Name: balloon-based injury to induce myointimal hyperplasia in the mouse abdominal aorta
Uploaded: 2018-02-07T12:30:00
Description: Watch this Scientific Journal Video about Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta at JoVE.com

The authors thank Christiane Pahrmann for her technical assistance.
D.W. was supported by the Max Kade Foundation. T.D. received grants from the Else Kro&#776;ner Fondation (2012_EKES.04) and the Deutsche Forschungsgemeinschaft (DE2133/2-1_. S. S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; SCHR992/3- 1, SCHR992/4-1).

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (&#39;&#39;Amt f&#252;r Gesundheit und Verbraucherschutz, Hansestadt (Office for Health and Consumer Protection) Hamburg&#39;&#39;).
1. Catheter Preparation
NOTE: Refer to the Table of...

<ol>
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This article demonstrates a murine model to study the development of myointimal hyperplasia and allows the exploration of the underlying pathological processes and the testing of new drugs or therapeutic options.
The most critical step in this protocol is the denudation of the aorta. Special care should be paid during this step as excessive denudation will lead to aneurysm formation and model failure. On the other hand, if denudation is performed insufficiently, too little myointima will devel...

Arterial stenosis in coronary and peripheral arteries has a large effect on the morbidity and mortality of patients1. One underlying pathological mechanism is myointima hyperplasia (MH), which is characterized by increased proliferation, migration, and synthesis of extracellular matrix proteins from vascular smooth muscle cells (SMC)2. SMC are located in the media layer of the vessel and migrate upon stimulation to the surface of the lumen. Stimulatory signals include growth factors, cytokines, cell-cell contact, lipids, extracellular matrix components, and mechanical shear and stretch forces3,4,5,6. Injuries of the vessel wall, pathological or iatrogenic, cause endothelial cell and smooth muscle cell damage and stimulate inflammatory reactions, and thus lead to MH7.
Different animal models are currently available to study arterial injury and myointima hyperplasia. Large animals like pigs or dogs have the advantage of sharing a similar artery and coronary anatomy with humans and are especially suitable for studies investigating angioplasty techniques, procedure, and devices8. However, pig models have the drawback of higher thrombogenicity9,10, while dogs only have a mild response to vessel injury11. In addition, all large animal models require special housing, equipment, and staff, which are connected with high costs and are not always available at an institution. Small animal models include rats and mice. Compared to rats, mice have the advantages of lower cost and the existence of a variety of knock out models. The model described in this video can be combined with ApoE-/- mice fed with a western diet to closely mimic the clinical setting of angioplasty in atherosclerotic vessels12. Previous models induced vascular injury via wire injury13, fluid desiccation14, spring15, or cuff injury16. Since the nature of the injury will greatly impact the development and constitution of MH, using a balloon catheter to induce vessel injury is the best way to mimic the clinical setting.
In this article we describe a novel method to induce MH with a balloon catheter in mice. The use of a balloon catheter (1.2 mm x 6 mm) with a RX-Port (Figure 1A) allows the scraping of the intimal layer and, at the same time, the induction of an overdistension of the vessel. Both of these factors are important triggers for the development of MH. The observation time for this model is 28 days17.

