This work was supported by Generalitat Valenciana (GV2015-062), Universitat de Val&#232;ncia (UV-INV-PRECOMP14-206372) to MZ, Generalitat Valenciana (PROMETEOII/2014/037) and Instituto de Salud Carlos III-FEDER funds (CIBERCV CB16/11/0486) to FJC.

Animal care and the experimental protocols used in this study complied with EU directive 2010/63 on the protection of animals used for scientific purposes, and were approved by the Institutional Animal Care and Use Committee (2015/VSC/PEA/00049).
NOTE: The protocol consists of the chronic administration of a high-fat, high-sucrose diet for 28 weeks, and the assessment of the main components of MetS. We used 11 adult male New Zealand White (NZW) rabbits weighing 4.39 &#177; 0.14 (s.d.) kg, whic...

<ol>
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A., 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=Harmonizing+the+metabolic+syndrome:+a+joint+interim+statement+of+the+International+Diabetes+Federation+Task+Force+on+Epidemiology+and+Prevention;+National+Heart,+Lung,+and+Blood+Institute;+American+Heart+Association;+World+Heart+Federation;+International+Atherosclerosis+Society;+and+International+Association+for+the+Study+of+Obesity.">Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity.</a> Circulation. 120 (16), 1640-1645 (2009).</li><li>Grundy, 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=Pre-diabetes,+metabolic+syndrome,+and+cardiovascular+risk.">Pre-diabetes, metabolic syndrome, and cardiovascular risk.</a> JACC. 59 (7), 635-643 (2012).</li><li>Verkest, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+metabolic+syndrome+a+useful+clinical+concept+in+dogs?+A+review+of+the+evidence.">Is the metabolic syndrome a useful clinical concept in dogs? A review of the evidence.</a> Vet J. 199 (1), 24-30 (2014).</li><li>Zhang, X., Lerman, L. 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B., Koike, T., Yu, Y., Inoue, T., Aoki, T., Liu, E., 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=High-fat+diet+without+excess+calories+induces+metabolic+disorders+and+enhances+atherosclerosis+in+rabbits.">High-fat diet without excess calories induces metabolic disorders and enhances atherosclerosis in rabbits.</a> Atherosclerosis. 213 (1), 148-155 (2010).</li><li>Fan, J., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol-fed+and+transgenic+rabbit+models+for+the+study+of+atherosclerosis.">Cholesterol-fed and transgenic rabbit models for the study of atherosclerosis.</a> J Atheroscler Thromb. 7 (1), 26-32 (2000).</li><li>Yin, W., Yuan, Z., Wang, Z., Yang, B., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+diet+high+in+saturated+fat+and+sucrose+alters+glucoregulation+and+induces+aortic+fatty+streaks+in+New+Zealand+White+rabbits.">A diet high in saturated fat and sucrose alters glucoregulation and induces aortic fatty streaks in New Zealand White rabbits.</a> Int J Exp Diabetes Res. 3 (3), 179-184 (2002).</li><li>Zhao, S., Chu, Y., Zhang, C., Lin, Y., Xu, K., Yang, 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=Diet-induced+central+obesity+and+insulin+resistance+in+rabbits.">Diet-induced central obesity and insulin resistance in rabbits.</a> J Anim Physiol Anim Nutr (Berl). 92 (1), 105-111 (2008).</li><li>Helfenstein, T., Fonseca, F. 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J., Marrachelli, V. G., Ruiz-Saur&#237;, A., Alberola, A., Morales, J. M., Such-Miquel, L., Monleon, D., Chorro, F. J., Such, L., Zarzoso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+experimental+model+of+diet-induced+metabolic+syndrome+in+rabbit.">Development and characterization of an experimental model of diet-induced metabolic syndrome in rabbit.</a> PLoS One. 12 (5), e0178315 (2017).</li><li>Nelson, R. W., Himsel, C. A., Feldman, E. C., Bottoms, G. 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The authors declare that they have no competing financial interests.

