Name: ultrasound assessment of flow-mediated dilation of the brachial and superficial femoral arteries in rats
Uploaded: 2016-11-03T13:00:00
Description: Watch this Scientific Journal Video about Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats at JoVE.com

All animal imaging was performed at the Small Animal Imaging Core Facility, University of Utah.
This study was funded in part by grants from the National Institutes of Health (R21 AG043952, R01 AG040297, K01 AG046326, K02 AG045339, and R01 DK100505).

所有动物的程序符合指南实验动物14的管理和使用以及犹他州大学和盐湖城的退伍军人医疗中心动物护理和使用获得批准。 1.动物的制备<ol><li>将动物含100％氧气3％异氟醚麻醉诱导室。留在感应室中的动物，直到它响应外部刺激。 </li><li>从感应腔室取出动物，并将其放置在配备有心电图（ECG）电极加热的检查表。维持在100％的氧气3％异氟醚麻醉。肱动脉和股浅动脉...

<ol>
	<li>Smits, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+release+of+nitric+oxide+contributes+to+the+vasodilator+effect+of+adenosine+in+humans.">Endothelial release of nitric oxide contributes to the vasodilator effect of adenosine in humans.</a> Circulation. 92, 2135-2141 (1995).</li><li>Forstermann, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+isozymes.+Characterization,+purification,+molecular+cloning,+and+functions.">Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.</a> Hypertension. 23, 1121-1131 (1994).</li><li>Gardiner, S. M., Compton, A. M., Bennett, T., Palmer, R. M., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+regional+blood+flow+by+endothelium-derived+nitric+oxide.">Control of regional blood flow by endothelium-derived nitric oxide.</a> Hypertension. 15, 486-492 (1990).</li><li>Harris, R. A., Nishiyama, S. K., Wray, D. W., Richardson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+assessment+of+flow-mediated+dilation.">Ultrasound assessment of flow-mediated dilation.</a> Hypertension. 55, 1075-1085 (2010).</li><li>Corretti, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+ultrasound+assessment+of+endothelial-dependent+flow-mediated+vasodilation+of+the+brachial+artery:+a+report+of+the+International+Brachial+Artery+Reactivity+Task+Force.">Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force.</a> J Am Coll Cardiol. 39, 257-265 (2002).</li><li>Celermajer, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+endothelial+dysfunction+in+children+and+adults+at+risk+of+atherosclerosis.">Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis.</a> Lancet. 340, 1111-1115 (1992).</li><li>Niebauer, J., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+effects+of+exercise:+role+of+endothelial+shear+stress.">Cardiovascular effects of exercise: role of endothelial shear stress.</a> J Am Coll Cardiol. 28, 1652-1660 (1996).</li><li>Sessa, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eNOS+at+a+glance.">eNOS at a glance.</a> J Cell Sci. 117, 2427-2429 (2004).</li><li>Wray, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+brachial+artery+flow-mediated+vasodilation+provide+a+bioassay+for+NO?.">Does brachial artery flow-mediated vasodilation provide a bioassay for NO?.</a> Hypertension. 62, 345-351 (2013).</li><li>Green, D. J., Dawson, E. A., Groenewoud, H. M., Jones, H., Thijssen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+flow-mediated+dilation+nitric+oxide+mediated?+A+meta-analysis.">Is flow-mediated dilation nitric oxide mediated? A meta-analysis.</a> Hypertension. 63, 376-382 (2014).</li><li>Green, D. J., Jones, H., Thijssen, D., Cable, N. T., Atkinson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-mediated+dilation+and+cardiovascular+event+prediction:+does+nitric+oxide+matter?.">Flow-mediated dilation and cardiovascular event prediction: does nitric oxide matter?.</a> Hypertension. 57, 363-369 (2011).</li><li>Brevetti, G., Silvestro, A., Schiano, V., Chiariello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction+and+cardiovascular+risk+prediction+in+peripheral+arterial+disease:+additive+value+of+flow-mediated+dilation+to+ankle-brachial+pressure+index.">Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease: additive value of flow-mediated dilation to ankle-brachial pressure index.