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, which were 20 - 22 weeks old at the beginning of the experimental protocol. They were housed in a room with humidity (50 &#177; 5%) and temperature (20 &#177; 1.5 &#176;C) controlled conditions with a 12-h light cycle. The words &#34;chow&#34; and &#34;diet&#34; may be used interchangeably in the protocol steps.
1. Diet Administration
<ol>
	<li>Obtain or prepare diets
	<ol>
		<li>Obtain a commercially available high fat diet with added hydrogenated coconut oil (10%) and lard (5%)19. This diet will provide 3.7 kcal&#183;g-1.</li>
		<li>Prepare 5 to 15% sucrose solutions by dissolving the appropriate amounts of sucrose in sterilized water (e.g., use 300 g sucrose in 2 L stock for a 15% sucrose solution). A 15% solution will provide 0.6 kcal&#183;mL-1.</li>
		<li>Obtain a commercially available control diet19, which provides 2.7 kcal&#183;g-1.</li>
	</ol>
	</li>
	<li>Acclimate the animals for 4 weeks
	<ol>
		<li>Feed each animal in the control group 120 g of control diet daily. Provide water ad libitum.</li>
		<li>Feed animals in MetS group 250 g chow starting with a 50% control and 50% high-fat chow, increasing progressively to 100% high-fat chow by the end of week 4. 
		NOTE: The aim would be to achieve: (i) 35% control and 65% high-fat chow by the end of week 1; (ii) 25% control and 75% high-fat chow by the end of week 2; (iii) 15% control and 85% high-fat chow by the end of week 3. (iv) 100% high-fat chow by the end of week 4.</li>
		<li>Give animals in the MetS group water with 5% sucrose at the start, and increase sucrose concentration to 15% by the end of the 4th week.</li>
		<li>Register the daily intake of chow and sucrose solution to calculate caloric intake as per values provided in 1.1.1. and 1.1.2.</li>
	</ol>
	</li>
	<li>Induce MetS (28 weeks)
	<ol>
		<li>Feed each animal in control group 120 g of control chow and water ad libitum daily.</li>
		<li>Feed the animals in the MetS group 250 g of high-fat chow and 15% sucrose in water. Replace chow daily and the sucrose solution every third day.</li>
		<li>Weigh the remaining chow and water daily to estimate daily intake.</li>
	</ol>
	</li>
</ol>
2. Morphological Assessment
<ol>
	<li>Measure animal bodyweight on a weekly basis.</li>
	<li>Measure height, length, abdominal contour, and tibial length, and estimate BMI before administration of the experimental diet and at weeks 14 and 28 in anesthetized animals.
	<ol>
		<li>Cannulate right ear marginal vein with a sterile disposable catheter (18 - 22 G) and inject propofol (8 mgkg-1) followed by 1.5 mL of 0.9% NaCl solution. In the anesthetized rabbit, perform the measurements listed in the subsequent steps.</li>
		<li>Measure height and length. Using measuring tape, measure and record the distance from the nose to the heel in lateral decubitus position (length). In the same position, take the distance from the acromion in the shoulder to the tip of the paw (height).</li>
		<li>Calculate Body mass index (BMI)20 as bodyweight (kg) &#183; [body length (m) &#215; height (m)]-1.</li>
		<li>Place the measuring tape gently around the abdominal contour and take a measurement with the animal in supine position.</li>
		<li>Measure tibial length from the lower part of the knee joint to the insertion of Achilles tendon.</li>
	</ol>
	</li>
</ol>
3. Fasting Glycemia and Intravenous Glucose Tolerance Test (IVGTT)
NOTE: It is advisable to start the procedures the same time of day (i.e., 2 - 3 PM).
<ol>
	<li>Prepare a glucose stock solution (60%) with 60 g glucose in 100 mL of 0.9% NaCl solution.&#160;</li>
	<li>Fast the animal for 7 h (removing food and maintaining water), then place the conscious rabbit in a restrainer in the prone position. Prepare the glucose meter (insert a new strip into the meter), and take the first sample from the left ear marginal vein using a lancet to get a drop of blood. Then touch the blood drop with the test strip and measure blood glucose levels using the glucose meter to determine fasting glycemia.</li>
	<li>Cannulate right ear marginal vein with a disposable catheter (18 - 22 G) and inject a bolus of a 60% glucose solution (0.6 g&#183;kg-1). 
	NOTE: To prepare the bolus, add 1 mL/kg of the glucose stock.</li>
	<li>Take blood samples using the lancet (one drop of blood) at 15, 30, 60, 90, 120, and 180 min after glucose injection and analyze them with the glucose meter as in 3.2.</li>
	<li>Remove the disposable catheter and pinch the site of catheter insertion with a gauze. Once blood has coagulated, remove the gauze and check the status of the animal.</li>
</ol>
4. Blood Pressure
<ol>
	<li>Prepare the acquisition system including a pressure transducer, a 10-mL syringe with 0.9% NaCl, a three-way stopcock, an amplifier, and a PC/laptop with the acquisition software (for blood pressure recording).</li>
	<li>Set up the equipment. First, place the three-way stopcock and the syringe in the pressure transducer, between the transducer and the catheter, and connect the pressure transducer to the amplifier. Then connect the amplifier to the PC/laptop.</li>
	<li>Perform the pressure transducer calibration according to the manufacturer&#39;s recommendations.</li>
	<li>Place the conscious animal in a rabbit restrainer in the prone position. Warm up the ear before cannulation, then topically apply a local anesthetic (2.5% lidocaine/prilocaine) 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 (18 - 22 G) in the left ear central artery. Loosen the restraints and allow the animal to stay quiet for 30 min.</li>
	<li>Record blood pressure continuously for 20 min directly from the arterial catheter, placing the pressure transducer positioned next to the animal at the heart level (sampling frequency: 1 KHz, see Figure 5B). 
	NOTE: To keep the blood pressure (BP) recording free from blood coagulation interference (BP signal loses amplitude or disappears), an NaCl (0.9%) injection should be made. Using the three-way stopcock, close the circuit that goes from the transducer to the catheter, open the circuit that goes from the syringe to the catheter, and inject 1 - 2 mL. This will remove blood clots that may form in the catheter. Then, open the circuit between the transducer and the catheter, and continue the recording once the signal has been recovered.</li>
	<li>Once the recording is finished, remove the catheter and pinch with a gauze in the site of catheter insertion to stop blood loss. Once the blood has coagulated, remove the gauze and check the status of the animal.</li>
</ol>
5. Plasma Measurements
NOTE: It is advisable to start the procedures the same time of day (i.e., 2 - 3 PM).
<ol>
	<li>Fast the animal for 7 h (removing food and maintaining water), then place the conscious animal in a restrainer in the prone position and insert a sterile 21 G needle in 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. Store the samples on ice.</li>
	<li>Centrifuge blood samples at 1,500 x g, 15 min, 4 &#176;C. After centrifugation, suction the plasma using a pipette and prepare aliquots of 250 &#181;L.</li>
	<li>Analyze the fresh samples immediately. Basic control parameters are as follows: triglycerides, total cholesterol, HDL, and LDL cholesterol. 
	NOTE: Samples not freshly analyzed should be stored immediately in a -80 &#176;C freezer. If interested in analyzing blood glucose from plasma samples, the blood glucose test should use tubes with Fluoride Oxalate instead of EDTA.</li>
</ol>

