A refined model of chronic cerebral hypoperfusion resulting in cognitive impairment and a low mortality rate in rats

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OBJECTIVE

The cognitive deficits of vascular dementia and the vasoocclusive state of moyamoya disease have often been mimicked with bilateral stenosis/occlusion of the common carotid artery (CCA) or internal carotid artery. However, the cerebral blood flow (CBF) declines abruptly in these models after ligation of the CCA, which differs from “chronic” cerebral hypoperfusion. While some modified but time-consuming techniques have used staged occlusion of both CCAs, others used microcoils for CCA stenosis, producing an adverse effect on the arterial endothelium. Thus, the authors developed a new chronic cerebral hypoperfusion (CCH) model with cognitive impairment and a low mortality rate in rats.

METHODS

Male Sprague-Dawley rats were subjected to unilateral CCA occlusion and contralateral induction of CCA stenosis (modified CCA occlusion [mCCAO]) or a sham operation. Cortical regional CBF (rCBF) was measured using laser speckle flowmetry. Cognitive function was assessed using a Barnes circular maze (BCM). MRI studies were performed 4 weeks after the operation to evaluate cervical and intracranial arteries and parenchymal injury. Behavioral and histological studies were performed at 4 and 8 weeks after surgery.

RESULTS

The mCCAO group revealed a gradual CBF reduction with a low mortality rate (2.3%). White matter degeneration was evident in the corpus callosum and corpus striatum. Although the cellular density declined in the hippocampus, MRI revealed no cerebral infarctions after mCCAO. Immunohistochemistry revealed upregulated inflammatory cells and angiogenesis in the hippocampus and cerebral cortex. Results of the BCM assessment indicated significant impairment in spatial learning and memory in the mCCAO group. Although some resolution of white matter injury was observed at 8 weeks, the animals still had cognitive impairment.

CONCLUSIONS

The mCCAO is a straightforward method of producing a CCH model in rats. It is associated with a low mortality rate and could potentially be used to investigate vascular disease, moyamoya disease, and CCH. This model was verified for an extended time point of 8 weeks after surgery.

ABBREVIATIONS BCCAO = bilateral CCA occlusion; BCCAS = bilateral CCA stenosis; BCM = Barnes circular maze; CBF = cerebral blood flow; CCA = common carotid artery; CCH = chronic cerebral hypoperfusion; CV = cresyl violet; DBP = diastolic blood pressure; DFn = degrees of freedom in the numerator; LFB = luxol fast blue; LSF = laser speckle flowmetry; mCCAO = modified CCA occlusion; MRA = MR angiography; PBS = phosphate-buffered saline; PR = pulse rate; rCBF = regional CBF; ROI = region of interest; SBP = systolic blood pressure; TOF = time of flight; VA = vertebral artery; WM = white matter.

Article Information

Correspondence Kuniyasu Niizuma: Tohoku University Graduate School of Biomedical Engineering, Sendai, Japan. niizuma@nsg.med.tohoku.ac.jp.

INCLUDE WHEN CITING Published online September 7, 2018; DOI: 10.3171/2018.3.JNS172274.

Disclosures This work was partially supported by JSPS KAKENHI Grant Number 17H01583 and the Project for Japan Translational and Clinical Research Core Centers (No. 17lm0203026h0001) from the Japan Agency for Medical Research and Development, AMED. The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Summary of the mCCAO model. A: Time chart showing the 2 protocols and the sequence of experiments conducted. B: Induction of severe CCA stenosis by the needle-ligature technique. C: The thinned skull as viewed through the surgical microscope. The left side of the skull is thinned in this image, showing a visible cortical vessel (arrow), while the right half of the skull is normal thickness. The right side would also be thinned before mean blood flow measurement. D and E: The temporal profile of the rCBF after mCCAO. The rCBF color map measured by LSF (D) and a graph of the mean rCBF values (from 10 animals) as percentage of baseline (E). The rCBF significantly decreased immediately after the surgery, decreased further a little more at 3 days, and then plateaued until the 2nd week. The rCBF increased again at 3 weeks, then reached a value near normal at 4 weeks after mCCAO. There was no significant difference in rCBF between the stenosis and occlusion sides (time difference, p < 0.001; group difference, p > 0.05). Error bars indicate standard deviations. “Pre” indicates baseline (before mCCAO); d = days; h = hours; w = weeks.