The establishment of an appropriate experimental model can provide a more consistent and reliable method to study the development of MetS, and it is also necessary to understand the basic mechanisms that underlie the organs and systems remodeling. Here, we describe the methods used to develop a relevant experimental model of diet-induced MetS and how to assess the main components of this cluster of metabolic and cardiovascular abnormalities that characterize this model: central obesity, hypertension, glucose intolerance,...

Obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits1. There are several definitions of MetS, but most of them describe it as a cluster of cardiovascular and metabolic alterations such as abdominal obesity, reduced HDL and elevated LDL cholesterol, elevated triglycerides, glucose intolerance, and hypertension2,3,4. Diagnosis requires that three out of these five criteria are present.
Owing to animal models, basic research has been able to investigate the mechanisms underlying pathological processes such as MetS. Several animal models have been used, but it is of crucial importance that the model of choice reproduces the main clinical manifestations of the human pathology (Figure 1). With this aim, animal models considered similar to humans, mainly canine and swine, have been developed (see Verkest5 and Zhang &#38; Lerman6 for review). However, canine models do not show all the components of MetS, given that the development of atherosclerosis or hyperglycemia in dogs by means of the diet is questionable5. Swine models present the most anatomical and physiological similarity with humans, and thus offer significant predictive power for elucidating the mechanisms underlying MetS, but their maintenance and the complexity of the experimental procedures make the use of this model very labor intensive and costly6.
On the other hand, rodent models (mouse and rat), diet-induced spontaneous and transgenic, have been extensively used in the literature for the study of obesity, hypertension, and MetS, and its pathological consequences in different organs and systems (see Wong et al.7 for review). Although the use of these models is more affordable than canine or swine, they have important drawbacks. Indeed, depending on the strain, animals develop some components of MetS, whereas others such as hypertension, hyperglycemia, and hyperinsulinemia are absent7. Furthermore, one of the main components of MetS, obesity, in some genetically modified strains, does not only depend on factors associated with the diet, rather it has been shown that some animals become obese with normal or even reduced food intake8. Finally, mice and rats show a natural deficiency in cholesteryl ester transfer protein (CETP) and use HDL as the major means of cholesterol transport, which makes them relatively resistant to the development of atherosclerosis. This is an important difference in lipid metabolism with humans, who express CETP and transport their cholesterol mainly in LDL9.
Conversely, the laboratory rabbit represents an intermediate stage between the larger animal and rodent experimental models. Thus, the rabbit can be easily submitted to different types of protocols with minimal requirements of personnel and maintenance, being more easily handled in experimental procedures than larger animal models. Furthermore, it has been reported that rabbits fed with a high-fat diet have similar hemodynamic and neurohumoral changes as obese humans8,10,11. Of note, regarding lipid metabolism, the rabbit has abundant CETP in plasma and their lipoprotein profile is LDL-rich12, which is also similar to humans. Additionally, rabbits develop hyperlipidemia quite rapidly given that, as herbivores, they are very sensitive to dietary fat13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57117/57117fig1.jpg" /> 