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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&lt;br/&gt; 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&lt;br/&gt; http://copele.com/es/herramientas/48-pala-para-pienso-de-aluminio.html</td></tr><tr><td>Balance</td><td>PCE Ib&amp;eacute;rica</td><td>PCE-TB 15</td><td>Balance&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; https://www.seca.com/es_es/productos/todos-los-productos/detalles-del-producto/seca201.html#referred</td></tr><tr><td>Sterican 21Gx1&quot; - 0,8x25mm verde</td><td>B Braun</td><td>4657543</td><td>Single Use Hypodermic Needle&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; https://www.adinstruments.com/products/labchart</td></tr><tr><td>Animal Bio Amp</td><td>AD Instruments</td><td>FE136</td><td>Amplifier&lt;br/&gt; 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&lt;br/&gt; http://catalog.bd.com/nexus-ecat/getProductDetail?productId=367861</td></tr><tr><td>Centrifuge</td><td>SciQuip</td><td>2-16KL</td><td>Centrifuge&lt;br/&gt; http://www.sigma-centrifuges.co.uk/store/products/refrigerated-sigma-2-16k-centrifuge/</td></tr><tr><td>Eppendorf Reference 2, 100 &amp;ndash; 1000 &amp;mu;L</td><td>Eppendorf</td><td>4920000083</td><td>Pipette&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; http://www.granjasanbernardo.com/en/welcome/</td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma</td><td>S0389-5KG</td><td>Sucrose for drinking solution&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=es&amp;amp;region=ES</td></tr><tr><td>Rabbit maintenance control diet</td><td>Ssniff</td><td>V2333-000</td><td>Control diet&lt;br/&gt; http://www.ssniff.com/</td></tr><tr><td>Rabbit high-fat diet</td><td>Ssniff</td><td>S9052-E020</td><td>High-fat diet&lt;br/&gt; http://www.ssniff.com/</td></tr><tr><td>Rabbit rack and drinker</td><td>Sodispan</td><td>not available</td><td>Rack for rabbits&lt;br/&gt; 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 ...