<table>
	<tbody>
		<tr>
			<td>10-0 Ethilon suture</td>
			<td>Ethicon</td>
			<td>2814G</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL Syringe</td>
			<td>BD Medical</td>
			<td>309658</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 prolene suture</td>
			<td>Ethicon</td>
			<td>EH7229H</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 prolene suture</td>
			<td>Ethicon</td>
			<td>8706H</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Fuchsin</td>
			<td>Sigma-Aldrich</td>
			<td>F8129-25G</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Antigen retrieval solution</td>
			<td>Dako</td>
			<td>S1699</td>
			<td></td>
		</tr>
		<tr>
			<td>Azophloxin</td>
			<td>Waldeck</td>
			<td>1B-103</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Bepanthen Eye and Nose ointment</td>
			<td>Bayer</td>
			<td>1578675</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Charles River</td>
			<td>Stock number 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp applicator</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen 3</td>
			<td>abcam</td>
			<td>ab7778</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fischer</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG AF555</td>
			<td>Invitrogen</td>
			<td>A21432</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF488</td>
			<td>Invitrogen</td>
			<td>A11055</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF555</td>
			<td>Invitrogen</td>
			<td>A31572</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Th. Geyer</td>
			<td>2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Th. Geyer</td>
			<td>2295</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Th. Geyer</td>
			<td>2246</td>
			<td></td>
		</tr>
		<tr>
			<td>FAP</td>
			<td>abcam</td>
			<td>ab28246</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Forceps fine</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps standard</td>
			<td>Fine Science Tools</td>
			<td>11023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>537020</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Rotexmedica</td>
			<td>PZN 3862340</td>
			<td>25.000 I.E./mL</td>
		</tr>
		<tr>
			<td>High temperature cautery kit</td>
			<td>Bovie</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-iT FX Signal Enhancer</td>
			<td>Invitrogen</td>
			<td>I36933</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Light Green SF</td>
			<td>Waldeck</td>
			<td>1B-211</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Microsurgical clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Micro-Serrefine - 4mm</td>
		</tr>
		<tr>
			<td>MINI TREK Coronary Dilatation Catheter 1.20 mm x 6 mm / Rapid-Exchange</td>
			<td>Abbott</td>
			<td>1012268-06U</td>
			<td></td>
		</tr>
		<tr>
			<td>Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>1.00532.0100</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>NaCl 0,9%</td>
			<td>B.Braun</td>
			<td>PZN 06063042 Art. Nr.: 3570160</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12075-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>Serva Electrophoresis</td>
			<td>33427</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Primary antibody diluent</td>
			<td>Dako</td>
			<td>S3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Mounting solution</td>
			<td>Thermo Fischer</td>
			<td>P36930</td>
			<td>Mounting solution for immunofluorescence stained slides</td>
		</tr>
		<tr>
			<td>Replaceable Fine Tip</td>
			<td>Bovie</td>
			<td>H101</td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcin-Fuchsin Weigert</td>
			<td>Waldeck</td>
			<td>2E-30</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>400684.00.00</td>
			<td>Carprofen</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Vannas-style</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast acting Adhesive MINIS 3x1g</td>
			<td>UHU</td>
			<td>45370</td>
			<td>Cyanoacrylate</td>
		</tr>
		<tr>
			<td>Slide Rack</td>
			<td>Ted Pella</td>
			<td>21057</td>
			<td></td>
		</tr>
		<tr>
			<td>SM22</td>
			<td>abcam</td>
			<td>ab10135</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>abcam</td>
			<td>ab21027</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Staining dish</td>
			<td>Ted Pella</td>
			<td>21075</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabotamp fibrillar</td>
			<td>Ethicon</td>
			<td>431962</td>
			<td>Absorbable hemostat</td>
		</tr>
		<tr>
			<td>Transpore Surgical Tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin syringe</td>
			<td>BD Medical</td>
			<td>324825</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Dilator</td>
			<td>Fine Science Tools</td>
			<td>18603-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitro-Clud</td>
			<td>Langenbrinck</td>
			<td>04-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigerts iron hematoxylin Kit</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Th. Geyer</td>
			<td>3410</td>
			<td></td>
		</tr>
	</tbody>
</table>

balloon-based injury to induce myointimal hyperplasia in the mouse abdominal aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

Balloon denudation is a suitable model to study the development of MH in mice. Animals recover well from the surgery and show an excellent physical condition post-operation. We established this model in 50 mice with less than 3% death rate due to the surgical procedure. Figures 1B-C show the key surgical steps. After a skin incision along the linea alba, identify the aorta abdominalis. Place microsurgical clamps (<strong cla...

Watch this Scientific Journal Video about Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta at JoVE.com

