Mumefural Improves Recognition Memory and Alters ERK-CREB-BDNF Signaling in a Mouse Model of Chronic Cerebral Hypoperfusion.

Cognitive impairment resulting from chronic cerebral hypoperfusion (CCH) is known as vascular dementia (VaD) and is associated with cerebral atrophy and cholinergic deficiencies. Mumefural (MF), a bioactive compound found in a heated fruit of Prunus mume Sieb. et Zucc, was recently found to improve cognitive impairment in a rat CCH model. However, additional evidence is necessary to validate the efficacy of MF administration for treating VaD. Therefore, we evaluated MF effects in a mouse CCH model using unilateral common carotid artery occlusion (UCCAO). Mice were subjected to UCCAO or sham surgery and orally treated with MF daily for 8 weeks. Behavioral tests were used to investigate cognitive function and locomotor activity. Changes in body and brain weights were measured, and levels of hippocampal proteins (brain-derived neurotrophic factor (BDNF), extracellular signal-regulated kinase (ERK), cyclic AMP-response element-binding protein (CREB), and acetylcholinesterase (AChE)) were assessed. Additionally, proteomic analysis was conducted to examine the alterations in protein profiles induced by MF treatment. Our study showed that MF administration significantly improved cognitive deficits. Brain atrophy was attenuated and MF treatment reversed the increase in AChE levels. Furthermore, MF significantly upregulated p-ERK/ERK, p-CREB/CREB, and BDNF levels after UCCAO. Thus, MF treatment ameliorates CCH-induced cognitive impairment by regulating ERK/CREB/BDNF signaling, suggesting that MF is a therapeutic candidate for treating CCH.

Peroxiredoxin 2 deletion impairs hippocampal-dependent memory via exacerbating transient ischemia-induced oxidative damage.

Peroxiredoxin 2 (Prx2) regulates oxidative stress response in neuronal injury. The present study examined the effects of Prx2 deletion on transient global ischemia-induced hippocampal-dependent memory impairment. First, 20-min bilateral common carotid artery occlusion (BCCAO)-reperfusion and sham-operated control procedures were conducted in 6- or 7-month-old Prx2 knockout and wild-type mice. The cognitive status of these mice was assessed using the Morris water maze task with a hidden platform and a novel object recognition task 7 days after the 20-min BCCAO. Next, to evaluate neuronal degeneration and oxidative stress in the CA1 subregion of the hippocampus critical for learning and memory, we measured immunoreactive Fluoro-jade C (FJC)-positive signals and 4-hydroxy-2-trans-nonenal (4-HNE) levels, respectively. The 20-min BCCAO induced cognitive impairments and increased the intensity of FJC-positive signals and 4-HNE levels of CA1 in Prx2 knockout mice but not in wild-type mice. These results suggest that Prx2 deficiency reduces resilience to transient global ischemia.

Impaired Cognitive Flexibility Induced by Chronic Cerebral Hypoperfusion in the 5XFAD Transgenic Mouse Model of Mixed Dementia.

Cerebrovascular lesions are widely prevalent in patients with Alzheimer's disease (AD), but their relationship to the pathophysiology of AD remains poorly understood. An improved understanding of the interaction of cerebrovascular damage with AD is crucial for the development of therapeutic approaches. Herein, we investigated the effects of chronic cerebral hypoperfusion (CCH) in a 5XFAD transgenic (Tg) mouse model of AD. We established CCH conditions in both Tg and non-Tg mice by inducing unilateral common carotid artery occlusion (UCCAO). Cognitive performance in mice was evaluated, and their brain tissue was examined for amyloid-beta (Aß) pathology to elucidate possible mechanisms. We found that UCCAO-operated Tg mice showed impaired cognitive flexibility in the reversal phase of the hidden-platform water maze task compared to sham-operated Tg mice. Interestingly, UCCAO-operated Tg mice used fewer spatial cognitive strategies than sham-operated Tg mice during reversal learning. These cognitive deficits were accompanied by increased Aß plaque burden and Aß42 levels in the hippocampus and prefrontal cortex, 2 regions that play essential roles in the regulation of cognitive flexibility. Furthermore, changes in cognitive flexibility are strongly correlated with the expression levels of enzymes related to Aß clearance, such as neprilysin and insulin-degrading enzymes. These findings suggest that, in 5XFAD mice, impaired cognitive flexibility is related to CCH, and that Aß clearance might be involved in this process.