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    Imaging findings 4 weeks after mCCAO. A: Maximum intensity projection of the 3D-TOF MRA. In sham-operated rats, the bilateral CCAs are clearly visible, whereas the VAs are faint and narrow. In contrast, in the mCCAO rat, the CCA signal at the occlusion side is not visualized (dotted arrow), and the CCA on the stenosis side shows a filling defect (arrow); the VAs (double-headed arrow) have thickened walls and increased diameter and tortuosity in the mCCAO group compared to shams. B: Quantification revealed a significant CCA stenosis (83.2% ± 3.2%, p = 0.004); the VA length and area were increased in mCCAO rats 4 weeks after the surgery (analyzed as percentage of the values obtained in the sham group; p = 0.013 and p = 0.009, respectively). The graphs show means of 4 rats for the sham group and 6 rats for the mCCAO group at 4 weeks; error bars indicate standard deviations. *p < 0.05. C: Coronal T2-weighted images obtained 4 weeks after surgery. No brain infarction was seen in the mCCAO or sham-operated rats.

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    Cognitive dysfunction after mCCAO evaluated using the BCM. A: Representative diagrams of paths recorded during animal trials to detect the target box. In each set of 2 images the day 1 and day 4 diagrams show the path taken by a single rat. Comparing the paths from the 1st and 4th days of training on the BCM demonstrates a more complex and longer path to reach the target in the 4- and 8-week mCCAO groups compared to the path of the rat from the sham groups. B: Graph of time latency to find the target box revealing that the latency was significantly longer for both operation and time at all training days in the mCCAO group (*p < 0.05). Analysis of multiple comparisons with Bonferroni correction revealed that the latency of the 8-week mCCAO rats was significantly longer than that of sham-operated rats on day 1 (*p < 0.05), the latency of 8-week mCCAO and 4-week mCCAO rats was significantly longer than that of sham-operated rats on day 2 (*p < 0.05 each), the latency of 8-week mCCAO rats was significantly longer than that of 4-week mCCAO rats on day 2 (*p < 0.05), the latency of 8-week mCCAO and 4-week mCCAO rats was significantly longer than that of sham-operated rats on day 3 (*p < 0.05 each), the latency of 8-week mCCAO rats was significantly longer than that of 4-week mCCAO rats on day 3 (*p < 0.05), and the latency of 8-week mCCAO and 4-week mCCAO rats was significantly longer than that of sham-operated rats on day 4 (*p < 0.05 each). n.s. = not statistically significant; s = seconds. C: The number of times that rats stayed at wrong holes, demonstrating a significantly increased number for both operation and time at all training days in mCCAO rats (*p < 0.05). D: Average movement speed. There was no significant difference between mCCAO and sham rats, indicating exclusive spatial learning and memory impairment. The graphs show the means of 6 rats for the sham group, 9 rats for the mCCAO group at 4 weeks, and 8 rats for the mCCAO group at 8 weeks; error bars indicate standard deviations. Figure is available in color online only.

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    White matter degeneration after mCCAO. LFB + CV staining of the mid–corpus callosum (A) and striatum (B) 1, 4, and 8 weeks after mCCAO demonstrates significantly less LFB staining density in the mCCAO group at 1 week than in the sham group (*p = 0.002). Four weeks after mCCAO, the LFB staining density tends to increase compared to the 1-week time point but is still low compared to the value in the sham group (*p = 0.001). Eight weeks after mCCAO, the LFB staining density tends to reverse to sham group values (p = 0.09). Photomicrographs, original magnification 200×; bar = 200 μm. The graph shows the means of 6 rats for the sham group, 6 rats for the mCCAO group at 4 weeks, and 8 rats for the mCCAO group at 8 weeks; error bars indicate standard deviations.

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    Morphological changes in the neuronal cells in the hippocampal CA1 subregion. A: CV staining of the hippocampal CA1 area. The number of damaged cells (pyknotic nuclei and dark, shrunken cytoplasm) was significantly increased in the hippocampal CA1 subregion in the mCCAO group compared to the sham group (*p < 0.001). Bar = 50 μm. B: Immunohistochemistry of cleaved caspase-3 at 4 and 8 weeks after mCCAO; the percentage of cleaved caspase-3–positive cells was increased in the hippocampal CA1 subregion 4 and 8 weeks after mCCAO (*p < 0.001). Photomicrographs, original magnification 200×; bar = 50 μm. The graph shows the means of 15 rats for the sham group, 21 rats for the mCCAO group at 4 weeks, and 8 rats for the mCCAO at 8 weeks; error bars indicate standard deviations.

