Impaired functional recovery of endothelial colony-forming cells from moyamoya disease in a chronic cerebral hypoperfusion rat model

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OBJECTIVE

Endothelial colony-forming cells (ECFCs) isolated from pediatric patients with moyamoya disease (MMD) have demonstrated decreased numbers and defective functioning in in vitro experiments. However, the function of ECFCs has not been evaluated using in vivo animal models. In this study, the authors compared normal and MMD ECFCs using a chronic cerebral hypoperfusion (CCH) rat model.

METHODS

A CCH rat model was made via ligation of the bilateral common carotid arteries (2-vessel occlusion [2-VO]). The rats were divided into three experimental groups: vehicle-treated (n = 8), normal ECFC-treated (n = 8), and MMD ECFC-treated (n = 8). ECFCs were injected into the cisterna magna. A laser Doppler flowmeter was used to evaluate cerebral blood flow, and a radial arm maze test was used to examine cognitive function. Neuropathological examinations of the hippocampus and agranular cortex were performed using hematoxylin and eosin and Luxol fast blue staining in addition to immunofluorescence with CD31, von Willebrand factor, NeuN, myelin basic protein, glial fibrillary acidic protein, and cleaved caspase-3 antibodies.

RESULTS

The normal ECFC-treated group exhibited improvement in the restoration of cerebral perfusion and in behavior compared with the vehicle-treated and MMD ECFC-treated groups at the 12-week follow-up after the 2-VO surgery. The normal ECFC-treated group showed a greater amount of neovasculogenesis and neurogenesis, with less apoptosis, than the other groups.

CONCLUSIONS

These results support the impaired functional recovery of MMD ECFCs compared with normal ECFCs in a CCH rat model. This in vivo study suggests the functional role of ECFCs in the pathogenesis of MMD.

ABBREVIATIONS 2-VO = 2-vessel occlusion; CBF = cerebral blood flow; CCH = chronic cerebral hypoperfusion; ECFC = endothelial colony-forming cell; GFAP = glial fibrillary acidic protein; LDF = laser Doppler flowmeter; LDL = low-density lipoprotein; LFB = Luxol fast blue; MBP = myelin basic protein; MCA = middle cerebral artery; MCAO = MCA occlusion; MMD = moyamoya disease; RAM = radial arm maze; SMPC = smooth muscle progenitor cell; vWf = von Willebrand factor.

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Article Information

Correspondence Seung-Ki Kim: Seoul National University Children’s Hospital, Seoul, Republic of Korea. nsthomas@snu.ac.kr.

INCLUDE WHEN CITING Published online November 9, 2018; DOI: 10.3171/2018.8.PEDS1883.

S.A.C. and S.C. contributed equally to this work.

Disclosures 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.

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Figures

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    Schematic diagram of the experimental design. Before the 2-VO surgery the rats underwent 6 sets of practice over 2 weeks to habituate to the maze test. After the 2-VO surgery CBF was estimated by LDF at 0, 8, and 12 weeks. Cognitive function was evaluated through a RAM test 1, 2, 4, 8, 10, and 12 weeks after the 2-VO surgery. Normal and MMD ECFCs were injected into the cisterna magna the 1st week after the 2-VO surgery.

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    Characterization of ECFCs from normal volunteers and MMD patients. A: Peripheral blood mononuclear cells from both normal and MMD ECFCs differentiated into a cobblestone-like morphology. Bar = 500 μm. B and D: The uptake of acetylated LDL of both normal and MMD ECFCs. Bar = 100 μm. C and E: Capillary network formation on Matrigel showing that normal ECFCs have better capillary formation abilities than the MMD ECFCs. ***p <0.001. Bar = 500 μm. F: Representative flow cytometry analysis of ECFCs revealing high expression of the surface markers KDR, CD34, and CD31, but low expression of CD45 and CD14.

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    Distribution of normal ECFCs in the hippocampal CA1 area and agranular cortex. Representative fluorescence images showing that CELL-STALKER-CSR-labeled normal ECFCs (red) were observed 4 weeks after injection of the cells. Nuclei were counterstained with DAPI (blue). Bars = 100 μm.

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    Measurement of CBF by LDF at the indicated time intervals. CBF was rapidly reduced in all rats to 54.5% ± 22.9% of the baseline level immediately after the 2-VO surgery and gradually recovered over 8 weeks in all rats. CBF was significantly higher in the normal ECFC-treated group than in the vehicle-treated group (61.8% ± 25.6%) and MMD ECFC-treated group (55.5% ± 17.3%) at 12 weeks. Values are presented as mean ± SD. ***p <0.001.

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    Effect of ECFCs on cognitive function. A: The running time after 2-VO surgery was found to be significantly lower in the normal ECFC-treated group than in the vehicle-treated group and MMD ECFC-treated group. ***p <0.001. B: The total number of errors was also significantly lower in the normal ECFC-treated group than in the vehicle-treated group and MMD ECFC-treated group. Values are presented as mean ± SD. ***p <0.001.

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    Effects of ECFCs on neurons and myelin in the hippocampal CA1 area and agranular cortex. H & E (A) and LFB (B) staining were performed on sections from the hippocampal CA1 region and the agranular cortex after injection of ECFCs. Compared with the normal ECFC-treated group, H & E staining showed a loss of hippocampal CA1 pyramidal neurons in the MMD ECFC-treated group. LFB staining revealed reduced demyelination in the normal ECFC-treated group. Blue areas indicate myelination, whereas pale areas indicate demyelination. Bar = 100 μm.

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    Effects of ECFCs on neovasculogenesis, neurogenesis, and apoptosis in the hippocampal CA1 region. A: Representative immunofluorescence images show the expression of CD31, vWF, NeuN, MBP, GFAP, and cleaved caspase-3. The normal ECFC-treated group was compared with the vehicle-treated group and MMD ECFC-treated group. B: Quantification of the number of positive cells is shown in the graphs. The number of CD31- (red), vWF- (red), NeuN- (green), and MBP-positive (red) cells was significantly higher in the normal ECFC-treated group. The GFAP expression (red) shows that there were no statistically significant differences among the groups. A decreased number of cleaved caspase-3–positive (red) cells was observed in the normal ECFC-treated group. Bar = 50 μm. Nuclei were counterstained with DAPI (blue). *p <0.05, **p <0.01, ***p <0.001.

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