</a> Circulation. , 2093-2098 (2003).</li><li>Gokce, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+value+of+noninvasively+determined+endothelial+dysfunction+for+long-term+cardiovascular+events+in+patients+with+peripheral+vascular+disease.">Predictive value of noninvasively determined endothelial dysfunction for long-term cardiovascular events in patients with peripheral vascular disease.</a> J Am Coll Cardiol. 41, 1769-1775 (2003).</li><li>National Research Council (U.S.). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. , (2011).</li><li>Alley, H., Owens, C. D., Gasper, W. J., Grenon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+assessment+of+endothelial-dependent+flow-mediated+vasodilation+of+the+brachial+artery+in+clinical+research.">Ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery in clinical research.</a> J Vis Exp. , e52070 (2014).</li><li>Ghiadoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+flow-mediated+dilation+reproducibility:+a+nationwide+multicenter+study.">Assessment of flow-mediated dilation reproducibility: a nationwide multicenter study.</a> J Hypertension. 30, 1399-1405 (2012).</li><li>Thijssen, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+conduit+artery+function+in+humans:+impact+of+arterial+size.">Heterogeneity in conduit artery function in humans: impact of arterial size.</a> Am J Physiol Heart Circ. 295, H1927-H1934 (2008).</li><li>Green, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+isn't+flow-mediated+dilation+enhanced+in+athletes?.">Why isn't flow-mediated dilation enhanced in athletes?.</a> Med Sci Sports. 45, 75-82 (2013).</li><li>Heiss, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+flow-mediated+vasodilation+in+living+rats+using+high-resolution+ultrasound.">In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound.</a> Am J Physiol Heart Circ. 294, H1086-H1093 (2008).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+S-nitrosoglutathione+reductase+improves+endothelial+vasodilatory+function+in+rats+in+vivo.">Pharmacological inhibition of S-nitrosoglutathione reductase improves endothelial vasodilatory function in rats in vivo.</a> J Appl Physiol. 114, 752-760 (2013).</li><li>Pinnamaneni, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+exposure+to+secondhand+smoke+reversibly+impairs+endothelial+vasodilatory+function.">Brief exposure to secondhand smoke reversibly impairs endothelial vasodilatory function.</a> Nicotine Tob Res. 16, 584-590 (2014).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+Endothelial+Function+by+Little+Cigar+Secondhand+Smoke.">Impairment of Endothelial Function by Little Cigar Secondhand Smoke.</a> Tob Regul Sci. 2, 56-63 (2016).</li><li>Schuler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+endothelium-dependent+vasodilation+in+mice--brief+report.">Measurement of endothelium-dependent vasodilation in mice--brief report.</a> Arterioscler Thromb Vasc Biol. 34, 2651-2657 (2014).</li><li>Erkens, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+diastolic+dysfunction+in+Nrf2+knock+out+mice+is+associated+with+cardiac+hypertrophy,+decreased+expression+of+SERCA2a,+and+preserved+endothelial+function.">Left ventricular diastolic dysfunction in Nrf2 knock out mice is associated with cardiac hypertrophy, decreased expression of SERCA2a, and preserved endothelial function.</a> Free Radic Biol Med. 89, 906-917 (2015).</li><li>Harris, S. A., Billmeyer, E. R., Robinson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+repeated+measurements+of+radon-222+concentrations+in+well+water+sampled+from+bedrock+aquifers+of+the+Piedmont+near+Richmond,+Virginia,+USA:+:+effects+of+lithology+and+well+characteristics.">Evaluation of repeated measurements of radon-222 concentrations in well water sampled from bedrock aquifers of the Piedmont near Richmond, Virginia, USA: : effects of lithology and well characteristics.</a> Environmental research. 101, 323-333 (2006).</li></ol>