Mumefural Improves Blood Flow in a Rat Model of FeCl3-Induced Arterial Thrombosis.

Mumefural (MF), a bioactive component of the processed fruit of Prunus mume Sieb. et Zucc, is known to inhibit platelet aggregation induced by agonists in vitro. In this study, we investigated the anti-thrombotic effects of MF using a rat model of FeCl3-induced arterial thrombosis. Sprague-Dawley rats were intraperitoneally injected with MF (0.1, 1, or 10 mg/kg) 30 min before 35% FeCl3 treatment to measure the time to occlusion using a laser Doppler flowmeter and to assess the weight of the blood vessels containing thrombus. MF treatment significantly improved blood flow by inhibiting occlusion and thrombus formation. MF also prevented collagen fiber damage in injured vessels and inhibited the expression of the platelet activation-related proteins P-selectin and E-selectin. Moreover, MF significantly reduced the increased inflammatory signal of nuclear factor (NF)-κB, toll-like receptor 4 (TLR4), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in blood vessels. After administration, MF was detected in the plasma samples of rats with a bioavailability of 36.95%. Therefore, we suggest that MF may improve blood flow as a candidate component in dietary supplements for improving blood flow and preventing blood circulation disorders.

4-Hydroxynonenal Immunoreactivity Is Increased in the Frontal Cortex of 5XFAD Transgenic Mice.

Oxidative stress was implicated in the functional impairment of the frontal cortex observed in early Alzheimer's disease (AD). To elucidate this role in an animal AD model, we assessed cognitive function of 4-month-old five familial AD (5XFAD) transgenic (Tg) mice using a learning strategy-switching task requiring recruitment of the frontal cortex and measuring levels of 4-hydroxy-2-trans-nonenal (4-HNE), a marker of oxidative stress, in their frontal cortex. Mice were sequentially trained in cued/response and place/spatial versions of the water maze task for four days each. 5XFAD and non-Tg mice exhibited equal performance in cued/response training. However, 5XFAD mice used spatial search strategy less than non-Tg mice in the spatial/place training. Immunoblot and immunofluorescence staining showed that 4-HNE levels increased in the frontal cortex, but not in the hippocampus and striatum, of 5XFAD mice compared to those in non-Tg mice. We report early cognitive deficits related to the frontal cortex and the frontal cortex's oxidative damage in 4-month-old 5XFAD mice. These results suggest that 4-month-old 5XFAD mice be a useful animal model for the early diagnosis and management of AD.

Combined MS/MS-NMR Annotation Guided Discovery of Iris lactea var. chinensis Seed as a Source of Viral Neuraminidase Inhibitory Polyphenols.

In this study, the chemical diversity of polyphenols in Iris lactea var. chinensis seeds was identified by combined MS/MS-NMR analysis. Based on the annotated chemical profile, the isolation of stilbene oligomers was conducted, and consequently, stilbene oligomers (1-10) were characterized. Of these, compounds 1 and 2 are previously undescribed stilbene dimer glycoside (1) and tetramer glycoside (2), respectively. Besides, to evaluate this plant seed as a rich source of stilbene oligomers, we quantified three stilbene oligomers of I. lactea var. chinensis seeds. The contents of three major stilbene oligomers-trans-ε-viniferin (3), vitisin A (6), and vitisin B (9)-in I. lactea var. chinensis seeds were quantified as 2.32 (3), 4.95 (6), and 1.64 (9) mg/g dry weight (DW). All the isolated compounds were tested for their inhibitory activities against influenza neuraminidase. Compound 10 was found to be active with the half maximal inhibitory concentration (IC50) values at 4.76 µM. Taken together, it is concluded that I. lactea var. chinensis seed is a valuable source of stilbene oligomers with a human health benefit.

Acute and Subacute Oral Toxicity of Mumefural, Bioactive Compound Derived from Processed Fruit of Prunus mume Sieb. et Zucc., in ICR Mice.

