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.
- Alzheimer disease
- brain atrophy
- chronic cerebral hypoperfusion
- reference memory
- subcortical vascular dementia
Subcortical ischemic vascular dementia (SIVD) is one of the major subtypes of vascular dementia in elderly people and accounts for at least 10% to 20% of all dementia cases in developed countries.1 SIVD is characterized by white matter changes and lacunar infarctions in which cerebral blood flow (CBF) is decreased over an extended period of time because of small vessel changes.1–3 Few studies have explored the molecular mechanisms of SIVD devising chronic cerebral hypoperfusion models that reproduce white matter damage and behavioral changes characteristic of SIVD in rats and gerbils.4–6 However, the rat chronic cerebral hypoperfusion model has significant drawbacks, such as the development of visual impairment and its nonapplicability to genetically engineered mice.7 To address this issue, we have recently developed a mouse model of chronic cerebral hypoperfusion8 that can be produced by placing microcoils bilaterally on the common carotid arteries, resulting in bilateral common carotid artery stenosis (BCAS) for 30 days. The white matter damage was associated with working memory deficits but not with reference memory deficits, suggesting that this method serves as a model of SIVD.9 However, the neurological deficits arising from human SIVD are known to progress over a period of decades. It is therefore apparent that the BCAS model should be applied for a longer period encompassing a significant proportion of life to replicate the condition in humans. Our study therefore examined histological, metabolic, and behavioral changes in older mice after long-term cerebral hypoperfusion by BCAS. This would determine whether the longer-term hypoperfusion replicates advanced stages of SIVD and possibly provide evidence linking chronic hypoperfusion and aging.
Materials and Methods
Surgical Procedure of Chronic Cerebral Hypoperfusion and Experimental Design
Male C57BL/6J mice (16-week-old, weighing 25 to 35 grams; Japan SLC, Hamamatsu, Japan) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and chronic cerebral hypoperfusion was induced by applying microcoils (0.18-mm internal diameter) to both common carotid arteries as we previously described.8 Mice in the control group underwent sham operation. After the operation, the mice were housed in cages with a 12-hour light/dark cycle (lights on at 7:00am) with access to food and water ad libitum. CBF in the frontal cortices was measured by laser speckle imaging (OmegaZone; Omegawave) and monitored at 2 hours and at 1, 2, and 3 months after BCAS. At 2 months after BCAS, animals were divided into 2 groups, 1 of which underwent comprehensive behavioral test battery (Figure 1) and the other was examined for CBF and histological changes. Two cohorts were used because procedures for CBF measurement were thought to affect behavioral performance. All procedures were performed in accordance with the guidelines for animal experimentation from the Ethical Committee of Kyoto University.
Comprehensive Behavioral Test Battery
We have previously described detailed procedures of each test battery.10–12 Comprehensive behavioral battery was performed per schedule in Figure 1 in a similar sequence to that performed in our previous report to minimize performance interference among tasks.9,13 Detailed procedures are described in the Supplemental Methods (available online at http://stroke.ahajournals.org).
18F-Fluorodeoxyglucose Positron Emission Tomography Analysis
Small animal positron emission tomography imaging was performed to assess temporal changes in 18F-fluorodeoxyglucose (FDG) uptake during the first 5 minutes and again between 45 and 90 minutes. Detailed procedures are described in the Supplemental Methods.
At 8 months after BCAS (12 months of age), the mice were euthanized under anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneal) by transcardiac perfusion fixation and the brains were removed and processed as previously described.8,11 Detailed procedures and antibody information are given in the Supplemental Methods.
Image Analysis and Statistical Analysis
The applications used for the behavioral studies were based on the public domain National Institutes of Health’s Image program (available at http://rsb.info.nih.gov/nih-image/) and ImageJ program (http://rsb.info.nih.gov/ij/), which were modified for each test by Tsuyoshi Miyakawa (available through O’Hara & Co, Tokyo, Japan). Statistical analysis was conducted using StatView (SAS Institute). Data were analyzed by 2-way analysis of variance or 2-way repeated measures analysis of variance, unless noted otherwise. Values in the graphs were expressed as mean±SEM. P<0.05 was considered statistically significant.
General Health and CBF
All procedures for BCAS were accomplished within 15 minutes, except for an interval of 30 minutes between operating on the right and left common carotid arteries. By day 3 after the BCAS operation, at least 85% of the mice had survived and, after that period, there was no difference in survival rate between the sham-operated and BCAS-operated mice. In the control group, the mean CBF after the sham operation varied from 96.0% to 103.2%, without any significant changes between time intervals (1 factorial analysis of variance, P>0.2). In contrast, the CBF values decreased significantly from the preoperative baseline after the surgery in the BCAS-operated group. The CBF values temporarily decreased to 62.9±18.5% (mean±SE) at 2 hours after BCAS as compared to the sham group but gradually recovered to 81.7%±4.0% at 1 month, 83.2%±1.8% at 2 months, and 85.0%±8.7% at 3 months (sham, n=5; BCAS, n=6).
