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浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2019, Vol. 48 Issue (6): 644-650    DOI: 10.3785/j.issn.1008-9292.2019.12.09
    
Correlation of cardiovascular risk factors with brain iron deposition: A magnetic resonance imaging study
HU Linlin1,2(),ZHANG Ruiting1,WANG Shuyue1,HONG Hui1,HUANG Peiyu1,*(),ZHANG Minming1,*()
1. Department of Radiology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
2. Department of Radiology, Jinhua Central Hospital, Jinhua 321000, Zhejiang Province, China
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Abstract  

Objective: To study the correlation of common cardiovascular risk factors with brain iron deposition. Methods: Eighty-four elderly subjects without neurological diseases or brain trauma were included in the study. The cardiovascular risk factors were comprehensively assessed. MRI examination was performed to obtain high-resolution T1-weighted images and enhanced susceptibility weighted angiography (ESWAN) images, and R2* figure was obtained by post-processing the ESWAN sequence. High definition T1 images were segmented using computer segmentation technique. After registration to the ESWAN image, R2* values of each region of interest were extracted. Multiple linear regression analysis was used to analyze the relationship of R2* values in each area of interest with gender, age and vascular risk factors. Results: Smoking was associated with increased R2* values in the hippocampus, white matter and cortex (β=0.244, 0.317, 0.277, P < 0.05 or P < 0.01). Hypertension was correlated with the increase of R2* in the putamen (β=0.241, P=0.027). Hyperglycemia was associated with the increase of R2* in the thalamus (β=0.234, P < 0.05). In the thalamus, the R2* value of males was higher than that of females (β=0.320, P < 0.05). Age was correlated with the R2* values of thalamus, caudate nucleus, pallidus, white matter and cortex (β=-0.218、-0.254、0.216、-0.280 and -0.238, P < 0.05 or P < 0.01). Conclusion: Common cardiovascular risk factors may lead to iron deposition in the brain, and the deposition patterns vary with the gender, age and different risk factors.

Key words： Cardiovascular diseases/etiology      Stroke/etiology      Risk factors      Iron/metabolism      Magnetic resonance imaging     
Received: 05 July 2019      Published: 19 January 2020
CLC:  R445.2  
Corresponding Authors: HUANG Peiyu,ZHANG Minming     E-mail: hu001987@163.com;huangpy@zju.edu.cn;zhangminming@zju.edu.cn
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HU Linlin
ZHANG Ruiting
WANG Shuyue
HONG Hui
HUANG Peiyu
ZHANG Minming
Cite this article:

HU Linlin,ZHANG Ruiting,WANG Shuyue,HONG Hui,HUANG Peiyu,ZHANG Minming. Correlation of cardiovascular risk factors with brain iron deposition: A magnetic resonance imaging study. J Zhejiang Univ (Med Sci), 2019, 48(6): 644-650.

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http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2019.12.09     OR     http://www.zjujournals.com/med/Y2019/V48/I6/644


心脑血管疾病高危因素对脑铁沉积影响的磁共振影像学研究

目的: 采用MRI方法研究常见心脑血管疾病高危因素与脑铁沉积的关系。方法: 纳入84名无神经系统疾病和脑外伤的社区中老年人，全面评估其心脑血管疾病高危因素，行MRI检查获取高清T1加权图像及T2*加权三维梯度回波序列（ESWAN）图像，ESWAN图像后处理得R2*图，采用计算机分割技术对高清T1加权图像进行组织分割，配准至ESWAN图像后，提取各脑区的R2*值。采用多元线性回归分析揭示中老年人性别、年龄、心脑血管疾病高危因素对各脑区R2*值的影响。结果: 海马、脑白质、脑皮质R2*值升高与吸烟有关（β值分别为0.244、0.317、0.277，P < 0.05或P < 0.01）；壳核R2*值升高与高血压有关（β值为0.241，P < 0.05）；丘脑R2*值升高与高血糖有关（β值为0.234，P < 0.05）；男性丘脑R2*值高于女性（β值为0.320，P < 0.05）；丘脑、尾状核、苍白球、脑白质、脑皮质等R2*值与年龄相关（β值分别为-0.218、-0.254、0.216、-0.280和-0.238，P < 0.05或P < 0.01）。结论: 常见心脑血管疾病高危因素可能导致脑铁沉积，且不同因素引起的铁沉积模式不同。


关键词： 心血管疾病/病因学,  卒中/病因学,  危险因素,  铁/代谢,  磁共振成像 
组别 n 脑白质 脑皮质 丘脑 尾状核 壳核 苍白球 海马 杏仁体
与男性比较，* *P＜0.01；与有该项风险因素组比较，#P＜0.05.