Figure 1: Comparison of MetS animal models. See Verkest5, Zhang and Lerman6, and Wong et al.7 for review. &#34;<img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" />&#34; indicates an advantage and &#34;<img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" />&#34; indicates a disadvantage. *controversial, depends on the study, **as pointed out by Carroll et al.8, some genetically modified strains become obese independently of food intake. CEPT: cholesteryl ester transfer protein. GTT: glucose tolerance test. <a href="//cloudflare.jove.com/files/ftp_upload/57117/57117fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In order to elucidate the basic mechanisms underlying the pathological remodeling produced by MetS in the different organs and systems, and to gain understanding of this complex pathology, the choice of an experimental model that reproduces the main components of human MetS is essential. The rabbit can provide many advantages given its similarity with human physiology and the affordability of use in chronic protocols and measurements. In this line, few diet-induced rabbit models using high-fat and high-sucrose diet have been used14,15,16,17,18,19 (Table 1), and a characterization of the different components of MetS is of great importance when relating a phenotype with organ remodeling. Thus, this article&#39;s main objective is to describe the methods to develop a model of diet-induced MetS in rabbits that allows the study of its pathophysiology and impact in organ remodeling.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Study</td>
			<td rowspan="2">Diet</td>
			<td rowspan="2">Duration</td>
			<td rowspan="2">Breed</td>
			<td colspan="4">MetS components</td>
		</tr>
		<tr>
			<td>Ob</td>
			<td>HT</td>
			<td>HG</td>
			<td>Dl</td>
		</tr>
		<tr>
			<td rowspan="2">Yin et al. (2002)14</td>
			<td>&#183;&#160;&#160;&#160; 10% fat</td>
			<td rowspan="2">24 weeks</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; Male NZW</td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="2">-</td>
			<td rowspan="2"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td rowspan="2"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160; 37% sucrose</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; 2 kg</td>
		</tr>
		<tr>
			<td rowspan="2">Zhao et al. (2007)15</td>
			<td>&#183;&#160;&#160;&#160; 10% fat</td>
			<td rowspan="2">36 weeks</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; Male JW</td>
			<td rowspan="2"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160; 30% sucrose</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; 16 weeks</td>
		</tr>
		<tr>
			<td rowspan="3">Helfestein et al. (2011)16</td>
			<td>&#183;&#160;&#160;&#160; 10% fat</td>
			<td rowspan="3">24 weeks</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; Male NZW</td>
			<td rowspan="3"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="3">-</td>
			<td rowspan="3"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td rowspan="3"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160; 40% sucrose</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; 12 weeks</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160; 0.5-0.1 cholesterol</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Ning et al. (2015)17</td>
			<td>&#183;&#160;&#160;&#160; 10% fat</td>
			<td rowspan="2">8-16 weeks</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; Male WHHL</td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="2">-</td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="2"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160; 30% fructose*</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; 12 weeks</td>
		</tr>
		<tr>
			<td rowspan="2">Liu et al. (2016)18</td>
			<td>&#183;&#160;&#160;&#160; 10% fat</td>
			<td rowspan="2">48 weeks</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; Male NZW</td>
			<td rowspan="2"><img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" /></td>
			<td rowspan="2">-</td>
			<td rowspan="2"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td rowspan="2"><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160; 30% sucrose</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; 12 weeks</td>
		</tr>
		<tr>
			<td>Arias-Mutis et al. (2017)19</td>
			<td>&#183;&#160;&#160;&#160; 15% fat</td>
			<td>28 weeks</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160; Male NZW</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
			<td><img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" /></td>
		</tr>
	</tbody>
</table>
Table 1: Diet-induced MetS rabbit models using high-fat, high-sucrose diet. The symbol &#34;<img alt="Equation 2" src="/files/ftp_upload/57117/57117eq2.jpg" />&#34; indicates absence, &#34;<img alt="Equation 1" src="/files/ftp_upload/57117/57117eq1.jpg" />&#34; presence, and &#34;-&#34; not evaluated. *restricted. WHHL, Watanabe heritable hiperlipidemic rabbits. JW, Japanese white rabbits. Ob, obesity. HT, hypertension. HG, hyperglycemia. Dl, dyslipidemia.