18F-FDG Positron Emission Tomography Analysis
The first 5-minute uptake of 18F-FDG in the cerebral cortex decreased to ≈70% of the pre-BCAS level at 2 hours after BCAS and recovered to 88% of the pre-BCAS level at 2 months after BCAS (Figure 2A, 2C). The early 18F-FDG uptake scan in the striatum showed a similar temporal profile to that of the cerebral cortex. By contrast, the first 5-minute uptake of 18F-FDG in the hippocampus did not decrease at 2 hours or 2 months after BCAS but decreased at 6 months. The late 18F-FDG uptake scans showed that the glucose uptake of the hippocampus did not decrease by 2 months after BCAS but decreased by 20% at 6 months after BCAS (Figure 2B, 2D).
Behavioral Tests: Working Memory Test
The number of different arm choices in the first 8 entries ranges from 5.3 for a “chance” performance to 8 for a “perfect” performance. Sham-operated mice improved their performance significantly more than did BCAS-operated mice with each consecutive training session (Figure 3A). The difference was significant in the 1 to 22 trials (P=0.0024; 2-way repeated measures analysis of variance) but not in the 23 to 28 trials (P=0.1768). In terms of the number of revisiting (errors), the sham-operated mice improved their performance over the course of training, whereas BCAS-operated mice did not and made significantly more errors than did the sham-operated control (Figure 3B). The difference was significant both in the 1 to 22 trials (P=0.0098) and in the 23 to 28 trials (P=0.0007).
Reference Memory Test
The mice with BCAS exhibited prolonged latency to reach the desired target (P=0.010; Figure 4A) and increased number of errors (P=0.0017; Figure 4B) compared to the sham-operated mice. The first probe test showed that time spent around each hole did not differ between the BCAS-operated and sham-operated mice (P=0.6839; Figure 4C). Consistent with this, there was a significant preference for the target hole vs hole at angle 30° both in the BCAS-operated and sham-operated mice (P<0.0001 each; P<0.0024 is statistically significant by Bonferroni/Dunn test). The second probe test showed that the time spent around each hole tended to be different between the 2 groups (Figure 4D; P=0.0520). There was a significant preference for the target hole in the sham-operated mice (P<0.0001), whereas such significant preference was absent in the BCAS-operated mice (P=0.0089; P<0.0024 is statistically significant). Thus, the probe test suggested that longer-term memory retention was preferably impaired in the BCAS-operated mice.
Other Behavioral Tests
The results of other behavioral tests (Figure 1) are described in Supplemental Materials, including Supplemental Figures I and II⇓ (locomotion and motor function, respectively; available online at http://stroke.ahajournals.org). Briefly, the BCAS-operated mice exhibited impairment in locomotion and motor function after 3 months.
Histological Findings: Hippocampal Atrophy in Mice With Longer-Term BCAS
At 8 months after BCAS, pyknotic neurons were frequently observed in the cerebral cortex and the hippocampus (Figure 5A). Furthermore, there was significant atrophy in the hippocampus (P=0.0064) but not in the cerebral cortex (P=0.8937) or the corpus callosum (P=0.8751; Figure 5B). The number of fragmented or shrunken nuclei stained for single-stranded DNA increased in the CA1 (P=0.0005) and CA3 sectors of the hippocampus (P=0.0086) but not in the dentate gyrus (P>0.05; Figure 5C). We did not detect amyloid β deposits or axonal amyloid precursor protein immunoreactive accumulation in the hippocampus or the cerebral cortex at 8 months after BCAS (data not shown).
Involvement of Cholinergic Fibers
The acetylcholine esterase-positive cholinergic fibers in the external capsule were stained similarly between the sham-operated and BCAS-operated mice, but the cholinergic fibers in the adjacent cortex became disarrayed and fewer, indicating morphological changes in cholinergic fibers (Figure 5D). Densitometric analysis showed a reduction in the cholinergic fibers of the BCAS-operated mice (n=6; % stained area, 20.0%±12.5%) compared to the sham-operated mice (n=4; 41.0%±4.8%). The cell count of choline acetyltransferase-positive cells in the nucleus basalis of Meynert neurons, however, did not differ significantly between the 2 groups but there was a tendency toward a decrease in the BCAS-operated group (64.9±13.2/mm2 in the BCAS-operated vs 74.4±11.1/mm2 in the sham-operated groups; mean±SE).