Mumefural is a bioactive compound derived from the processed fruit of Prunus mume Sieb. et Zucc., a traditional health food; however, its safety has not been evaluated. We investigated the toxicity of mumefural through single and repeated oral administration at doses of 1250, 2500, and 5000 mg/kg in Institute of Cancer Research (ICR) mice. The acute toxicity assessment was not associated with adverse effects or death. Similarly, the subacute (four weeks) toxicity assessment did not reveal any mumefural-associated mortality, abnormal organ damage, or altered clinical signs, body weight, food consumption, or hematological parameters. However, albumin/globulin ratio and chloride ion levels were significantly increased in male mice treated with mumefural at ≥ 2500 mg/kg. Female mice exhibited significantly higher levels of chloride, sodium, and potassium ions, at a dose of 5000 mg/kg. Furthermore, the administration of 2500 and 5000 mg/kg mumefural decreased the absolute weight of spleen in male mice. These findings indicated that the approximate lethal dose of mumefural in ICR mice was > 5000 mg/kg. No significant mumefural toxicity was observed at ≤ 5000 mg/kg. Our findings provide a basis for conducting future detailed studies to evaluate reproductive, neurological, genetic, and chronic toxicity of mumefural.

Recognition memory impairments and amyloid-beta deposition of the retrosplenial cortex at the early stage of 5XFAD mice.

Early diagnosis and treatment of AD are critical for delaying its progression. The present study, therefore, examined the cognitive status and neuropathological characteristics of 4-month-old 5X familial AD (5XFAD) transgenic (Tg) mice, as an early stage of AD animal model. The novel object recognition task was performed with retention tests at varying intervals (i.e., 10 min, 1 h, 4 h, and 24 h) to measure the retention capacity of recognition memory of 5XFAD mice. At the 4h retention interval, 5XFAD mice exhibited worse performances than non-Tg control mice. Therefore, using amyloid-beta (Aß) 42- and 4G8-immunoreactive plaques, the accumulation of Aß was examined in the gray and white matter of the system that was necessary for the retention of recognition memory, with a focus on the hippocampus and retrosplenial cortex. The expression of ionized calcium-binding adapter molecule-1 (Iba-1) was also examined to measure microglial activation. The immunohistological analysis of Aß and Iba-1 revealed that the retrosplenial cortex was the most affected region in the brains of 4-month-old 5XFAD mice. These findings indicate that the cognitive and neuropathological characteristics of 4-month-old 5XFAD mice would provide a research platform for studying early diagnosis and treatment of AD.

The Involvement of Canonical Wnt Signaling in Memory Impairment Induced by Chronic Cerebral Hypoperfusion in Mice.

Chronic cerebral hypoperfusion (CCH) has been proposed to contribute to the progression of memory loss, which is the main symptom of vascular cognitive impairment (VCI). Accumulating evidence indicates that underlying pathophysiology, such as neurodegeneration, may lead to memory loss. However, the underlying molecular basis of memory loss in CCH remains unclear. Here, we investigated the roles of canonical Wnt signaling, which modulates hippocampal function, in a CCH model. CCH was induced by unilateral common carotid artery occlusion (UCCAO). Mice were randomly divided into a sham-operated group or one of three UCCAO groups with different endpoints (1.5, 2.5, and 3.5 months) after UCCAO. Memory function and hippocampal levels of Wnt-related proteins were measured. A Wnt activator, lithium, was administered intraperitoneally to assess memory improvements. In the groups examined 2.5 and 3.5 months after UCCAO, impaired object recognition memory was accompanied by inhibition of Wnt signaling and decreased expression of synaptic/neural activity-related proteins. Recognition memory and Wnt signaling were significantly positive correlated. Moreover, activation of Wnt signaling with lithium significantly attenuated memory loss and recovered synaptic/neural marker expression after UCCAO. Our results suggest that CCH may affect synaptic plasticity via dysregulation of signaling pathways, including canonical Wnt signaling, which could be partly involved in memory loss. As Wnt activator administration alleviated the effects of CCH on memory loss, modulation of Wnt signaling may be a promising therapeutic strategy for VCI.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

Mumefural Ameliorates Cognitive Impairment in Chronic Cerebral Hypoperfusion via Regulating the Septohippocampal Cholinergic System and Neuroinflammation.

