Name: a modified surgical model of hind limb ischemia in apoe-/- mice using a miniature incision
Uploaded: 2021-05-13T12:30:00
Description: Watch this Scientific Journal Video about A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision at JoVE.com

Authors thank Viktoria Skude, Alexander Schlund, and Felix H&#246;rner for the excellent technical support.

NOTE: All experimental procedures were performed according to the EC guideline EC 2010/63/EU and have been approved by the local German legislation (35-9185.81/G[1]239/18). Ten male ApoE-/- mice with the C57BL/6J background, weighing 29.6-38.0 g, were housed on a 12 h light/dark cycle and fed a western diet (1.25% cholesterol and 21% fat) and water ad libitum for 12 weeks from the age of 8 weeks. HLI was performed on 20-week-old mice as described below.
1. Induction of HLI in...

This study reports a modified, simplified, and surgically efficient approach to establish an HLI model in ApoE-/- mice using double ligation in the proximal and distal regions of the FA through a 3-4 mm incision without any required laboratory upgrades. The main characteristic of this method is the smaller size of the incision compared to previously reported studies describing mouse HLI models8,9,10,<sup cl...

There is an unmet need for preclinical animal models for research in vascular diseases such as peripheral artery disease (PAD). Despite the advanced developments in diagnosis and treatment, there were more than 200 million patients with PAD in 20181, and their number is constantly increasing. Although several novel therapeutic approaches2,3,4,5,6,7 have been described, successful translation of these therapeutic modalities into clinical application remains a daunting task. Therefore, reliable and relevant in vivo experimental models simulating the human disease condition are required to investigate the potential mechanism and efficiency of these new therapeutic approaches to treat PAD6,7.
Hyperlipidemia and atherosclerosis (AS) are the main risk factors for the development of PAD. ApoE-/- mice (on a high-fat diet) display abnormal fat metabolism and hyperlipidemia and subsequently develop atherosclerotic plaques rendering ApoE-/- mice as the best choice to simulate the clinically relevant PAD. Preclinical HLI animal models are generated through double ligation of the femoral artery (DLFA), which is the most widely used approach in laboratories all over the world8,9,10,11,12,13,14,15 to simulate acute-on-chronic ischemia. However, this approach usually requires a relatively large and invasive incision. Furthermore, it inevitably leads to the animals (especially mice) suffering from increased pain injury and inflammation, which also influences the subsequent experimental results5,6,16,17. This paper describes an acute-on-chronic HLI model in APOE-/- mice by using a very small incision.