Histological and behavioral differences between the previous shorter-term BCAS model8,9 and the present longer-term model are summarized in the Table. In the shorter-term model, specific white matter changes were observed without any apparent cerebral cortical or hippocampal changes, which might explain why working memory, but not reference memory, is impaired. By contrast, in the present longer-term BCAS model, not only the white matter changes but also the hippocampal changes (atrophy and cell death) were documented by 8 months after BCAS. Consistent with these histological changes, the series of behavioral batteries demonstrated deficits in both working and reference memory. Thus, mild cerebral ischemia of an insufficient magnitude appears to induce subacute pathologies, which may lead to subsequent changes in the gray matter, including the cerebral cortex and hippocampus.
Laser speckle flowmetry showed that in the cerebral cortex, the CBF decreased at 2 hours after BCAS but recovered by 1 to 3 months. Such temporal profile of CBF was similar to that of the first 5-minute 18F-FDG uptake in the cerebral cortex. Such a similarity and linear correlation of the early uptake of 18F-FDG with 15O-measured blood flow14 suggest that the early 18F-FDG uptake scan can serve as an estimate of CBF. This may also be supported by the substantially different patterns of 18F-FDG uptake between the early and late 18F-FDG images. Intriguingly, in the hippocampus, the CBF estimated from the first 5-minute 18F-FDG uptake did not decrease at 2 months after BCAS probably because the hippocampus is supplied by both anterior and posterior circulations.15 However, the hippocampal CBF was found to be decreased at 6 months after BCAS. This temporally correlated with reduced glucose metabolism in the hippocampus and global memory impairment. Given that the shorter-term BCAS mice demonstrate white matter damage without any apparent hippocampal damage at 1 month after BCAS,8 hippocampal degeneration may be secondary to the preceding white matter damage. This may then subsequently contribute to the dementia syndrome, partly overlapping with Alzheimer disease (AD) in their cognitive profiles and histological changes. These findings are intriguing given the widely accepted fact that vascular dementia and AD both increase in prevalence with age, frequently occur concomitantly, and overlap considerably in their symptomatology, pathophysiology, and comorbidity.16
Consistent with this notion, patients with AD and SIVD are reported to show a similar pattern of pyramidal neuronal loss and atrophy in the hippocampal CA1 sector.17,18 Moreover, most AD patients show amyloid angiopathy19 and decreased capillary beds in the cerebral cortex,20 thereby leading to chronic cerebral hypoperfusion. These results, as well as those presented here, suggest a bidirectional relationship between AD and SIVD.
By contrast, a recent report from the Honolulu Asia Aging Study suggests that the burden of vascular-type and AD-type lesions are independent.21 However, there is a possibility that the studies of vascular dementia contain some proportion of large vessel disease or infarction.22 Our longer-term BCAS model does not develop overt cerebral infarction despite the global memory deficits. This strengthens the potential significance of chronic noninfarctional hypoperfusion as a cause of the dementia syndrome.2 The reasoning is also consistent with the hypothesis that hippocampal sclerosis is often accompanied by leukoencephalopathy, and that occult hypoxic–ischemic episodes may represent its pathogenic factors.3,23 Thus, chronic “noninfarctional” hypoperfusion may accelerate hippocampal neurodegeneration in a latent manner, as is reproduced in the present mouse model. Nevertheless, amyloid β deposits, which are a pathological hallmark of AD, are not detectable in this model at least up to 8 months after the BCAS operation. This is probably because, in contrast with their human counterpart, murine amyloid β proteins do not have propensity to form oligomers and fibrils. In support of this notion, AD model mice overexpressing the mutant form of human amyloid precursor protein have greater amounts of amyloid β fibrils in their brains after 30 days of cerebral hypoperfusion by BCAS,24 implying a causal link between vascular and human amyloid β neuropathology.
A limitation of this study is that the effectiveness of the first 5-minute 18F-FDG blood flow analysis needs to be further confirmed. In tumor diagnosis, blood flow estimated from the early uptake of 18F-FDG is linearly correlated with 15O-measured blood flow25 (the current gold standard method for measuring blood flow in humans). However, because the mean positron range of 15O is far larger than that of 18F (2.5 mm vs 0.6 mm), 15O-water is insufficient to apply to a small animal positron emission tomography scanner in terms of the spatial resolution.14 In addition, 15O-water is a short-lived tracer with a 2-minute half-life, necessitating an on-site cyclotron.25 Therefore, 18F-FDG imaging may be practical and feasible to estimate CBF noninvasively in rodent models for the simple first 5-minute, despite the incomplete extraction of 18F-FDG compared to 15O-water. Although appropriate kinetic models of 18F-FDG will be required to separate the flow component and K1 from the metabolic component of uptake, a method of measuring blood flow and metabolism from a single injection of 18F-FDG uptake may be an important addition to functional imaging of rodent disease models with 18F-FDG positron emission tomography.