性别 男性 42 20.9±1.0 20.2±1.5 20.2±1.8 19.5±2.1 25.2±3.2 37.5±6.1 18.3±6.1 18.5±9.0
女性 42 20.0±1.0** 19.7±0.9 19.0±1.6** 18.7±2.0 24.8±3.1 38.6±7.5 16.8±1.7 17.0±2.2
高血压 有 62 20.3±1.1 19.9±1.0 19.6±2.0 19.2±2.2 25.4±3.4 38.2±7.6 17.2±1.9 17.2±2.1
无 22 20.6±2.0 20.0±1.9 19.3±1.1 18.7±1.7 23.7±2.1# 38.0±4.7 18.6±8.6 19.2±12.8
高血糖 有 21 20.2±1.0 19.8±0.9 20.2±1.9 19.2±2.4 25.3±3.5 40.4±7.7 17.0±1.4 16.6±1.3
无 63 20.5±1.5 20.0±1.4 19.3±1.8 19.0±2.0 24.9±3.1 37.4±6.5 17.8±5.3 18.2±7.7
高血脂 有 32 20.4±1.0 20.0±0.9 20.1±1.9 19.0±2.1 25.0±3.5 37.9±7.4 17.4±1.4 17.2±1.8
无 52 20.4±1.6 19.9±1.5 19.2±1.7# 19.1±2.1 25.0±3.0 38.3±6.7 17.7±5.5 18.1±8.4
吸烟史 有 25 21.2±1.9 20.5±1.8 20.3±1.9 19.7±1.8 25.2±3.1 36.9±6.0 19.2±7.9 19.7±11.6
无 59 20.1±0.9# 19.7±0.9# 19.2±1.7# 18.8±2.2# 24.9±3.2 38.7±7.2 16.8±1.6# 16.9±2.0
Tab 1 Differences in R2* values of brain regions between genders and groups with/without cardiovascular risk factors  ($\bar x \pm s$, s-1)
脑区 心脑血管疾病高危因素 未标准化系数 标准化系数β P值
β 标准误
脑皮质 吸烟史 0.769 0.287 0.277 ＜0.01
年龄 -0.035 0.015 -0.238 ＜0.05
脑白质 吸烟史 0.972 0.307 0.317 ＜0.01
年龄 -0.046 0.016 -0.280 ＜0.01
丘脑 性别 1.164 0.366 0.320 ＜0.01
高血糖 0.982 0.419 0.234 ＜0.05
年龄 -0.046 0.021 -0.218 ＜0.05
尾状核 年龄 -0.062 0.026 -0.254 ＜0.05
壳核 高血压 1.724 0.766 0.241 ＜0.05
苍白球 年龄 0.174 0.087 0.216 ＜0.05
海马 吸烟史 2.460 1.081 0.244 ＜0.05
Tab 2 Factors correlated with R2* value in different brain regions
[1]   WARD R J , ZUCCA F A , DUYN J H et al. The role of iron in brain ageing and neurodegenerative disorders[J]. Lancet Neurol, 2014, 13 (10): 1045- 1060
doi: 10.1016/S1474-4422(14)70117-6
[2]   GUAN X, XUAN M, GU Q, et al. Regionally progressive accumulation of iron in Parkinson's disease as measured by quantitative susceptibility mapping[J/OL]. NMR Biomed, 2017, 30(4): e3489.