Chronic cerebral hypoperfusion (CCH) causes cognitive impairment and neurogenic inflammation by reducing blood flow. We previously showed that Fructus mume (F. mume) improves cognitive impairment and inhibits neuroinflammation in a CCH rat model. One of the components of F. mume, Mumefural (MF), is known to improve blood flow and inhibit platelet aggregation. Whether MF affects cerebral and cognitive function remains unclear. We investigated the effects of MF on cognitive impairment and neurological function-related protein expression in the rat CCH model, established by bilateral common carotid arterial occlusion (BCCAo). Three weeks after BCCAo, MF (20, 40, or 80 mg/kg) was orally administrated once a day for 42 days. Using Morris water maze assessment, MF treatment significantly improved cognitive impairment. MF treatment also inhibited cholinergic system dysfunction, attenuated choline acetyltransferase-positive cholinergic neuron loss, and regulated cholinergic system-related protein expressions in the basal forebrain and hippocampus. MF also inhibited myelin basic protein degradation and increased the hippocampal expression of synaptic markers and cognition-related proteins. Moreover, MF reduced neuroinflammation, inhibited gliosis, and attenuated the activation of P2X7 receptor, TLR4/MyD88, NLRP3, and NF-κB. This study indicates that MF ameliorates cognitive impairment in BCCAo rats by enhancing neurological function and inhibiting neuroinflammation.

Assessment of Cognitive Phenotyping in Inbred, Genetically Modified Mice, and Transgenic Mouse Models of Alzheimer's Disease.

Genetically modified mouse models are being used predominantly to understand brain functions and diseases. Well-designed and controlled behavioral analyses of genetically modified mice have successfully led to the identification of gene functions, understanding of brain diseases, and development of treatments. Recently, complex and higher cognitive functions have been examined in mice with genetic mutations. Therefore, research strategies for cognitive phenotyping should be sophisticated and evolve to convey the exact meaning of the findings and provide robust translational tools for testing hypotheses and developing treatments. This review addresses issues of experimental design and discusses studies that have examined cognitive function using mouse strain differences, genetically modified mice, and transgenic mice for Alzheimer's disease.

Superior Place Learning of C57BL/6 vs. DBA/2 Mice Following Prior Cued Learning in the Water Maze Depends on Prefrontal Cortical Subregions.

The participation of the prefrontal cortex (PFC), hippocampus, and dorsal striatum in switching the learning task from cued to place learning were examined in C57BL/6 and DBA/2 mice, by assessing changed levels of phosphorylated CREB (pCREB). Mice of both strains first received cued training in a water maze for 4 days (4 trials per day), and were then assigned to one of four groups, one with no place training, and three with different durations of place training (2, 4, or 8 days). Both strains showed equal performance in cued training. After the switch to place training, C57BL/6 mice with 2 or 4 days of training performed significantly better than DBA/2 mice, but their superiority disappeared during the second half of an 8 days-place training period. The pCREB levels of these mice were measured 30 min after place training and compared with those of mice that received only cued training. Changes in pCREB levels of C57BL/6 mice were greater in the hippocampal CA3, hippocampal dentate gyrus, orbitofrontal and medial PFC than those of DBA/2 mice, when mice of both received the switched place training for 2 days. We further investigated the roles of orbitofrontal and medial PFC among these brain regions showing strain differences, by destroying each region using selective neurotoxins. C57BL/6 mice with orbitofrontal lesions were slower to acquire the place learning and continued to use the cued search acquired during the cued training phase. These findings indicate that mouse orbitofrontal cortex (OFC) pCREB is associated with behavioral flexibility such as the ability to switch a learning task.

Chronic Cerebral Hypoperfusion Induces Alterations of Matrix Metalloproteinase-9 and Angiopoietin-2 Levels in the Rat Hippocampus.