Another limitation of this study is that the long-term BCAS mice do not have lacunar infarcts, which are a key feature of the advanced stages of SIVD1 and are often linked with development of motor dysfunction. However, despite absence of lacunar infarcts, the BCAS mice exhibited impaired motor function at 3 months after BCAS, recapitulating one of the key features of SIVD. The frontal–subcortical circuit disruption may have resulted not only in the cognitive impairment but also in the motor dysfunction. Therefore, this longer-term BCAS model will provide evidence linking chronic hypoperfusion and aging-associated neurological deficits such as cognitive and motor impairment. However, this model does not (no current models can) describe all features of SIVD.26
In conclusion, we describe a mouse model of longer-term cerebral hypoperfusion that at least partially replicates an advanced stage of SIVD in which hippocampal neuronal loss becomes significant. This mouse model exhibits global memory disturbances, which may help further elucidate the mechanism by which neurodegeneration and dementia progress in the elderly, and enhances strategies to tackle these disorders. Further characterization of our model, particularly in view of the hippocampal circuitry, may help to decipher the substrates associated with impaired memory.
The authors express their cordial gratitude to Dr Ahmad Khundakar for critical reading of the manuscript, Dr Yoko Okamoto for helpful discussions, and Dr Makoto Kinoshita for his valuable advice. The authors also thank Hitomi Nakabayashi for histochemistry, Kazuo Nakanishi for statistical analysis, Taeko Ogura and all technical staff for animal care, and Dr Akiyoshi Nishio for continuous encouragement.
Sources of Funding
This work was supported in part by a research grant from the Astellas Foundation for Research on Metabolic Disorders (to M.I.) and by grants from BIRD of Japan Science and Technology Agency and Integrative Brain Research (IBR-shien) from the MEXT of Japan (to T.M.).
- Received February 11, 2010.
- Accepted February 27, 2010.
Ihara M, Tomimoto H, Ishizu K, Mukai T, Yoshida H, Sawamoto N, Inoue M, Doi T, Hashikawa K, Konishi J, Shibasaki H, Fukuyama H. Decrease in cortical benzodiazepine receptors in symptomatic patients with leukoaraiosis: a positron emission tomography study. Stroke. 2004; 35: 942–947.
Kudo T, Tada K, Takeda M, Nishimura T. Learning impairment and microtubule-associated protein 2 decrease in gerbils under chronic cerebral hypoperfusion. Stroke. 1990; 21: 1205–1209.
Nakaji K, Ihara M, Takahashi C, Itohara S, Noda M, Takahashi R, Tomimoto H. Matrix metalloproteinase-2 plays a critical role in the pathogenesis of white matter lesions after chronic cerebral hypoperfusion in rodents. Stroke. 2006; 37: 2816–2823.
Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke. 2004; 35: 2598–2603.
Shibata M, Yamasaki N, Miyakawa T, Kalaria RN, Fujita Y, Ohtani R, Ihara M, Takahashi R, Tomimoto H. Selective impairment of working memory in a mouse model of chronic cerebral hypoperfusion. Stroke. 2007; 38: 2826–2832.
Ihara M, Yamasaki N, Hagiwara A, Tanigaki A, Kitano A, Hikawa R, Tomimoto H, Noda M, Takanashi M, Mori H, Hattori N, Miyakawa T, Kinoshita M. Sept4, a component of presynaptic scaffold and Lewy bodies, is required for the suppression of alpha-synuclein neurotoxicity. Neuron. 2007; 53: 519–533.
Crawley JN. What’s wrong with my mouse: Behavioral phenotyping of transgenic and knockout mice. Wilmington, DE: Wiley-Liss; 2007.
Kalaria R. Similarities between Alzheimer’s disease and vascular dementia. J Neurol Sci. 2002; 203–204: 29–34.
Kril JJ, Patel S, Harding AJ, Halliday GM. Patients with vascular dementia due to microvascular pathology have significant hippocampal neuronal loss. J Neurol Neurosurg Psychiatry. 2002; 72: 747–751.
Du AT, Schuff N, Laakso MP, Zhu XP, Jagust WJ, Yaffe K, Kramer JH, Miller BL, Reed BR, Norman D, Chui HC, Weiner MW. Effects of subcortical ischemic vascular dementia and AD on entorhinal cortex and hippocampus. Neurology. 2002; 58: 1635–1641.