[3]   罗骁, 邱甜甜, 荚耘路 et al. 磁共振脑铁成像技术在阿尔茨海默病中的应用进展[J]. 临床放射学杂志, 2016, 35 (4): 645- 648
LUO Xiao , QIU Tiantian , JIA Yunlu et al. Advances in the application of magnetic resonance encephalon imaging in alzheimer's disease[J]. Journal of Clinical Radiology, 2016, 35 (4): 645- 648
[4]   SUN Y , GE X , HAN X et al. Characterizing brain iron deposition in patients with subcortical vascular mild cognitive impairment using quantitative susceptibility mapping:a potential biomarker[J]. Front Aging Neurosci, 2017, 9 81
[5]   PIRPAMER L , HOFER E , GESIERICH B et al. Determinants of iron accumulation in the normal aging brain[J]. Neurobiol Aging, 2016, 43 149- 155
doi: 10.1016/j.neurobiolaging.2016.04.002
[6]   VALDéS HERNáNDEZ M , ALLERHAND M , GLATZ A et al. Do white matter hyperintensities mediate the association between brain iron deposition and cognitive abilities in older people?[J]. Eur J Neurol, 2016, 23 (7): 1202- 1209
doi: 10.1111/ene.13006
[7]   DEL C , VALDéS HERNáNDEZ M , RITCHIE S , GLATZ A et al. Brain iron deposits and lifespan cognitive ability[J]. Age (Dordr), 2015, 37 (5): 100
doi: 10.1007/s11357-015-9837-2
[8]   LANGKAMMER C , KREBS N , GOESSLER W et al. Quantitative MR imaging of brain iron:a postmortem validation study[J]. Radiology, 2011, 257 (2): 455- 462
[9]   中国心血管病预防指南(2017)写作组, 中华心血管病杂志编辑委员会 . 中国心血管病预防指南(2017)[J]. 中华心血管病杂志, 2018, 46 (1): 10- 25
China cardiovascular disease prevention guidelines (2017) writing group , Editorial board of Chinese Journal of Cardiovascular Disease . China cardiovascular disease prevention guidelines (2017)[J]. Chinese Journal of Cardiovascular Disease, 2018, 46 (1): 10- 25
doi: 10.3760/cma.j.issn.0253-3758.2018.01.004
[10]   HALLGREN B , SOURANDER P . The effect of age on the non-haemin iron in the human brain[J]. J Neurochem, 1958, 3 (1): 41- 51
doi: 10.1111/j.1471-4159.1958.tb12607.x
[11]   ZECCA L , YOUDIM M B , RIEDERER P et al. Iron, brain ageing and neurodegenerative disorders[J]. Nat Rev Neurosci, 2004, 5 (11): 863- 873
doi: 10.1038/nrn1537
[12]   CONDE J R , STREIT W J . Microglia in the aging brain[J]. J Neuropathol Exp Neurol, 2006, 65 (3): 199- 203
doi: 10.1097/01.jnen.0000202887.22082.63
[13]   FARRALL A J , WARDLAW J M . Blood-brain barrier:ageing and microvascular disease-systematic review and meta-analysis[J]. Neurobiol Aging, 2009, 30 (3): 337- 352
doi: 10.1016/j.neurobiolaging.2007.07.015
[14]   王波, 龚霞蓉, 张洁 et al. R2*值评价健康成年人脑铁含量与年龄的相关性研究[J]. 中国现代医学杂志, 2016, 26 (1): 82- 88
WANG Bo , GONG Xiarong , ZHANG Jie et al. R2* value to evaluate the correlation between iron content and age in healthy adults[J]. China Journal of Modern Medicine, 2016, 26 (1): 82- 88
doi: 10.3969/j.issn.1005-8982.2016.01.015
[15]   JIN L , WANG J , ZHAO L et al. Decreased serum ceruloplasmin levels characteristically aggravate nigral iron deposition in Parkinson's disease[J]. Brain, 2011, 134 (Pt 1): 50- 58
[16]   XU X , WANG Q , ZHANG M . Age, gender, and hemispheric differences in iron deposition in the human brain:an in vivo MRI study[J]. Neuroimage, 2008, 40 (1): 35- 42
doi: 10.1016/j.neuroimage.2007.11.017
[17]   AQUINO D , BIZZI A , GRISOLI M et al. Age-related iron deposition in the basal ganglia:quantitative analysis in healthy subjects[J]. Radiology, 2009, 252 (1): 165- 172