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JoVE Journal

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10.6K Views.  Instituto de Salud Carlos III. Methoden voor het ontwikkelen van een experimenteel model van dieet-geïnduceerde metabool syndroom (MetS) beschrijven we bij konijnen met behulp van een high-fat, hoog-sacharose dieet. Dieren ontwikkeld centrale obesitas, milde hypertensie, pre diabetes en dyslipidemia, dus het reproduceren van de belangrijkste componenten van menselijke MetS. Deze chronische model zal het toestaan van overname van de onderliggende mechanismen van de kennis van progressie van de ziekte.

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

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity.&#160;While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...

Murine Model for Diabetic Cardiomyopathy via High-Fat Diet and STZ Injection

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...

A Chronic High-Intensity Interval Training and Diet-Induced Obesity Model to Maximize Exercise Effort and Induce Physiologic Changes in Rats

Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that results in similar physiologic benefits. This paper presents a HIIT protocol that can be used to assess various health markers in a Sprague-Dawley rat model of diet-induced obesity. Female Sprague Dawley rats aged 21 days old were randomly assigned to the following groups: control (CON, n = 10), exercise-trained (TRN, n = 10), high-fat diet (HFD, n = 10), and high-fat diet/exercise training (HFD/TRN, n = 10). The control diets consisted of commercial laboratory chow with 10% kilocalories (kcal) from fat (3.82 kcal/g), and the high-fat diets (HFD) consisted of 45% kcal from fat (4.7 kcal/g). The animals had ad libitum access to their assigned diet throughout the study. After an 8 week diet induction period, the exercise cohorts completed four HIIT sessions per week for 8 weeks. Each HIIT session consisted of 10 intervals of 1 min sprints/2 min rest using a rodent treadmill with a motor-driven belt. After the 8 weeks of training, the animals were sacrificed for tissue collection. The results revealed no differences in the distance run between the TRN and HFD/TRN groups, and the training speed steadily increased over the duration of the study, with a final running speed of 115 cm/s and 111 cm/s for the TRN and HFD/TRN groups, respectively. The weekly caloric intake was decreased (p &#60; 0.05) in the TRN group relative to the CON group but increased (p &#60; 0.05) in the HFD/TRN group relative to the HFD group. Lastly, the animals on the HFD had greater (p &#60; 0.05) adiposity, and the trained animals had reduced (p &#60; 0.05) adiposity relative to controls. This protocol demonstrates an efficient method to evaluate the effects of HIIT on various physiologic outcomes in a diet-induced obesity model.

This paper presents the morphometric responses and training performance outcomes of a high-intensity interval training (HIIT) protocol in a Sprague-Dawley rat model of diet-induced obesity. The purpose of this protocol was to maximize exercise intensity and determine the physiologic responses to HIIT in lean and obese rats.

Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that ...

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

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