Attems J, Jellinger KA. Hippocampal sclerosis in Alzheimer disease and other dementias. Neurology. 2006; 66: 775.
Mullani NA, Herbst RS, O'Neil RG, Gould KL, Barron BJ, Abbruzzese JL. Tumor blood flow measured by PET dynamic imaging of first-pass 18F-FDG uptake: a comparison with 15O-labeled water-measured blood flow. J Nucl Med. 2008; 49: 517–523.
PET Acquisition and Image Analysis
Small animal positron emission tomography (PET) imaging was performed with the GE eXplore Vista (GE Medical Systems, Milwaukee, Wisc, USA).1 In brief, the effective transverse and axial field of view are 6.7 cm and 4.7 cm, respectively. The spatial resolution of eXplore VISTA with 2D ordered subset expectation maximization (OSEM) is 1.2 mm at the center of field.
Five male C57BL/6J mice (body weight range at the start of the experiment 20 to 30 g; Japan SLC, Hamamatsu) were given free access to food and water ad libitum and were deprived of food but allowed free access to water for at least 12 hours before PET scanning. Before small animal PET imaging, the mice were anesthetized with 1.5% isoflurane and air (3 1/min) and placed on the scanner bed in the prone position. All mice were breathing spontaneously throughout the entire experiment. Rectal temperatures were monitored and a far-infrared lamp was used for keeping body temperature around 37.0°C. Dynamic PET studies were performed for 90 minutes after the intravenous application of 18.5 to 30.0 MBq 18F-fluorodeoxyglucose (18F-FDG) via a tail vein using a 23-frame protocol (5 frames of 1 minute, 2 frames of 2.5 minutes, and 16 frames of 5 minutes). The PET studies were performed a week before and 2 hours after BCAS in a mouse, 2 months after BCAS in a mouse, and 6 months after BCAS in 3 mice. All PET procedures were performed in accordance with the guidelines for animal experimentation from the ethical committee of Kyoto University.
PET data were reconstructed by 2D OSEM (2 iterations and 16 subsets) and no corrections were made for attenuation or scatter since a rodent brain size is relatively small compared to that of humans.2 The images consisted of a 175×175×61 matrix with actual voxel size of 0.3875×0.3875×0.775 mm. First 5 minute scans of 18F-FDG uptake were used as an indicator of cerebral blood, which is suggested in tumor studies.3 18F-FDG scans obtained between 45 and 90 minutes after injection were used as an indicator of cerebral glucose metabolism. Volumes of interest (VOIs) were defined on a representative magnetic resonance (MR) image obtained by male C57BL/6J mice using a 7 T system (Bruker BioSpec 70/20 USR, Bruker, Karlsruhe, Germany): cortex (frontal area around from -1.5 mm posterior to the bregma), striatum (caudate-putamen), hippocampus, and cerebellum. Each reconstructed PET image during 45 to 90 minutes was rigidly coregistered to the MR image using the mutual information maximization algorithm4 implemented in Statistical Parametric Mapping (SPM) version 2 (Wellcome Department of Cognitive Neuroscience, London, UK). Each PET image during first 5 minutes was transformed by the same parameters obtained by a 45 to 90 minute image. Average counts under each VOI were calculated and PET images and counts under the three VOIs (cortex, striatum and hippocampus) were normalized to an average count of the cerebellum as a reference because ischemic changes were not observed in the cerebellum in mice with BCAS.5,6
Neurological screening was conducted with 6-month-old male mice (2 months after operation). Ear twitch, whisker touch, and righting reflexes were evaluated.
Eight-Arm Radial Maze Test for Working Memory
The eight-arm radial maze test was conducted with 9-month-old male mice (5 months post-BCAS) essentially as described previously.7 Each arm (9×40 cm) radiated from an octagonal central starting platform. Identical food wells with pellet sensors were placed at the distal end of each arm. After initial pretraining, each mouse was placed in the central starting platform and allowed to explore and to consume food pellets scattered on the whole maze for a 5-minute period. Subsequently, these mice received further pretraining to take a pellet from each food well after being placed at the distal end of each arm. A trial was terminated after the subject consumed the pellet. This was repeated eight times, using eight different arms, for each mouse. All eight arms were baited with food pellets. Mice were placed on the central platform and allowed to obtain all eight pellets within 25 minutes. A trial was terminated immediately after all eight pellets were consumed or 25 minutes had elapsed. For each trial, choices of arms, latency to obtain all pellets, distance traveled, and number of different arms chosen within the first eight choices were recorded. The number of revisits, and omission errors were automatically recorded.