<table>
	<tbody>
		<tr>
			<td>Veterinary scale</td>
			<td>SOEHNLE</td>
			<td>7858</td>
			<td>Scale 
			https://www.soehnle-professional.com/en/productgroup/details/103/veterinary-scale</td>
		</tr>
		<tr>
			<td>Shovel for aluminum feed</td>
			<td>COPELE</td>
			<td>10308</td>
			<td>Shovel for aluminum feed 
			http://copele.com/es/herramientas/48-pala-para-pienso-de-aluminio.html</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>PCE Ib&#233;rica</td>
			<td>PCE-TB 15</td>
			<td>Balance 
			http://www.pce-iberica.es/medidor-detalles-tecnicos/balanzas/balanza-compacta-pce-bdm.htm</td>
		</tr>
		<tr>
			<td>Strainer (20 cm diam.)</td>
			<td>ZWILLING</td>
			<td>39643-020-0</td>
			<td>Strainer 
			https://es.zwilling-shop.com/Menaje-del-hogar/Menaje-de-cocina/Menaje-especial/Accesorios/Colador-20-cm-ZWILLING-39643-020-0.html</td>
		</tr>
		<tr>
			<td>Bowl</td>
			<td>ZWILLING</td>
			<td>40850-751-0</td>
			<td>Scale 
			https://www.soehnle-professional.com/en/productgroup/details/103/veterinary-scale</td>
		</tr>
		<tr>
			<td>Funnel</td>
			<td>BT Ingenieros</td>
			<td>not available</td>
			<td>Funnel 
			http://www.bt-ingenieros.com/fluidos-y-combustibles/961-juego-de-4-embudos-de-plastico.html?gclid=EAIaIQobChMIuInui_y-1QIVASjTCh28Zwf-EAQYBSABEgK7xPD_BwE</td>
		</tr>
		<tr>
			<td>Introcan Certo 22G blue</td>
			<td>B Braun</td>
			<td>4251318</td>
			<td>Peripheral intravenous catheter 
			http://www.bbraun-vetcare.es/producto/introcan-</td>
		</tr>
		<tr>
			<td>Propofol Lipuro 10 mg/ml vial 20 ml</td>
			<td>B Braun</td>
			<td>3544761VET</td>
			<td>General intravenous anesthetic 
			http://www.bbraun-vetcare.es/producto/propofol-lipuro-1-</td>
		</tr>
		<tr>
			<td>FisioVet serum solution 500ml</td>
			<td>B Braun</td>
			<td>472779</td>
			<td>Scale 
			https://www.soehnle-professional.com/en/productgroup/details/103/veterinary-scale</td>
		</tr>
		<tr>
			<td>Askina Film Vet 1,25cm x 5m</td>
			<td>B Braun</td>
			<td>OCT13501</td>
			<td>Plastic Plaster 
			http://www.bbraun-vetcare.es/producto/askina-film-vet</td>
		</tr>
		<tr>
			<td>Askina Film Vet 2,50cm x 5m</td>
			<td>B Braun</td>
			<td>OCT13502</td>
			<td>Plastic Plaster 
			http://www.bbraun-vetcare.es/producto/askina-film-vet</td>
		</tr>
		<tr>
			<td>Injekt siringe 10ml luer</td>
			<td>B Braun</td>
			<td>4606108V</td>
			<td>Injection-aspiration syringe of two single-use bodies 
			http://www.bbraun-vetcare.es/producto/injekt-</td>
		</tr>
		<tr>
			<td>Seca 201</td>
			<td>seca</td>
			<td>seca 201</td>
			<td>Ergonomic tape for measuring perimeters 
			https://www.seca.com/es_es/productos/todos-los-productos/detalles-del-producto/seca201.html#referred</td>
		</tr>
		<tr>
			<td>Sterican 21Gx1&#34; - 0,8x25mm verde</td>
			<td>B Braun</td>
			<td>4657543</td>
			<td>Single Use Hypodermic Needle 
			http://www.bbraun-vetcare.es/producto/agujas-hipodermicas-sterican-</td>
		</tr>
		<tr>
			<td>CONTOURNEXT-Meter</td>
			<td>BAYER</td>
			<td>84413470</td>
			<td>Blood glucose analysis system 
			http://www.contournextstore.com/en/contour-next-meter-2</td>
		</tr>
		<tr>
			<td>CONTOUR NEXT test strips</td>
			<td>BAYER</td>
			<td>83624788</td>
			<td>Blood glucose test strips 
			http://www.contournextstore.com/en/contour-next-test-strips-100-ct-package</td>
		</tr>
		<tr>
			<td>MICROLET NEXT LANCING DEVICE</td>
			<td>BAYER</td>
			<td>6702</td>
			<td>Lancing device 
			http://www.contournextstore.com/en/new-microlet-next-lancing-device</td>
		</tr>
		<tr>
			<td>MICROLET 2 Colored Lancets</td>
			<td>BAYER</td>
			<td>81264857</td>
			<td>Ultra-thin sterile lancet for capillary puncture 
			http://www.contournextstore.com/en/microlet2-colored-lancets-100s</td>