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

The overall goal of this mouse based model system is to study the development of myointimal hyperplasia after aortic balloon injury. This model system can help answer key questions about myointimal hyperplasia such as what factors contribute to its development. By studying the development of myointimal hyperplasia in transgenic mice we hope to gain a better understanding of the pathomechanism of the disease.Another advantage of this animal model, is that the disease progresses quickly. In just weeks, we're studied in mice. First, prepare the required catheter.Remove it from the holder and pull the guide wire out through the lumen through the distal port. Next put in a drop of cyanoacrylate on the distal end of the catheter and feed the guide wire through the RX port, then through the lumen and out the distal port. Next pull the guide wire back slightly to leave about five millimieters sticking out and let the adhesive dry.Later fill a three milliliter syringe with 1.5 milliliters of 0.9%saline and connect it to the ballon inflation port of the catheter. Then push the plunger to test the balloon inflation. Leave about 0.6 milliliters of saline in the syringe.Next, prepare the mouse for surgery. Anesthetize it in an isoflurane chamber and later, during surgery, deliver isoflurane via a nose cone. Before proceeding, confirm a sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.Then remove the abdominal hair using a hair trimmer and fix the position of the hind legs using tape. Lastly, disinfect the abdominal area using three alternating scrubs of povidone iodine followed by 80%ethanol. Begin with dissecting open the skin and muscle layers along the linea alba to expose the abdominal organs.Then move aside the intestines, wrap them in a saline soaked gauze to keep them moist. Now using two fine forceps to remove the fatty tissue above the abdominal aorta. Then from an insulin syringe, inject about 12 units of heparin sulfate in to the IVC and wait three minutes for systemic distribution.Next use forceps to dissect the infrarenal aorta down to the bifurcation and to reveal the outgoing branches. Then using cauterization, ligate the side vessels along the infrarenal aorta within the area that will be clamped. Then, stop the blood flow by clamping the vessel directly beneath the renal arteries and clamping at a distal position just above the aortic bifurcation.Now perform a small horizontal incision along the vessel at the midpoint between the clamps. The incision should be about 1/3 the circumference of the vessel. Then, using an insulin syringe, flush the aorta with heparin solution as before.Next place 10-0 sutures with single knots on each side of the incision. To proceed, dilate the aorta. Place a vessel dilator in to the incision and spread the vessel slightly.Do this two or three times. Then prepare the balloon catheter by wetting it with saline. Then insert the balloon catheter deflated in to the aorta and advance it towards the proximal clamp.Carefully open the proximal clamp and inflate the balloon to prevent blood leakage by injecting about 0.6 milliliters of saline. Next, advance the catheter retrograde about two centimeters. Pull the catheter back while deflating the balloon slightly.When the catheter reaches the incision of the aorta, reattach the proximal clamp. Then, deflate the balloon completely and remove it. Now rinse the aorta with heparin solution.Then proceed by closing the aortic incision using interrupted 10-0 sutures on each lateral side. Followed by one or two stitches on the ventral side. Next open the distal clamp, check for bleeding and place additional sutures as needed.Once there's no bleeding, carefully open the proximal clamp. Use swabs on the sutures for support and to slow bleeding. Then place absorbable hemostats on the sutures to sustain them.With both clamps opened, an aortic pulse should be visible distally from the incision. Now place the intestines back in to the abdomen and rinse the abdominal cavity with saline warmed to 37 degrees Celsius. Next close the abdominal muscle layer using 6-0 running sutures.And then close the skin with 5-0 running sutures. Next immediately inject an analgesic subcutaneously before the animal wakes up. Now monitor the mouse until it has gained consciousness and can maintain sternal recumbency.Once conscious, house the animal alone until it has completely recovered. Provide Metamizole to the drinking water for three days. Be sure to check on the mouse every day to ensure a good recovery.Balloon denudation is a suitable method to study the development of myointimal hyperplasia in mice. Animals recovered well from the surgery and were in excellent physical condition. Less than 3%died in over 50 surgeries.Myointimal hyperplasia developed progressively in the graft. Histological staining with Masson's trichrome showed the myointima formation inside the internal elastic lamina. The myointimal lesions consisted mainly of cellular components positive for SM22 indicating smooth muscle cells and ECM cells like myofibroblasts also shown in red.After watching this video you should have a good understanding how to perform the balloon injuries in mice. Once mastered, this technique can be done in 20 minutes if it's performed properly.

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Research

JoVE Journal

Medicine

The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL varies between 2.5 and 20% 2-5. Over the years, many strategies aimed at lowering the incidence of anastomotic leakage have been examined6, 7. 
The cause of AL is probably multifactorial. Etiological factors include insufficient arterial blood supply, tension on the anastomosis, hematoma and/or infection at the anastomotic site, and co-morbid factors of the patient as diabetes and atherosclerosis8. Furthermore, some anastomoses may be insufficient from the start due to technical failure.
Currently a new device is developed in our institute aimed at protecting the colorectal anastomosis and lowering the incidence of AL. This so called C-seal is a biofragmentable drain, which is stapled to the anastomosis with the circular stapler. It covers the luminal side of the colorectal anastomosis thereby preventing leakage.
The C-seal is a thin-walled tube-like drain, with an approximate diameter of 4 cm and an approximate length of 25 cm (figure 1). It is a tubular device composed of biodegradable polyurethane. Two flaps with adhesive tape are found at one end of the tube. These flaps are used to attach the C-seal to the anvil of the circular stapler, so that after the anastomosis is made the C-seal can be pulled through the anus. The C-seal remains in situ for at least 10 days. Thereafter it will lose strength and will degrade to be secreted from the body together with the gastrointestinal natural contents.
The C-seal does not prevent the formation of dehiscences. However, it prevents extravasation of faeces into the peritoneal cavity. This means that a gap at the anastomotic site does not lead to leakage.
Currently, a phase II study testing the C-seal in 35 patients undergoing (colo-)rectal resection with stapled anastomosis is recruiting. The C-seal can be used in both open procedures as well as laparoscopic procedures. The C-seal is only applied in stapled anastomoses within 15cm from the anal verge. In the video, application of the C-seal is shown in an open extended sigmoid resection in a patient suffering from diverticular disease with a stenotic colon.

The C-seal is a biofragmentable drain protecting the stapled colorectal anastomosis. In this video, details of the manufacturing, deployment and use are shown.

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL ...

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...