafumi Ihara

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.

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.

Does vascular pathology contribute to Alzheimer changes

In recent years there has been increased interest in whether vascular disease contributes to Alzheimers disease (AD). This review considers how modifiable risk factors such as hypertension, atherosclerosis, diabetes, dyslipidaemia and adiposity may impact on vascular structure and function to promote neurodegenerative processes and instigate AD. The presence of vascular pathology involving arterial stiffness, arteriolosclerosis, endothelial degeneration and blood-brain barrier dysfunction leads to chronic cerebral hypoperfusion. Pathological changes in human brain and animal studies suggest cerebral hypoperfusion which in turn induces several features of AD pathology including selective brain atrophy, white matter changes and accumulation of abnormal proteins such as amyloid β. Cerebral pathological changes may be further modified by genetic factors such as the apoliopoprotein E ε4 allele. Although tau hyperphosphorylation and tangle formation still needs robust explanation further support for the notion that vascular pathology influences AD changes is provided by the evidence that interventions which improve vascular function attenuate AD pathology.

Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat.

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.

Sept4, a Component of Presynaptic Scaffold and Lewy Bodies, Is Required for the Suppression of α-Synuclein Neurotoxicity

In Parkinson disease (PD), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PD model, insoluble deposits of Ser129-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser129 phosphorylation. Taken together, these data show that Sept4 may be involved in PD as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.

Association of the Cytoskeletal GTP-binding Protein Sept4/H5 with Cytoplasmic Inclusions Found in Parkinson's Disease and Other Synucleinopathies

α-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and α-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged α-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and α-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, α-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in α-synuclein-associated neurodegenerative disorders.

Quantification of myelin loss in frontal lobe white matter in vascular dementia, Alzheimer’s disease, and dementia with Lewy bodies

The aim of this study was to characterize myelin loss as one of the features of white matter abnormalities across three common dementing disorders. We evaluated post-mortem brain tissue from frontal and temporal lobes from 20 vascular dementia (VaD), 19 Alzheimer’s disease (AD) and 31 dementia with Lewy bodies (DLB) cases and 12 comparable age controls. Images of sections stained with conventional luxol fast blue were analysed to estimate myelin attenuation by optical density. Serial adjacent sections were then immunostained for degraded myelin basic protein (dMBP) and the mean percentage area containing dMBP (%dMBP) was determined as an indicator of myelin degeneration. We further assessed the relationship between dMBP and glutathione S-transferase (a marker of mature oligodendrocytes) immunoreactivities. Pathological diagnosis significantly affected the frontal but not temporal lobe myelin attenuation: myelin density was most reduced in VaD compared to AD and DLB, which still significantly exhibited lower myelin density compared to ageing controls. Consistent with this, the degree of myelin loss was correlated with greater %dMBP, with the highest %dMBP in VaD compared to the other groups. The %dMBP was inversely correlated with the mean size of oligodendrocytes in VaD, whereas it was positively correlated with their density in AD. A two-tier regression model analysis confirmed that the type of disorder (VaD or AD) determines the relationship between %dMBP and the size or density of oligodendrocytes across the cases. Our findings, attested by the use of three markers, suggest that myelin loss may evolve in parallel with shrunken oligodendrocytes in VaD but their increased density in AD, highlighting partially different mechanisms are associated with myelin degeneration, which could originate from hypoxic–ischaemic damage to oligodendrocytes in VaD whereas secondary to axonal degeneration in AD.

Matrix Metalloproteinase-2 Plays a Critical Role in the Pathogenesis of White Matter Lesions After Chronic Cerebral Hypoperfusion in Rodents

Background and Purpose— Cerebrovascular white matter (WM) lesions contribute to cognitive impairment and motor dysfunction in the elderly. A disruption of the blood–brain barrier (BBB) is believed to be a critical early event leading to these WM lesions. Previous studies have suggested the involvement of matrix metalloproteinase-2 (MMP-2) in BBB disruptions and the upregulation of MMP-2 after chronic cerebral hypoperfusion in a rat model. In the present study, we asked whether MMP-2 is involved in the BBB disruption and the subsequent WM lesions after chronic cerebral hypoperfusion. Methods— We compared the severity of white matter lesions in rats after chronic cerebral hypoperfusion with or without an MMP inhibitor. Then, we also induced the chronic cerebral hypoperfusion in wild-type and MMP-2-null mice. Results— In the rats treated with a relatively selective MMP-2 inhibitor, AG3340, the WM lesions after chronic cerebral hypoperfusion were significantly less severe, and the number of activated astroglia and microglia were also significantly lower as compared with the vehicle-treated rats. Gene knockout of MMP-2 also reduced the severity of the WM lesions and the number of activated astroglia and microglia in a mice system. In both rodents, the disruption of BBB function, as assessed by IgM staining and the Evans blue extravasation test, was less severe when MMP-2 activity was attenuated. Conclusions— These findings indicate that MMP-2 plays a critical role in the BBB disruption, glial cell activation, and WM lesions after chronic cerebral hypoperfusion and suggest the potential value of MMP-2 inhibitors as a therapeutic tool in cerebrovascular WM lesions.

