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

an-experimental-model-diet-induced-metabolic-syndrome-rabbit

Research

JoVE Journal

Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

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 ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

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 ...