doi: 10.1148/radiol.2522081399
[18]   GONG N J , WONGA C S , HUIA E S et al. Hemisphere, gender and age-related effects on iron deposition in deep gray matter revealed by quantitative susceptibility mapping[J]. NMR Biomed, 2015, 28 1267- 1274
doi: 10.1002/nbm.3366
[19]   BARTZOKIS G , TISHLER T A , LU P H et al. Brain ferritin iron may influence age-and gender-related risks of neurodegeneration[J]. Neurobiol Aging, 2007, 28 (3): 414- 423
doi: 10.1016/j.neurobiolaging.2006.02.005
[20]   FREY B N , DIAS R S . Sex hormones and biomarkers of neuroprotection and neurodegeneration:implications for female reproductive events in bipolar disorder[J]. Bipolar Disord, 2014, 16 (1): 48- 57
doi: 10.1111/bdi.12151
[21]   ABBRUSCATO T J , LOPEZ S P , MARK K S et al. Nicotine and cotinine modulate cerebral microvascular permeability and protein expression of ZO-1 through nicotinic acetylcholine receptors expressed on brain endothelial cells[J]. J Pharm Sci, 2002, 91 (12): 2525- 2538
doi: 10.1002/jps.10256
[22]   HOSSAIN M , SATHE T , FAZIO V et al. Tobacco smoke:a critical etiological factor for vascular impairment at the blood-brain barrier[J]. Brain Res, 2009, 1287 192- 205
doi: 10.1016/j.brainres.2009.06.033
[23]   ABBRUSCATO T J , LOPEZ S P , RODER K et al. Regulation of blood-brain barrier Na, K, 2Cl-cotransporter through phosphorylation during in vitro stroke conditions and nicotine exposure[J]. J Pharmacol Exp Ther, 2004, 310 (2): 459- 468
doi: 10.1124/jpet.104.066274
[24]   RAIJ L , DEMASTER E G , JAIMES E A . Cigarette smoke-induced endothelium dysfunction:role of superoxide anion[J]. J Hypertens, 2001, 19 (5): 891- 897
doi: 10.1097/00004872-200105000-00009
[25]   TSUCHIYA M , ASADA A , KASAHARA E et al. Smoking a single cigarette rapidly reduces combined concentrations of nitrate and nitrite and concentrations of antioxidants in plasma[J]. Circulation, 2002, 105 (10): 1155- 1157
doi: 10.1161/hc1002.105935
[26]   TUON T , VALVASSORI S S , LOPES-BORGES J et al. Effects of moderate exercise on cigarette smoke exposure-induced hippocampal oxidative stress values and neurological behaviors in mice[J]. Neurosci Lett, 2010, 475 (1): 16- 19
doi: 10.1016/j.neulet.2010.03.030
[27]   LEE H M , REED J , GREELEY G H J R et al. Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke[J]. Toxicol Appl Pharmacol, 2009, 235 (2): 208- 215
doi: 10.1016/j.taap.2008.12.010
[28]   YU R , DEOCHAND C , KROTOW A et al. Tobacco smoke-induced brain white matter myelin dysfunction:potential co-factor role of smoking in neurodegeneration[J]. J Alzheimers Dis, 2016, 50 (1): 133- 148
[29]   ALKONDON M , ALBUQUERQUE E X . The nicotinic acetylcholine receptor subtypes and their function in the hippocampus and cerebral cortex[J]. Prog Brain Res, 2004, 145 109- 120
doi: 10.1016/S0079-6123(03)45007-3
[30]   BURGMANS S , VAN BOXTEL M P , GRONENSCHILD E H et al. Multiple indicators of age-related differences in cerebral white matter and the modifying effects of hypertension[J]. Neuroimage, 2010, 49 (3): 2083- 2093
doi: 10.1016/j.neuroimage.2009.10.035
[31]   管勇, 吕少萍, 孙淼 et al. 壳核动脉的显微解剖研究及其临床意义[J]. 中国临床神经外科杂志, 2006, 11 (4): 213- 215
GUAN Yong , LV Shaoping , SUN Miao et al. Microanatomy of the putamen artery and its clinical significance[J]. Chinese Journal of Clinical Neurosurgery, 2006, 11 (4): 213- 215