A Mouse Model Characterizing Features of Vascular Dementia With Hippocampal Atrophy

Background and Purpose— We have previously described effects of chronic cerebral hypoperfusion in mice with bilateral common carotid artery stenosis (BCAS) using microcoils for 30 days. These mice specifically exhibit working memory deficits attributable to frontal-subcortical circuit damage without apparent gray matter changes, indicating similarities with subcortical ischemic vascular dementia. However, as subcortical ischemic vascular dementia progresses over time, the longer-term effects that characterize the mouse model are not known. Methods— Comprehensive behavioral test batteries and histological examinations were performed in mice subjected to BCAS for up to 8 months. Laser speckle flowmetry and 18F-fluorodeoxyglucose positron emission tomography were performed to assess cerebral blood flow and metabolism at several time points. Results— At 2 hours after BCAS, cerebral blood flow in the cerebral cortex temporarily decreased to as much as 60% to 70% of the control value but gradually recovered to >80% at 1 to 3 months. At 5 to 6 months after BCAS, reference and working memory were impaired as demonstrated by the Barnes and radial arm maze tests, respectively. Furthermore, 18F-fluorodeoxyglucose positron emission tomography demonstrated that hippocampal glucose utilization was impaired at 6 months after BCAS. Consistent with these behavioral and metabolic abnormalities, histological analyses demonstrated hippocampal atrophy with pyknotic and apoptotic cells at 8 months after BCAS. Conclusions— These results suggest that the longer-term BCAS model replicates advanced stages of subcortical ischemic vascular dementia when hippocampal neuronal loss becomes significant.

Nonhypotensive Dose of Telmisartan Attenuates Cognitive Impairment Partially Due to Peroxisome Proliferator-Activated Receptor-γ Activation in Mice With Chronic Cerebral Hypoperfusion

Background and Purpose— The effect of telmisartan, an angiotensin II Type 1 receptor blocker with peroxisome proliferator-activated receptor-&ggr;-modulating activity, was investigated against spatial working memory disturbances in mice subjected to chronic cerebral hypoperfusion. Methods— Adult C57BL/6J male mice were subjected to bilateral common carotid artery stenosis using external microcoils. Mice received a daily oral administration of low-dose telmisartan (1 mg/kg per day), high-dose telmisartan (10 mg/kg per day), or vehicle with or without peroxisome proliferator-activated receptor-&ggr; antagonist GW9662 (1 mg/kg per day) for all treatments for 30 days after bilateral common carotid artery stenosis. Cerebral mRNA expression of monocyte chemoattractant protein-1 and tumor necrosis factor-&agr; was measured 30 days after bilateral common carotid artery stenosis, and postmortem brains were analyzed for demyelinating change with Klüver-Barrera staining and immunostained for glial, oxidative stress, and vascular endothelial cell markers. Spatial working memory was assessed by the Y-maze test. Results— Mean systolic blood pressure and cerebral blood flow did not decrease with low-dose telmisartan but significantly decreased with high-dose telmisartan. Low-dose telmisartan significantly attenuated, but high-dose telmisartan provoked, spatial working memory impairment with glial activation, oligodendrocyte loss, and demyelinating change in the white matter. Such positive effects of low-dose telmisartan were partially offset by cotreatment with GW9662. Consistent with this, low-dose telmisartan reduced the degree of oxidative stress of vascular endothelial cells and the mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-&agr; compared with vehicle. Conclusions— Anti-inflammatory and antioxidative effects of telmisartan that were exerted in part by peroxisome proliferator-activated receptor-&ggr; activation, but not its blood pressure-lowering effect, have protective roles against cognitive impairment and white matter damage after chronic cerebral hypoperfusion.