Angiogenic factors contribute to cerebral angiogenesis following cerebral hypoperfusion, and understanding these temporal changes is essential to developing effective treatments. The present study examined temporal alterations in angiogenesis-related matrix metalloproteinase-9 (MMP-9) and angiopoietin-2 (ANG-2) expression in the hippocampus following bilateral common carotid artery occlusion (BCCAo). Male Wistar rats (12 weeks of age) were randomly assigned to sham-operated control or experimental groups, and expression levels of MMP-9 and ANG-2 were assessed after BCCAo (1 week, 4 weeks, and 8 weeks), using western blotting. Protein expression increased 1 week after BCCAo and returned to control levels at 4 and 8 weeks. In addition, immunofluorescence staining demonstrated that the MMP-9- and ANG-2-positive signals were primarily observed in the NeuN-positive neurons with very little labeling in non-neuronal cells and no labeling in endothelial cells. In addition, these cellular locations of MMP-9- and ANG-2-positive signals were not altered over time following BCCAo. Other angiogenic factors such as vascular endothelial growth factor and hypoxia-inducible factor did not differ from controls at 1 week; however, expression of both factors increased at 4 and 8 weeks in the BCCAo group compared to the control group. Our findings increase understanding of alterations in angiogenic factors during the progression of cerebral angiogenesis and are relevant to developing effective temporally based therapeutic strategies for chronic cerebral hypoperfusion-associated neurological disorders such as vascular dementia.

Chemical Constituents of Apios americana Tubers and Their Inhibitory Activities on Nitric Oxide Production in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

Apios americana is an important food crop producing edible tubers with high nutritional and medicinal values and is widely cultivated in many countries. Despite its usefulness, research on its secondary metabolites and biological activities has been limited. In the present study, a new coumaronochromone, (2 R,3 S)-3,7,4'-trihydroxy-5-methoxycoumaronochromone (1), and two new isoflavone glucosides, 7,2',4'-trihydroxy-5-methoxyisoflavone-4'- O-ß-d-glucopyranoside (3) and 5,7,4'-trihydroxyisoflavone-7- O-ß-d-gentiotrioside (5), were isolated from the tubers of A. americana via chromatographic separation. Seventeen known compounds (2, 4, and 6-20) were also obtained from this plant part. The chemical structures of 1, 3, and 5 were determined by the interpretation of spectroscopic data. The absolute structure of the new compound 1 was established from experimental and calculated electronic circular dichroism spectra. This is the first study to determine the absolute configuration of a 3-hydroxycoumaronochromone derivative. The potential anti-inflammatory activity of the 20 isolates obtained was evaluated by measuring their inhibitory effects on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages. Among the isolates, seven compounds (1, 3, 6-8, 15, and 20) showed substantial inhibition of nitric oxide production in RAW 264.7 cells, with the most active being compound 1 (IC50 value of 0.38 ± 0.04 µM).

Terminalia chebula extract prevents scopolamine-induced amnesia via cholinergic modulation and anti-oxidative effects in mice.

BACKGROUND: Terminalia chebula Retz. (Combretaceae) is a traditional herbal medicine that is widely used in the treatment of diabetes, immunodeficiency diseases, and stomach ulcer in Asia. However, the anti-amnesic effect of T. chebula has not yet been investigated. The present study was designed to determine whether T. chebula extract (TCE) alleviates amnesia induced by scopolamine in mice. We also investigated possible mechanisms associated with cholinergic system and anti-oxidant effects. METHODS: TCE (100 or 200 mg/kg) was orally administered to mice for fourteen days (days 1-14), and scopolamine was intraperitoneally injected to induce memory impairment for seven days (days 8-14). Learning and memory status were evaluated using the Morris water maze. Hippocampal levels of acetylcholine (ACh), acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) were measured ex vivo. Levels of reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) in the hippocampus were also examined. RESULTS: In the Morris water maze task, TCE treatment reversed scopolamine-induced learning and memory deficits in acquisition and retention. TCE reduced hippocampal AChE activities and increased ChAT and ACh levels in the scopolamine-induced model. Moreover, TCE treatment suppressed scopolamine-induced oxidative damage by ameliorating the increased levels of ROS, NO, and MDA. CONCLUSION: These findings suggest that TCE exerts potent anti-amnesic effects via cholinergic modulation and anti-oxidant activity, thus providing evidence for its potential as a cognitive enhancer for amnesia.

Ginkgo biloba L. extract protects against chronic cerebral hypoperfusion by modulating neuroinflammation and the cholinergic system.