		</tr>
		<tr>
			<td>Injekt 20ml luer siringe</td>
			<td>B Braun</td>
			<td>4606205V</td>
			<td>Scale 
			https://www.soehnle-professional.com/en/productgroup/details/103/veterinary-scale</td>
		</tr>
		<tr>
			<td>Askina Mullkompressen 7,5x7,5cm - sterile</td>
			<td>B Braun</td>
			<td>9031219N</td>
			<td>Sterile gauze packets in envelopes 
			http://www.bbraun-vetcare.es/producto/askina-mullkompressen-esteril</td>
		</tr>
		<tr>
			<td>Emla lidocaine/prilocaine</td>
			<td>AstraZeneca</td>
			<td>not available</td>
			<td>Local anesthetics 
			https://www.astrazeneca.es/areas-terapeuticas/neurociencias.html</td>
		</tr>
		<tr>
			<td>Introcan Certo 18G short</td>
			<td>B Braun</td>
			<td>4251342</td>
			<td>Peripheral intravenous catheter 
			http://www.bbraun-vetcare.es/producto/introcan-</td>
		</tr>
		<tr>
			<td>Introcan Certo 20G</td>
			<td>B Braun</td>
			<td>4251326</td>
			<td>Peripheral intravenous catheter 
			http://www.bbraun-vetcare.es/producto/introcan-</td>
		</tr>
		<tr>
			<td>Blood Pressure Transducers-MA1 72-4497</td>
			<td>Harvard Apparatus</td>
			<td>724497</td>
			<td>Transducer for monitoring blood pressure 
			http://www.harvardapparatus.com/physiology/physiological-measurements/transducers/pressure-transducers/research-grade-pressure-transducers.html</td>
		</tr>
		<tr>
			<td>PowerLab 2/26</td>
			<td>AD Instruments</td>
			<td>ML826</td>
			<td>Amplifier 
			https://www.adinstruments.com/products/powerlab</td>
		</tr>
		<tr>
			<td>LabChart ver. 6</td>
			<td>AD Instruments</td>
			<td>not available</td>
			<td>Acquisition software 
			https://www.adinstruments.com/products/labchart</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE136</td>
			<td>Amplifier 
			https://www.adinstruments.com/products/bio-amps#product-FE136</td>
		</tr>
		<tr>
			<td>K2EDTA 7.2mg</td>
			<td>BD</td>
			<td>367861</td>
			<td>Blood collection tubes 
			http://catalog.bd.com/nexus-ecat/getProductDetail?productId=367861</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SciQuip</td>
			<td>2-16KL</td>
			<td>Centrifuge 
			http://www.sigma-centrifuges.co.uk/store/products/refrigerated-sigma-2-16k-centrifuge/</td>
		</tr>
		<tr>
			<td>Eppendorf Reference 2, 100 &#8211; 1000 &#956;L</td>
			<td>Eppendorf</td>
			<td>4920000083</td>
			<td>Pipette 
			https://online-shop.eppendorf.es/ES-es/Pipeteo-44563/Pipetas-44564/Eppendorf-Reference2-PF-42806.html</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 0.5 mL</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td>Tubes 
			https://online-shop.eppendorf.es/ES-es/Puntas-tubos-y-placas-44512/Tubos-44515/Eppendorf-Safe-Lock-Tubes-PF-8863.html</td>
		</tr>
		<tr>
			<td>NZW rabbits (16-18 weeks old)</td>
			<td>Granja San Bernardo</td>
			<td>not available</td>
			<td>New Zealand White rabbits 
			http://www.granjasanbernardo.com/en/welcome/</td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Sigma</td>
			<td>S0389-5KG</td>
			<td>Sucrose for drinking solution 
			http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=es&#38;region=ES</td>
		</tr>
		<tr>
			<td>Rabbit maintenance control diet</td>
			<td>Ssniff</td>
			<td>V2333-000</td>
			<td>Control diet 
			http://www.ssniff.com/</td>
		</tr>
		<tr>
			<td>Rabbit high-fat diet</td>
			<td>Ssniff</td>
			<td>S9052-E020</td>
			<td>High-fat diet 
			http://www.ssniff.com/</td>
		</tr>
		<tr>
			<td>Rabbit rack and drinker</td>
			<td>Sodispan</td>
			<td>not available</td>
			<td>Rack for rabbits 
			https://www.sodispan.com/jaulas-y-racks/racks-conejo-y-cobaya/</td>
		</tr>
		<tr>
			<td>Rabbit restrainer</td>
			<td>Zoonlab</td>
			<td>3045601</td>
			<td>http://www.zoonlab.de/en/index.html</td>
		</tr>
	</tbody>
</table>