Barnes Maze Test for Reference Memory
The Barnes maze test was performed with 10-month-old male mice (6 months post-BCAS). The task was conducted on ‘dry land,’ a white circular surface, 1.0 m in diameter, with 12 holes equally spaced around the perimeter.8 The circular open field was elevated 75 cm from the floor. A black Plexiglas escape box (17×13×7 cm), which contained paper cage bedding on its floor, was located under one of the holes. The hole above the escape box represented the target, analogous to the hidden platform in the Morris task. The location of the target was consistent for a given mouse but was randomized across mice. The maze was rotated daily, with the spatial location of the target unchanged with respect to the visual room cues, to prevent a bias based on olfactory or proximal cues within the maze. Three trials per day were conducted for ten successive days preliminarily (on days 5 and 6, no trial was undertaken). A probe trial was conducted 24 hours after the last training without the escape box in order to confirm that this spatial task was acquired based on navigation using distal environment room cues. Time of latency to reach the target hole, number of errors, distance to reach the target hole, and time spent around each hole were recorded by video tracking software (Image BM). To assess long-term retention, a second probe trial was applied a week after probe test 1 and additional three sessions of retraining.
Locomotor Activity Tests
In order to examine locomotor activity, the open field test, the light/dark transition test and the elevated plus maze test were conducted with 6- to 7-month-old male mice (2 to 3 months after operation) each in a single session. In the open field test, each animal was placed in the center of the open field apparatus (40×40×30 cm; Accuscan Instruments, Columbus, Ohio, USA). Total distance traveled (in cm), vertical activity (rearing measured by counting the number of photobeam interruptions), and time spent in the center were recorded. Data were collected for 120 minutes. The light/dark transition test apparatus consisted of a cage (21×42×25 cm) divided into two chambers, one of which was brightly illuminated (390 lux), and the other dark (2 lux). Mice were placed into the dark side and allowed to move freely for 10 minutes. The total number of transitions, time spent in each side, first latency to light side, and distance traveled were recorded by Image LD software (see ‘Image Analysis’). The elevated plus-maze (O'Hara & Co., Tokyo, Japan) consisted of two open arms (25×5 cm) and two enclosed arms of the same size, with 15-cm high transparent walls. The arms and central square were made of white plastic plates and were elevated to a height of 55 cm above the floor. To minimize the likelihood of animals falling from the apparatus, 3-mm high plastic ledges were provided for the open arms. Arms of the same type were arranged at opposite sides to each other. Each mouse was placed in the central square of the maze (5×5 cm), facing one of the closed arms. Mouse behavior was recorded during a 10-minute test period. The number of entries into, and the time spent in open and enclosed arms, were recorded. For data analysis, the following four measures were used: the percentage of entries into the open arms, the time spent in the open arms (s), the number of total entries, and total distance traveled (cm). Data acquisition and analysis were performed automatically using Image EP software (see ‘Image Analysis’).
Motor Function Tests
Motor coordination and balance were tested with the rotarod and beam tests with 7-month-old male mice (3 months after operation), and neuromuscular strength with wire hang test and grip strength test with 6-month-old male mice (4 months after operation). In addition, gait analysis was performed to examine possible deficits. In the wire hang test, the mouse was placed on a wire cage lid apparatus (O'hara & Co) to assess balance and grip strength. The mouse was placed on a wire mesh, which was then inverted, and latency to fall was recorded. A grip strength meter (O'hara & Co) was used to assess forelimb grip strength, when mice were pulled back. The rotarod test was performed by placing a mouse on a rotating drum (UGO Basile Accelerating Rotarod), and the time to maintain its balance on the rod was measured. The speed of the rotarod was accelerated from 4 to 40 rpm over a 5-minute period while a beam test was performed, which measured the ability of mice to traverse a narrow beam to reach a dark box. The beams, with a rough painted surface, consisted of two different strips of iron (each measuring 100 cm long; one measuring 2.8 cm [thick bar] and the other measuring 1.1 cm [thin bar] in diameter) placed horizontally 50 cm above the bench surface. One session of three trials was performed using the 2.8-cm beam. Mice were then tested using the 1.1-cm beam. Animals were allowed up to 90 seconds to traverse each beam. The number of sideslips and the moving speed were recorded for each trial by the Image OF software package (see ‘Image Analysis’). Gait analysis was performed using a DigiGait Imaging System (Mouse Specifics, Boston, MA) with 7-month-old male mice (3 months after operation). Eight variables were considered (stride duration (ms), stride length (cm), stance duration (ms), stance/stride ratio, braking duration (ms), brake/stance ratio, propulsion duration (ms), and propulsion/stance ratio) for each paw at two running speeds (24.7 and 30 cm/s).