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    Inflammatory changes in the parietal cortex and the hippocampal CA1 subregion. The number of cells positive for GFAP (A, *p = 0.001) and Iba-1 (B, *p = 0.002) was significantly increased in the bilateral parietal cortex and hippocampal CA1 subregion 4 and 8 weeks after mCCAO compared to findings in sham-operated rats. Photomicrographs, original magnification 200×; bar = 100 μm. The graph shows the means of 15 rats for the sham group, 21 rats for the mCCAO group at 4 weeks, and 8 rats for the mCCAO at 8 weeks; error bars indicate standard deviations.

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    Immunohistochemistry of CD34. The number of CD34-positive microvessels was significantly increased in the parietal cortex 4 and 8 weeks after mCCAO compared to findings in the sham group (*p = 0.004). Photomicrographs, original magnification 200×; bar = 100 μm. The graph shows the means of 15 rats for the sham group, 21 rats for the mCCAO group at 4 weeks, and 8 rats for the mCCAO group at 8 weeks, 3 sections per animal averaged from the same location of the parietal cortex; error bars indicate standard deviations.

References

  • 1

    Cechetti FPagnussat ASWorm PVElsner VRBen Jda Costa MS: Chronic brain hypoperfusion causes early glial activation and neuronal death, and subsequent long-term memory impairment. Brain Res Bull 87:1091162012

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 2

    Choi DHLee KHLee J: Effect of exercise-induced neurogenesis on cognitive function deficit in a rat model of vascular dementia. Mol Med Rep 13:298129902016

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3

    Dias Fiuza Ferreira EValério Romanini CCypriano PEWeffort de Oliveira RMMilani H: Sildenafil provides sustained neuroprotection in the absence of learning recovery following the 4-vessel occlusion/internal carotid artery model of chronic cerebral hypoperfusion in middle-aged rats. Brain Res Bull 90:58652013

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4

    Du SQWang XRXiao LYTu JFZhu WHe T: Molecular mechanisms of vascular dementia: What can be learned from animal models of chronic cerebral hypoperfusion? Mol Neurobiol 54:367036822017

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    Farkas ELuiten PGBari F: Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Brain Res Rev 54:1621802007

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6

    Hai JWan JFLin QWang FZhang LLi H: Cognitive dysfunction induced by chronic cerebral hypoperfusion in a rat model associated with arteriovenous malformations. Brain Res 1301:80882009

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7

    Hattori YEnmi JKitamura AYamamoto YSaito STakahashi Y: A novel mouse model of subcortical infarcts with dementia. J Neurosci 35:391539282015

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8

    Hiramatsu MHishikawa TTokunaga KKidoya HNishihiro SHaruma J: Combined gene therapy with vascular endothelial growth factor plus apelin in a chronic cerebral hypoperfusion model in rats. J Neurosurg 127:6796862017

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9

    Huang YFan SLi JWang YL: Bilateral common carotid artery occlusion in the rat as a model of retinal ischaemia. Neuroophthalmology 38:1801882014

  • 10

    Jing ZShi CZhu LXiang YChen PXiong Z: Chronic cerebral hypoperfusion induces vascular plasticity and hemodynamics but also neuronal degeneration and cognitive impairment. J Cereb Blood Flow Metab 35:124912592015

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Kim HSLee HJYeu ISYi JSYang JHLee IW: The neovascularization effect of bone marrow stromal cells in temporal muscle after encephalomyosynangiosis in chronic cerebral ischemic rats. J Korean Neurosurg Soc 44:2492552008

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12

    Kitamura ASaito SMaki TOishi NAyaki THattori Y: Gradual cerebral hypoperfusion in spontaneously hypertensive rats induces slowly evolving white matter abnormalities and impairs working memory. J Cereb Blood Flow Metab 36:159216022016

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Kusaka NSugiu KTokunaga KKatsumata ANishida ANamba K: Enhanced brain angiogenesis in chronic cerebral hypoperfusion after administration of plasmid human vascular endothelial growth factor in combination with indirect vasoreconstructive surgery. J Neurosurg 103:8828902005

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Leardini-Tristão MBorges JPFreitas FRangel RDaliry ATibiriçá E: The impact of early aerobic exercise on brain microvascular alterations induced by cerebral hypoperfusion. Brain Res 1657:43512017

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Li NGu ZLi YFu XWang JBai H: A modified bilateral carotid artery stenosis procedure to develop a chronic cerebral hypoperfusion rat model with an increased survival rate. J Neurosci Methods 255:1151212015