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	<tbody>
		<tr >
			<td >10x Phosphate buffer saline</td>
			<td >Roth</td>
			<td >9143.1</td>
			<td >Used for haematoxylin and eosin stain and immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >30% H2O2</td>
			<td >Roth</td>
			<td >9681.2</td>
			<td>Used for immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >6-0 absorbable sutures</td>
			<td>PROLENE</td>
			<td>8776H</td>
			<td>Used for stitching the skin</td>
		</tr>
		<tr >
			<td >6-0 absroable suture</td>
			<td >PROLENE</td>
			<td >EP8706</td>
			<td>Used in Surgery</td>
		</tr>
		<tr >
			<td >7-0 absorbable sutures</td>
			<td>PROLENE</td>
			<td >EH8021E</td>
			<td>Used for ligating the artery</td>
		</tr>
		<tr >
			<td >7-0 absroable suture</td>
			<td >PROLENE</td>
			<td >EP8755</td>
			<td>Used in Surgery</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td >Roth</td>
			<td>6755.1</td>
			<td>Used for haematoxylin and eosin stain</td>
		</tr>
		<tr >
			<td >Albumin Fraktion V</td>
			<td >Roth</td>
			<td >8076.2</td>
			<td>Used for immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td >Systec GmbH</td>
			<td >Systec VX-150</td>
			<td>Used for the sterilisation of the surgical instruments</td>
		</tr>
		<tr >
			<td >Axio vert A1 microscope</td>
			<td >Carl Zeiss</td>
			<td >ZEISS Axio Vert.A1</td>
			<td>Used for viewing and taking the pictures from haematoxylin and eosin stain and immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Bruker BioSpec 94/20 AVIII</td>
			<td >Bruker Biospin MRI GmbH</td>
			<td >N/A</td>
			<td>Scan the femoral artery blockage</td>
		</tr>
		<tr >
			<td >Buprenovet Sine 0,3mg/ml</td>
			<td >Bayer AG</td>
			<td >2542 (WDT)</td>
			<td>Used in post operative pain-management. Dose - 0.1 mg/kg body weight every 8 hours for 48 h after operation</td>
		</tr>
		<tr >
			<td >CD31 antibody</td>
			<td >Abcam</td>
			<td >ab28364</td>
			<td>Used for immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Eosin Y solution 0.5 % in water</td>
			<td >Roth</td>
			<td >X883.1</td>
			<td>Used for haematoxylin and eosin stain</td>
		</tr>
		<tr >
			<td >Epitope Retrieval Solution pH 6</td>
			<td >Leica Biosystems</td>
			<td >6046945</td>
			<td>Used for immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Ethanol &#8805; 99,5 %</td>
			<td >Roth</td>
			<td >5054.1</td>
			<td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Fentanyl</td>
			<td>Cayman Chemical</td>
			<td >437-38-7</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >Fine point forceps</td>
			<td >Medixplus</td>
			<td >93-4505S</td>
			<td>Used for separating the artery from nerve and vein</td>
		</tr>
		<tr >
			<td >Glass bead sterilisator</td>
			<td >Simon Keller</td>
			<td >Type 250</td>
			<td>Used for sterilisation of the surgical instruments</td>
		</tr>
		<tr >
			<td >Graefe iris forceps curved</td>
			<td >VUBU</td>
			<td >VUBU-02-72207</td>
			<td>Used for blunt separation of skin and subcutaneous tissue</td>
		</tr>
		<tr >
			<td >Hair Remover cream, Veet (with aloe vera)</td>
			<td >Reckitt Benckiser</td>
			<td >108972</td>
			<td>Remove hair from mice hind limbs</td>
		</tr>
		<tr >
			<td >Heating plate</td>
			<td >ST&#214;RK-TRONIC</td>
			<td >7042092</td>
			<td>Keep the satble temperature of mice</td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td >Roth</td>
			<td >T865.2</td>
			<td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Leica surgical microscope</td>
			<td >Leica</td>
			<td >M651</td>
			<td>Enlarge the field of view to facilitate the operation</td>
		</tr>
		<tr >
			<td >Liquid DAB+Substrate Chromogen System</td>
			<td >Dako</td>
			<td >K3468</td>
			<td>Used for immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Male ApoE-/- mice</td>
			<td >Charles River Laboratories</td>
			<td >N/A</td>
			<td>Used for establish the Peripheral artery disease mice model</td>
		</tr>
		<tr >
			<td >Medetomidine</td>
			<td>Cayman Chemical</td>
			<td>128366-50-7</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >Micro Needle Holder</td>
			<td >Black &#38; Black Surgical</td>
			<td >B3B-18-8</td>
			<td>Holding the needle</td>
		</tr>
		<tr >
			<td >Micro suture tying forceps</td>
			<td >Life Saver Surgical Industries</td>
			<td >PS-MSF-145</td>
			<td>Used to assist in knotting during surgery</td>
		</tr>
		<tr >
			<td >Microtome</td>
			<td >Biobase</td>
			<td >Bk-Mt268m</td>
			<td>Used for tissue sectioning</td>
		</tr>
		<tr >
			<td >Midazolam</td>
			<td>Ratiopharm</td>
			<td>44856.01.00</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >MR-compatible Small Animal Monitoring and Gating System Model 1025</td>
			<td>SA Instruments</td>
			<td>N/a</td>
			<td>monitoring vital signs of animal during MRI scan</td>
		</tr>
		<tr >
			<td >Octeniderm farblos</td>
			<td >Sch&#252;lke &#38; Mayr GmbH</td>
			<td >180212</td>
			<td>used for disinfection of the skin</td>
		</tr>
		<tr >
			<td >Ointment for the eyes and nose</td>
			<td >Bayer AG</td>
			<td >1578675</td>
			<td>Keep the eyes wet under the anesthesia</td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Roth</td>
			<td >0335.1</td>
			<td>Used for fixation of the tissue</td>
		</tr>
		<tr >
			<td >Pentobarbital</td>
			<td>Nembutal</td>
			<td >76-74-4</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >Saline</td>
			<td>DeltaSelect</td>
			<td >1299.99.99</td>
			<td>Used for anesthesia</td>
		</tr>
		<tr >
			<td >Spring handle scissors with fine, sharp tips</td>
			<td >Black &#38; Black Surgical</td>
			<td >B66167</td>
			<td>Used for cutting the artery</td>
		</tr>
		<tr >
			<td >SuperCut Scissors</td>
			<td >Black &#38; Black Surgical</td>
			<td >B55992</td>
			<td>Used for cutting the skin</td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Roth</td>
			<td >9002-93-1</td>
			<td>Used for immunohistochemistry stain</td>
		</tr>
		<tr >
			<td >Western diet, 1.25% Cholesterol</td>
			<td >ssniff Spezialdi&#228;ten GmbH</td>
			<td >E15723-34</td>
			<td>Diet for the mice</td>
		</tr>
		<tr >
			<td >Xylene</td>
			<td >Roth</td>
			<td >4436.3</td>
			<td>Used for haematoxylin and eosin stain and immunohistochemistry stain</td>
		</tr>
	</tbody>
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a modified surgical model of hind limb ischemia in apoe-/- mice using a miniature incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

Characteristics of ApoE-/- mice 
DLFA surgeries were successfully performed on 10 mice to establish the HLI model, and none of the mice died after the procedure. To follow changes in body weight, mice were weighed before the DLFA procedure (Pre-DLFA) and 7 days after the DLFA surgery (Post-DLFA). Pre-DLFA weights ranged from 29.6 to 38.0 g (mean 34.74 &#177; 2.47 g), and post-DLFA weights ranged from 26.5 to 34.1 g (mean 30.77 &#177; 2.15 g), which were s...

Watch this Scientific Journal Video about A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision at JoVE.com

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...

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