Startle Response/Prepulse Inhibition Tests
Startle Response/Prepulse Inhibition Test was performed on 7-month-old male mice (3 months after operation). A startle reflex measurement system was used (O'Hara & Co). The test session was started by placing a mouse in a plexiglasss cylinder for 10 minutes. The duration of white noise as the startle stimulus was 40 ms for all trial types. The startle response was recorded for 140 ms (measuring the response every 1 ms) starting with the onset of the prepulse stimulus. The peak startle amplitude recorded during the 140-ms sampling window was used as the dependent variable. A test session consisted of 6 trial types (ie, 2 types for startle stimulus only trials, and 4 types for prepulse inhibition trials). The intensity of startle stimulus was 110 or 120 dB. The prepulse sound was presented 100 ms before the startle stimulus, and its intensity was 74 or 78 dB. Four combinations of prepulse and startle stimuli were used (74/110, 78/110, 74/120, and 78/120). Six blocks of the 6 trial types were presented in pseudorandom order such that each trial type was presented once within a block. The average intertrial interval was 15 s (range: 10 to 20 s).
Hot Plate Test
The hot plate test was performed on 6-month-old male mice (2 months after operation). For nociception it was used to evaluate sensitivity to a thermal stimulus. Mice were placed on a 55.0 (±0.3)°C hot plate (Columbus Instruments), and latency to the first hind-paw response (a foot shake or a paw lick) was recorded.
Test for Depressive State
To examine the depressive state Porsolt forced swim test was performed on 7-month-old male mice (3 months after operation). The apparatus consisted of 4 plexiglass cylinders (20 cm height ×10 cm diameter). The cylinders were filled with water (23°C), up to a height of 7.5 cm. Mice were placed into the cylinders, and their behavior was recorded over a 10-minute test period (day 1, 2). Data acquisition and analysis were performed automatically, using Image PS software (see ‘Image Analysis’).
Social Interaction Test in a Novel Environment
Social interaction test in a novel environment was performed for 7-month-old male mice (3 months after operation). Two mice of identical genotypes that were previously housed in different cages, were placed into a box together (40×40×30 cm) and allowed to explore freely for 10 minutes. Social behavior was monitored by a CCD camera, which was connected to a Macintosh computer. Analysis was performed automatically using Image SI software. Total duration of contact, the number of contacts, the number of active contacts, mean duration per contact, and total distance traveled were measured. The number of active contacts was defined as follows. Images were captured at 1 frame per second, and distance traveled between two successive frames was calculated for each mouse. If the two mice contacted each other and the distance traveled by either mouse was longer than 5 cm, the behavior was considered as ‘active contact.’
At 8 months post-BCAS (12 months of age), the mice were euthanized under anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneal) by transcardiac perfusion fixation, the brains removed and processed as previously described.5,9 Frontal, middle, and posterior (hippocampal) portion of each brain was dissected in consultation with the respective brain maps.10 The coordinates measured from the bregma were as follows: from 0.15 to 0.3 mm for the frontal, from 0 to −1.0 mm for the middle, and from −1.8 to −2.2 mm for the posterior portion. The frontal and posterior portion were embedded in paraffin and sliced into 6 μm-thick coronal sections and then subjected to Klüver-Barrera staining and hematoxylin and eosin (H&E) staining. The middle portion of the brain was cut into serial sections (20-μm-thick) on a cryostat for immunohistochemistry and histochemical staining. In the H&E-stained coronal sections, the area of the cortex dorsal to the anterior commissure at +0.15 mm of bregma, the area of the hippocampus at −2.0 mm of bregma, and the area of the whole hemispheres and ventricular spaces at both positions were digitized and measured using the BZ-II Analyzer (KEYENCE, Co. Ltd., Kyoto, Japan) as an index of brain atrophy. The severity of the white matter lesions was evaluated at the corpus callosum using graded score, as previously described.11 For immunohistochemistry, the serial sections were incubated at 4°C for 48 hours with a rabbit antiglial fibrillary acidic protein (GFAP) antibody (diluted 1:20,000; DAKO, Denmark), a rabbit antiglutathione S-transferase-π (GST-π) antibody (1:100; Stressgen, MI), a rat anti-Iba 1 antibody (1:2000; Wako, Pure Chemicals, Osaka, Japan), a rabbit anti-amyloid precursor protein (APP) antibody (1:200; Millipore, Billerica, MA), a rabbit anti-single stranded DNA (ss DNA) antibody (1:100; Dako, Carpinteria, Calif), and a goat anticholine acetyltransferase (ChAT) antibody (1:100; Chemicon). Subsequently, these sections were treated with the appropriate biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, Calif) and were visualized with 0.01% diaminobenzidine tetrahydrochloride and 0.005% H2O2 in 50 mmol/L Tris HCl (pH 7.6). Immunohistochemistry for amyloid β (1–40) and amyloid β (1–42) was performed using Aβ staining kit (Dakoppatts, Denmark) according to the manufacturer’s recommendation. We also performed acetylcholine esterase (AChE) histochemistry, as previously described.12 The cholinergic fibers were evaluated by densitometric analysis of AChE histochemical staining; several regions of interest were set randomly on the deep layers 5 to 6 of the cortex close to the external capsule. The ChAT immunopositive neurons in the basal nucleus (Meynert) at −0.5 mm of bregma were counted.