doi: 10.3969/j.issn.1009-153X.2006.04.008
[32]   GREENBERG S M , VERNOOIJ M W , CORDONNIER C et al. Cerebral microbleeds:a guide to detection and interpretation[J]. Lancet Neurol, 2009, 8 (2): 165- 174
doi: 10.1016/S1474-4422(09)70013-4
[33]   SEALS D R , MOREAU K L , GATES P E et al. Modulatory influences on ageing of the vasculature in healthy humans[J]. Exp Gerontol, 2006, 41 (5): 501- 507
doi: 10.1016/j.exger.2006.01.001
[34]   AY H , KOROSHETZ W J , VANGEL M et al. Conversion of ischemic brain tissue into infarction increases with age[J]. Stroke, 2005, 36 (12): 2632- 2636
doi: 10.1161/01.STR.0000189991.23918.01
[35]   URICH H. Malformations of the nervous system, perinatal damage and related conditions in early life[M]//BLACKWOOD W, CORSELLIS J A N. Greenfield's neuropathology. London: Edward Arnold Publishers, 1973: 361-469.
[36]   CORDONNIER C , AL-SHAHI SALMAN R , WARDLAW J . Spontaneous brain microbleeds:systematic review, subgroup analyses and standards for study design and reporting[J]. Brain, 2007, 130 (Pt 8): 1988- 2003
[37]   LIU Y , LIU J , LIU H et al. Investigation of cerebral iron deposition in aged patients with ischemic cerebrovascular disease using susceptibility-weighted imaging[J]. Ther Clin Risk Manag, 2016, 12 1239- 1247
doi: 10.2147/TCRM.S107783
[38]   SONG E C , CHU K , JEONG S W et al. Hyperglycemia exacerbates brain edema and perihematomal cell death after intracerebral hemorrhage[J]. Stroke, 2003, 34 (9): 2215- 2220
doi: 10.1161/01.STR.0000088060.83709.2C
[39]   OUBIDAR M , BOQUILLON M , MARIE C et al. Ischemia-induced brain iron delocalization:effect of iron chelators[J]. Free Radic Biol Med, 1994, 16 (6): 861- 867
doi: 10.1016/0891-5849(94)90205-4
[40]   HUNT J V , DEAN R T , WOLFF S P . Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing[J]. Biochem J, 1988, 256 (1): 205- 212
doi: 10.1042/bj2560205
[41]   WOLFF S P , BASCAL Z A , HUNT J V . "Autoxidative glycosylation":free radicals and glycation theory[J]. Prog Clin Biol Res, 1989, 304 259- 275
[42]   WOLFF S P , JIANG Z Y , HUNT J V . Protein glycation and oxidative stress in diabetes mellitus and ageing[J]. Free Radic Biol Med, 1991, 10 (5): 339- 352
doi: 10.1016/0891-5849(91)90040-A
[43]   LI P A , LIU G J , HE Q P et al. Production of hydroxyl free radical by brain tissues in hyperglycemic rats subjected to transient forebrain ischemia[J]. Free Radic Biol Med, 1999, 27 (9-10): 1033- 1040
doi: 10.1016/S0891-5849(99)00152-5
[44]   FENG X , DEISTUNG A , REICHENBACH J R . Quantitative susceptibility mapping (QSM) and R2* in the human brain at 3T:Evaluation of intra-scanner repeatability[J]. Z Med Phys, 2018, 28 (1): 36- 48
[45]   HAMETNER S , ENDMAYR V , DEISTUNG A et al. The influence of brain iron and myelin on magnetic susceptibility and effective transverse relaxation-A biochemical and histological validation study[J]. Neuroimage, 2018, 179 117- 133
doi: 10.1016/j.neuroimage.2018.06.007
[46]   WANG R , XIE G , ZHAI M et al. Stability of R2* and quantitative susceptibility mapping of the brain tissue in a large scale multi-center study[J]. Sci Rep, 2017, 7 45261
doi: 10.1038/srep45261
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