an experimental model of diet-induced metabolic syndrome in rabbit: methodological considerations, development, and assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

MetS represents a cluster of metabolic and cardiovascular abnormalities whose study can be facilitated by the use of experimental models. Indeed, to elucidate the mechanisms underlying the pathological remodeling produced by MetS, the choice of an experimental model that appropriately resembles the human condition and is suitable for research is of crucial importance. Here, we present the methods to induce MetS in rabbit using a diet high in saturated fat and sucrose, and a detailed chara...

Watch this Scientific Journal Video about An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment at JoVE.com

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

The overall goal of this procedure is to develop and characterize a metabolic syndrome model in rabbits using a high fat, high sucrose diet, which is of great importance when associating a phenotype with organ remodeling. This method can help answer key questions in the pathophysiology field, such as understanding organ remodeling and the progression of the disease, the detection of paraclinical and clinical markers to identify patients at risk, or testing new therapeutic approaches. The rabbit can provide many advantages over other animal models, including murine, canine, or porcine, given its similarity to human physiology, and the affordability of use in chronic protocols and measurements.Demonstrating this procedure will be Oscar Arias, a PhD student from our laboratory. To acclimate the animals to their diet, feed each animal in the control group 120 grams of control diet daily for four weeks, and provide water ad libitum. Feed the animals in the MetS group 250 grams of chow starting with 50%control and 50%high fat chow.Then, progressively increase the diet to 100%high fat chow by the end of week four. In addition, give the animals in the MetS group water with 5%sucrose at the start, and increase the sucrose concentration to 15%by the end of the fourth week. Register the daily intake of chow and sucrose solution to calculate the caloric intake.Once the animals have been acclimated to their diets, feed each animal in the control group 120 grams of control chow, and water ad libitum daily. Feed the animals in the MetS group 250 grams of high-fat chow, and 15%sucrose in water. Replace the chow daily, and the sucrose solution every third day.Weigh the remaining chow and water daily to estimate daily intake. To carry out morphological assessment, weigh each animal on a weekly basis. Before administration of the experimental diet and at weeks 14 and 28, anesthetize the animal by using a sterile disposable catheter to cannulate the right ear marginal vein.Then, inject eight milligrams per kilogram of propofol, followed by 1.5 ml of 0.9%sodium chloride solution. Once anesthetization has been confirmed, place the animal in the lateral decubitus position and use a measuring tape to measure the distance from the nose to the heel. Record this measurement as the length.With the animal in the same position, measure the distance from the acromion in the shoulder to the tip of the paw and record this as the height. Now, with the animal with a supine position, gently place the measuring tape around the abdominal contour and take a measurement. Then measure tibial length from the lower part of the knee joint to the insertion of the Achilles tendon.Prepare a 60%glucose stock solution with 60 grams of glucose in 100 milliliters of 0.9%sodium chloride. Allow the animals to fast with only water for seven hours. Then, to determine fasting glycemia, place a conscious rabbit in a restrainer in the prone position.Insert a new strip into a glucose meter, then, use a lancet on the left marginal ear vein to get a drop of blood and touch the drop to the test strip. Use the glucose meter to measure the blood glucose level. With a disposable catheter, cannulate the right ear marginal vein, and inject a bolus of a 60%glucose solution.Use the lancet as before to take blood samples at 15, 30, 60, 90, 120, and 180 minutes after glucose injection and use the glucose meter to analyze them. Remove the disposable catheter, and use gauze to pinch the site of catheter insertion. Once blood is coagulated, remove the gauze and check the status of the animal.For