Physical Characteristics, Sensory Motor Reflexes, and Nociception
There were no significant differences between BCAS- and sham-operated mice in terms of their physical characteristics such as body weight, temperature and blood pressure. Body weight in BCAS- and sham-operated mice was not significantly different between the groups. There were also no significant differences in sensory-motor reflexes (percent with quick response of ear twitch, normal response of whisker twitch and righting reflex, and acoustic startle response), sensory-motor gating (prepulse inhibition), nociception (hot plate test), and physical strength (wire hang).
Locomotor Activity and Anxiety
In the open field test, the mice at 2 months post-BCAS exhibited a significant increase of total distance traveled (P=0.0244). The vertical activity and the center time tended to increase in the BCAS-operated mice, suggesting that the BCAS-operated mice were hyperactive. In the light/dark transition test, the BCAS-operated mice displayed an increase in the distance traveled in the light chamber and time spent in light. In the elevated plus maze test, the BCAS-operated mice showed no tendency to move a longer distance but significantly increased time spent on open arms. These behavioral phenotypes collectively indicated that mice were likely to become hyperactive following certain emotional changes such as a decrease in the levels of anxiety induced by BCAS for 2 months (Supplemental Figure I, available online at http://stroke.ahajournals.org).
Motor function was mainly examined at 3 months after BCAS. There were no significant differences between the BCAS- and sham-operated mice in the rotarod test. In the beam test, however, the BCAS-operated mice exhibited significantly reduced moving speed both on the bold and the narrow beams (P=0.002), suggesting impaired motor function of the BCAS-operated mice after 3 months. The gait analysis also showed that the brake-stance ratio of the forepaws were significantly increased, and the propulsion duration and the propulsion-stance ratio of the forepaws were significantly decreased in BCAS-operated mice, indicating impaired locomotion in BCAS-operated mice (Supplemental Figure II, available online at http://stroke.ahajournals.org).
Depression and Social Interaction
There were no significant differences between the sham- and BCAS-operated mice in the Porsolt forced swim test and social interaction test.
Wang Y, Seidel J, Tsui BM, Vaquero JJ, Pomper MG. Performance evaluation of the GE healthcare explore vista dual-ring small-animal pet scanner. J Nucl Med. 2006; 47: 1891–1900.
Casteels C, Vermaelen P, Nuyts J, Van Der Linden A, Baekelandt V, Mortelmans L, Bormans G, Van Laere K. Construction and evaluation of multitracer small-animal pet probabilistic atlases for voxel-based functional mapping of the rat brain. J Nucl Med. 2006; 47: 1858–1866.
Mullani NA, Herbst RS, O'Neil RG, Gould KL, Barron BJ, Abbruzzese JL. Tumor blood flow measured by pet dynamic imaging of first-pass 18F-FDG uptake: a comparison with 15O-labeled water-measured blood flow. J Nucl Med. 2008; 49: 517–523.
Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke. 2004; 35: 2598–2603.
Shibata M, Yamasaki N, Miyakawa T, Kalaria RN, Fujita Y, Ohtani R, Ihara M, Takahashi R, Tomimoto H. Selective impairment of working memory in a mouse model of chronic cerebral hypoperfusion. Stroke. 2007; 38: 2826–2832.
Miyakawa T, Yamada M, Duttaroy A, Wess J. Hyperactivity and intact hippocampus-dependent learning in mice lacking the m1 muscarinic acetylcholine receptor. J Neurosci. 2001; 21: 5239–5250.
Ihara M, Yamasaki N, Hagiwara A, Tanigaki A, Kitano A, Hikawa R, Tomimoto H, Noda M, Takanashi M, Mori H, Hattori N, Miyakawa T, Kinoshita M. Sept4, a component of presynaptic scaffold and lewy bodies, is required for the suppression of alpha-synuclein neurotoxicity. Neuron. 2007; 53: 519–533.
Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. San Diego, Calif: Academic Press; 2001.