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Long QHei YLuo QTian YYang JLi J: BMSCs transplantation improves cognitive impairment via up-regulation of hippocampal GABAergic system in a rat model of chronic cerebral hypoperfusion. Neuroscience 311:4644732015

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17

    Lu YLi CZhou MLuo PHuang PTan J: Clonidine ameliorates cognitive impairment induced by chronic cerebral hypoperfusion via up-regulation of the GABABR1 and GAD67 in hippocampal CA1 in rats. Pharmacol Biochem Behav 132:961022015

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    Luo PLu YLi CZhou MChen CLu Q: Long-lasting spatial learning and memory impairments caused by chronic cerebral hypoperfusion associate with a dynamic change of HCN1/HCN2 expression in hippocampal CA1 region. Neurobiol Learn Mem 123:72832015

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Matin NFisher CJackson WFDorrance AM: Bilateral common carotid artery stenosis in normotensive rats impairs endothelium-dependent dilation of parenchymal arterioles. Am J Physiol Heart Circ Physiol 310:H1321H13292016

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20

    Miki KIshibashi SSun LXu HOhashi WKuroiwa T: Intensity of chronic cerebral hypoperfusion determines white/gray matter injury and cognitive/motor dysfunction in mice. J Neurosci Res 87:127012812009

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    Nakaji KIhara MTakahashi CItohara SNoda MTakahashi R: Matrix metalloproteinase-2 plays a critical role in the pathogenesis of white matter lesions after chronic cerebral hypoperfusion in rodents. Stroke 37:281628232006

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22

    Neto CJPaganelli RABenetoli ALima KCMilani H: Permanent, 3-stage, 4-vessel occlusion as a model of chronic and progressive brain hypoperfusion in rats: a neurohistological and behavioral analysis. Behav Brain Res 160:3123222005

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    Nishino ATajima YTakuwa HMasamoto KTaniguchi JWakizaka H: Long-term effects of cerebral hypoperfusion on neural density and function using misery perfusion animal model. Sci Rep 6:250722016

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24

    Sarti CPantoni LBartolini LInzitari D: Cognitive impairment and chronic cerebral hypoperfusion: what can be learned from experimental models. J Neurol Sci 203–204:2632662002

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25

    Shibata MOhtani RIhara MTomimoto H: White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke 35:259826032004

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26

    Soria GTudela RMárquez-Martín ACamón LBatalle DMuñoz-Moreno E: The ins and outs of the BCCAo model for chronic hypoperfusion: a multimodal and longitudinal MRI approach. PLoS One 8:e746312013

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    Ueno YKoike MShimada YShimura HHira KTanaka R: L-carnitine enhances axonal plasticity and improves white-matter lesions after chronic hypoperfusion in rat brain. J Cereb Blood Flow Metab 35:3823912015

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28

    Venkat PChopp MChen J: Models and mechanisms of vascular dementia. Exp Neurol 272:971082015

  • 29

    Wakita HTomimoto HAkiguchi IMatsuo ALin JXIhara M: Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat. Brain Res 924:63702002

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30

    Wang JFu XJiang CYu LWang MHan W: Bone marrow mononuclear cell transplantation promotes therapeutic angiogenesis via upregulation of the VEGF-VEGFR2 signaling pathway in a rat model of vascular dementia. Behav Brain Res 265:1711802014

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31

    Wang JFu XYu LLi NWang MLiu X: Preconditioning with VEGF enhances angiogenic and neuroprotective effects of bone marrow mononuclear cell transplantation in a rat model of chronic cerebral hypoperfusion. Mol Neurobiol 53:605760682016

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32

    Wang ZFan JWang JLi YDuan DDu G: Chronic cerebral hypoperfusion induces long-lasting cognitive deficits accompanied by long-term hippocampal silent synapses increase in rats. Behav Brain Res 301:2432522016

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33

    Yata KTomimoto H: Chronic cerebral hypoperfusion and dementia. Neurol Clin Neurosci 2:1291342014

  • 34

    Yoshizaki KAdachi KKataoka SWatanabe ATabira TTakahashi K: Chronic cerebral hypoperfusion induced by right unilateral common carotid artery occlusion causes delayed white matter lesions and cognitive impairment in adult mice. Exp Neurol 210:5855912008

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 35

    Zhou ZZhang YZhu CSui JWu GMeng Z: Cognitive functions of carotid artery stenosis in the aged rat. Neuroscience 219:1371442012

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