在本研究中，FMD的一种非侵入性测量表现在肱动脉和大鼠股浅动脉。类似于人类6，以下5分钟闭塞期内，在血流速度（即，反应性充血），从而在动脉壁上导致动脉的后续血管舒张增加的剪切速率迅速增加。口蹄疫是在肱动脉和股浅动脉两个观察。此外，还有在FMD动脉之间有很强的关系。虽然最高剪切速率在股浅动脉较高，口蹄疫归到最高剪切速率保持动脉之间牢固的关系。总之，这些结...

血管内皮是蜂窝单层该线条动脉的内腔并且是血管功能的重要调节剂。有从内皮释放众多分子导致血管直径的调制。在这些分子，一氧化氮（NO），似乎是在响应于刺激从血管内皮释放的主血管舒张分子（例如，胰岛素，乙酰胆碱，或剪切应力的变化）1。在血管内皮细胞，编号是由酶产生内皮NO合酶（eNOS）和随后从血管内皮细胞2释放。否扩散到血管平滑肌，其中它引起松弛和增加的血管直径3。 内皮功能障碍可以在利用血流介导的舒张（FMD）技术4,5-人类非侵入性进行评估。口蹄疫已经提出来表示一个功能生物测定内皮衍生在人类中NO的生物利用度，且通常评估在下列一〜5分钟肢闭塞6响应于反应性充血肱或股浅动脉。反应性充血增加了被转导至内皮细胞的7层的剪切力，信令NO 8的释放。虽然近年来，血管舒张由NO释放发起的比重一直争论不休9,10，口蹄疫表明内皮依赖性扩张，并一直被证明预测心血管事件11-13。 迄今为止，成千上万的研究已经使用的口蹄疫技术用于人类血管内皮功能的非侵入性测量。考虑到在重点转化研究近期的转变，对口蹄疫的啮齿类动物的非侵入性测量准则将是非常有价值的。与平移方法保持一致，该协议是建立在肱动脉和苏佩FMD的测量大鼠rficial股动脉，由于这些网站都是人类最常用的测量。在大鼠一个强大的和可重复的口蹄疫响应这个协议的结果，然而，在大鼠口蹄疫测量技术要求高，可能是困难的其他研究者没有视频演示复制。因此，下面的文章将演示口蹄疫肱和大鼠股浅动脉血管无创测量的方法。

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="58">
			<td height="58" style="height:58px;width:203px;">Vevo 2100 High Resolution Micro-Ultrasound Imaging System</td>
			<td style="width:169px;">VisualSonics, Toronto, ON, CAN</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:203px;">MicroScan Ultra-High Frequency Linear Array Transducer - MS-700 30-70 MHz</td>
			<td style="width:169px;">VisualSonics, Toronto, ON, CAN</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Vevo Imaging Station</td>
			<td style="width:169px;">VisualSonics, Toronto, ON, CAN</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Thermasonic gel warmer</td>
			<td style="width:169px;">Parker Laboratories, Fairfield, NJ, USA</td>
			<td style="width:169px;">82-03</td>
			<td style="width:169px;">Optional</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Signacreme electrode cream</td>
			<td style="width:169px;">Parker Laboratories, Fairfield, NJ, USA</td>
			<td style="width:169px;">17-05</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Transpore surgical tape</td>
			<td style="width:169px;">3M, Maplewood, MN, USA</td>
			<td style="width:169px;">1527-1</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Depilatory cream (e.g., Nair)</td>
			<td style="width:169px;">General supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Cotton swabs</td>
			<td style="width:169px;">General supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Ultrasound gel</td>
			<td style="width:169px;">General supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Standard vascular occluder, 10 mm lumen diameter</td>
			<td style="width:169px;">Harvard Apparatus, Holliston, MA, USA</td>
			<td style="width:169px;">62-0115</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">10 ml syringe with Luer-Lok tip</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Paperclip</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Hypodermic needle &#8211; 18 gauge&#160;</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Medium binder clip</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
	</tbody>
</table>

ultrasound assessment of flow-mediated dilation of the brachial and superficial femoral arteries in rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

于8 Wistar大鼠的肱动脉和股浅动脉进行血流介导的扩张。的大鼠的定位，如图1所示。 股浅动脉的代表超声图像示于图2。 <img alt="图1" src="/files/ftp_upload/54762/54762fig1.jpg" /> 图1. 鼠和超声定位。 大鼠的定位臂丛（A）的测量和?...

Watch this Scientific Journal Video about Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats at JoVE.com

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

在人类血管内皮功能的非侵入性的评估可以通过血流介导的扩张的技术来确定。虽然千研究中使用了该技术，没有研究在大鼠进行这种技术非侵入。下面的文章介绍肱血流介导的扩张和大鼠股浅动脉无创测量。

肱血流介导的扩张和大鼠股浅动脉超声评估

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

Research

JoVE Journal

Medicine

A Novel Application of Musculoskeletal Ultrasound Imaging

肌肉骨骼超声成像的一种新应用

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

科研

医学

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

小鼠膈肌功能诊断超声成像

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

肱动脉的临床研究超声评价血管内皮依赖性血流介导的血管舒张功能

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

血管内皮功能的超声评估：血流介导的舒张试验的技术指导原则

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

聚丙烯酸/纳米水的气管内灌注积液模型

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Brachial Artery Catheterization in Swine

猪的臂动脉插管

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

成人大鼠心脏解剖与功能的超声心动图评估

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...