BACKGROUND: Ginkgo biloba extract (GBE)-a widely used nutraceutical-is reported to have diverse functions, including positive effects on memory and vasodilatory properties. Although numerous studies have assessed the neuroprotective properties of GBE in ischemia, only a few studies have investigated the neuro-pharmacological mechanisms of action of GBE in chronic cerebral hypoperfusion (CCH). PURPOSE: In the present study, we sought to determine the effects of GBE on CCH-induced neuroinflammation and cholinergic dysfunction in a rat model of bilateral common carotid artery occlusion (BCCAo). METHODS: Chronic BCCAo was induced in adult male Wistar rats to reflect the CCH conditions. On day 21 after BCCAo, the animals were treated orally with saline or GBE (5, 10, 20, and 40mg/kg) daily for 42 days. After the final treatment, brain tissues were isolated for the immunohistochemical analysis of glial markers and choline acetyltransferase (ChAT), as well as for the western blot analysis of proinflammatory cytokines, toll-like receptor (TLR)-related pathway, receptor for advanced glycation end products (RAGE), angiotensin-II (Ang-II), and phosphorylated mitogen-activated protein kinases (MAPKs). RESULTS: BCCAo increased glial proliferation in the hippocampus and white matter, whereas proliferation was significantly attenuated by GBE treatment. GBE also attenuated the BCCAo-related increases in the hippocampal expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6), TLR4, myeloid differentiation primary response gene 88, RAGE, Ang-II, and phosphorylated MAPKs (ERK, p38, and JNK). Furthermore, GBE treatment restored the ChAT expression in the basal forebrain following BCCAo. CONCLUSIONS: These findings suggest that GBE has specific neuroprotective effects that may be useful for the treatment of CCH. The pharmacological mechanism of GBE partly involves the modulation of inflammatory mediators and the cholinergic system.

Fructus mume extracts alleviate cognitive impairments in 5XFAD transgenic mice.

BACKGROUND: Fructus mume (F. mume) has been used as a traditional treatment for ulcer, cough, and digestive problems for many years in Asian countries. Previous studies have demonstrated that F. mume extracts alleviate cognitive deficits in rats with chronic cerebral hypoperfusion and in mice with scopolamine treatments. The present experiment was conducted to examine the effects of F. mume on cognitive impairments in 5XFAD transgenic mice with five familial Alzheimer's disease (AD) mutations. METHODS: F. mume was administered daily to 5XFAD mice at 12 weeks of age and continued for 90 days. Cognitive function was evaluated using a spatial memory version of the Morris water maze task, the object/location novelty recognition test, and contextual fear conditioning at 24 weeks of age. To elucidate the possible mechanisms underlying the memory improving effects of F. mume in 5XFAD mice, we examined alterations in hippocampal cholinergic function. RESULTS: Vehicle-treated 5XFAD mice exhibited hippocampus-dependent memory impairments compared with non-transgenic littermates, which was reversed in F. mume-treated 5XFAD mice. In addition, reduced hippocampal choline acetyltransferase (ChAT) levels in 5XFAD mice were reversed by F. mume treatment, indicating that F. mume enhances the effects of cholinergic neuronal function. CONCLUSIONS: F. mume may have therapeutic effects on cognitive impairments in AD.

Impairment of intradimensional shift in an attentional set-shifting task in rats with chronic bilateral common carotid artery occlusion.

Studies of rats with chronic bilateral common carotid artery occlusion (BCCAo), an animal model for vascular dementia (VaD), have reported hippocampus-dependent memory impairment and associated neuropathologies. Patients with VaD also experience attentional shifting dysfunction. However, animal models of VaD have not been used to study attentional function. Therefore, the present study examined attentional function in rats with BCCAo, using attentional set-shifting task (ASST) that required rats to choose a food-baited pot from 2 possible pots. ASST included 6 consecutive sessions including simple discrimination, compound discrimination, intradimensional shifting, extradimensional shifting, and reversals. The BCCAo rats were significantly slower at learning the intradimensional set-shifting task compared to control rats. Previous studies have demonstrated that the cingulate cortex and medial prefrontal cortex are critical to intradimensional and extradimensional set-shifting, respectively. Additionally, inflammatory responses and neuronal dysfunction were observed in rats with chronic BCCAo. In addition, OX-6 positive microglia significantly increased in the forceps minor white matter of BCCAo rats, and glutamate decarboxylase signals co-localized with NeuN were reduced in the anterior cingulate cortex of BCCAo rats, compared to control rats. Impaired neuronal and GABAergic neuronal integrity in the anterior cingulate cortex, damage to white matter, and attentional impairments observed in BCCAo rats suggest dysfunction of brain structures that are associated with attentional impairments observed in patients with VaD.