blood pressure recording, prepare the acquisition system that includes a pressure transducer, a 10 milliliter syringe with 0.9%sodium chloride, a three-way stopcock, an amplifier, and a PC laptop with the acquisition software. To set up the equipment, place the three-way stopcock and the syringe in the pressure transducer between the transducer in the catheter, and connect the pressure transducer to the amplifier. Then, connect the amplifier to the PC laptop.Place the conscious animal that a rabbit restrainer in the prone position. Warm up the ear before cannulation, then topically apply a local anesthetic in the ear around the site of insertion. Gently tap the area where the vascular package runs to easily identify the artery.Then insert a sterile catheter in the left ear central artery. Now, loosen the restraints, and allow the animal to stay quiet for 30 minutes. Position the pressure transducer next to the animal at heart level, and record the blood pressure continuously for 20 minutes, directly from the arterial catheter.Once the recording is finished, remove the catheter and use gauze to pinch the catheter insertion site to stop blood loss. Once the blood is coagulated, remove the gauze and check the status of the animal. To take plasma measurements, allow the animals to fast with water only for seven hours.Then place a conscious animal in a restrainer in the prone position, and insert a sterile 21 gauge needle into the left ear marginal vein. Once blood begins to drip, discard the first drop and collect the blood samples in EDTA tubes up to the level indicated in the tube. Then store the samples on ice.Centrifuge the blood samples at 1500 G and four degrees Celsius for 15 minutes. Following centrifugation, use a pipette to collect the plasma and prepare 250 microliter aliquots. Immediately analyze the fresh samples for total cholesterol, HDL, LDL, and triglycerides.As shown in this table, the weight of the rabbits in the MetS group increased progressively on the high-fat diet until the end of the experimental protocol. The behavior of energy intake and its fluctuations is illustrated in this graph, ranging from 250 to 815 kilocalories in the MetS group. In this graph, the relative contribution of the different sources of energy is depicted.There are critical periods in weeks 14 and 28, because, given the stress produced by the experimental procedures, rabbits might decrease chow and water intake. Mild hyperglycemia is observed at week 14, which reaches a plateau, and maintains similar values at week 28. The area under the curve also increases in the MedS group.Hypertension is closely and directly related to the severity of obesity. Rabbits fed a high fat, high sucrose diet for 28 weeks showed an increase in systolic, diastolic, and mean blood pressure already at week 14, and this increase in blood pressure is maintained at week 28. Finally, in this chronic model, an alteration in the lipid profile was observed as early as week 14, and this alteration remained stable until week 28.This included an increase in triglycerides and LDL, a decrease in HDL, and no changes in total cholesterol in MedS animals versus controls at both time points. Using this chronic model, other studies could be performed to answer additional questions like how the pathology are morphing in a similar way in human metabolic syndrome, allowing new perspectives in the pathophysiological mechanisms involved in the progression of the disease. This experimental model paved the way for researchers in the field of interactive physiology and pathophysiology to explore the mechanisms underlying organ remodeling.The detection of biomarkers to identify patients at risk, or testing new therapeutic approaches for the treatment of metabolic syndrome.

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Research

JoVE Journal

Medicine

10.5K Görüntüleme.  Instituto de Salud Carlos III. The overall goal of this procedure is to develop and characterize a metabolic syndrome model in rabbits using a high fat, high sucrose diet, which is of great importance when associating a phenotype with organ remodeling. This method can help answer key questions in the pathophysiology field, such as understanding organ remodeling and the progression of the disease, the detection of paraclinical and clinical markers to identify patients at risk, or testing new therapeutic approaches. The rabb...