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 Materials for information regarding the catheter.
<ol>
	<li>Take catheter from the catheter holder.</li>
	<li>Pull the guidewire from the lumen through the distal port out.</li>
	<li>Put in a drop of cyanoacrylate adhesive on the distal end of catheter. 
	Caution: Wear latex or nitrile gloves.</li>
	<li>Place the guidewire at the RX port of the catheter and advance it through the lumen to the distal port. Afterwards, pull the guidewire back slightly to leave a space of ~5 mm to the end.</li>
	<li>Wait 5 min to allow the adhesive to dry.</li>
	<li>Move the guidewire, it should be fixed. If it is still mobile, repeat steps 1.3 - 1.6.</li>
	<li>Fill a 3-mL syringe with 1.5 mL 0.9% saline and connect it to the balloon inflation port.</li>
	<li>Push the syringe plunger to test balloon inflation. Leave 0.6 mL saline in the syringe.</li>
</ol>
2. Mouse preparation
<ol>
	<li>Obtain male mice at the age of 14 weeks weighing approximately 30 g. 
	NOTE: We used animals obtained from the Institute of Laboratory Animals. We used C57BL/6J here.</li>
	<li>Use an induction chamber to anaesthetize the mouse with 2.0-2.5% isofurane (500 mL/min oxygen flow rate).</li>
	<li>Place the mouse on its back on a heating pad and maintain anesthesia with a facemask covering the mouth and nose of the mouse. Check for sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.</li>
	<li>Remove the abdominal hair using a hair trimmer.</li>
	<li>Spread the hind legs and fix their position using tape.</li>
	<li>Disinfect the abdominal area using povidone-iodine, followed by 80% ethanol. Repeat this step two more times.</li>
	<li>Use a surgical drape to ensure surgical site does not get contaminated.&#160;Open the skin and muscle layers along the linea alba with a scissor (or scalpel) to expose the abdominal organs.</li>
	<li>Lay the intestines in a 0.9% saline moisturized glove and wrap to keep them moist.</li>
	<li>Use two fine forceps to remove the fatty tissue above the abdominal aorta.</li>
	<li>Using an insulin syringe (30G), inject 250 &#181;L of heparin solution (50 U/mL) into the Inferior Vena Cava (IVC) and wait 3 min for its systemic distribution.&#160;Heparin will suppress hemostasis and prevent undesired clotting during surgery.</li>
	<li>Using two forceps, dissect the infrarenal aorta down to its bifurcation and its outgoing branch vessels.</li>
	<li>Ligate the side vessels, which are expected to be placed in the clamped area, with a high temperature cauterizer.</li>
	<li>Stop blood flow by clamping the infrarenal aorta directly beneath the renal arteries.</li>
	<li>Place a second clamp at a distal position just above the aortic bifurcation.</li>
	<li>Perform a small horizontal incision using scissors at the midpoint between the clamps along the vessel. 
	NOTE: The size of the incision should be equivalent to 1/3 of the circumference of the vessel.</li>
	<li>Insert a syringe with a (30G) needle into the incision and flush the aorta with 250 &#181;L heparin solution (50 U/mL).</li>
	<li>Using 10-0 sutures, place one single knot on each side of the incision.</li>
	<li>Dilate the aorta by inserting a vessel dilator into the incision and spread the vessel slightly. Repeat the dilation 2 to 3 times.</li>
	<li>Moisturize the balloon-catheter with 0.9% saline.</li>
	<li>Insert the flattened balloon-catheter into the aorta and advance it towards the proximal clamp on the aorta.</li>
	<li>When reaching the proximal clamp, carefully open the proximal clamp and inflate the balloon to prevent blood leakage, by injecting ~0.6 mL of saline. 
	NOTE: The ratio of the inflated balloon to vessel is 1.5:1.</li>
	<li>Advance the catheter retrograde for approximately 2 cm.</li>
	<li>Pull the expanded catheter back, while deflating the balloon slightly by releasing the syringe.</li>
	<li>Reattach the proximal clamp when the catheter reaches the incision of the aorta. Deflate the balloon completely and remove it.</li>
	<li>Rinse the aorta with 250 &#181;L heparin solution (50 U/mL) using a 30G syringe.</li>
	<li>Close the aortic incision using 10-0 sutures. Place interrupted stitches on each lateral side, followed by one or two stitches on the ventral side.</li>
	<li>Open the distal clamp. In case of bleeding, close the clamp again and place additional stitches.</li>
	<li>Open the proximal clamp carefully.</li>
	<li>Place two swabs on the suture to support it and stop any bleeding.</li>
	<li>Place absorbable hemostats on the suture to sustain it. 
	NOTE: An aortic pulse should be visible distally from the incision.</li>
	<li>Place the intestines back into the abdomen.</li>
	<li>Rinse the abdominal cavity with sterile 0.9% saline, which has been pre-warmed to 37 &#176;C.</li>
	<li>Close the abdominal muscle layer using 6-0 running sutures.</li>
	<li>Close the skin with 5-0 running sutures.</li>
	<li>Inject 4-5 mg/kg Carprofen subcutaneously before allowing the mouse to wake up. Monitor the animal until it has gained consciousness, maintain sternal recumbency. Keep the animal alone in a cage until complete recovery.</li>
	<li>Add Metamizole to the drinking water (50 mg/100 mL) as pain medication for 3 days and monitor the animal daily.&#160;Usually mice like the sweet taste of metamizole and start drinking immediately after the surgery. If preferred, sustained-release injectable agents can be used instead of metamizole. 
	NOTE: The observation period for this model is 28 days.</li>
</ol>
3. Histopathology
<ol>
	<li>Harvest the balloon-injured aorta after 28 days by preparing the mice as described in steps 2.2 to 2.9.</li>
	<li>Use scissors to remove the balloon-injured aorta (between the bifurcation and 0.3 mm above the renal vessels) and euthanize the mouse by cutting out its heart.</li>
	<li>Flush the lumen of the vessel with 0.9% NaCl.</li>
	<li>Fix the harvested vessel in 4% paraformaldehyde (PFA) overnight and dehydrate it in increasing concentrations of ethanol, starting with 70% ethanol for 2 hours, 80% ethanol for 1 hour, 95% ethanol for 2 hours, and 100% ethanol for 5 hours. Then, incubate the samples in xylene for 2 hours 3 times, before infiltrating the samples with paraffin. 
	NOTE: Instead of flushing the harvested vessel with 0.9% NaCl, it can be flushed with 4% PFA. 
	Caution: PFA and xylene are toxic and should be handled with special care.</li>
	<li>Embed the sample in paraffin and cut into slices of 5 &#181;m thickness using a microtome.</li>
	<li>Deparaffinize the slides with xylene 3 times for 5 minutes.</li>
	<li>Rehydrate tissue slides using a decreasing series of ethanol. Start with 100% ethanol 2 times for 5 min, followed by 3 minutes of 95%, 80% and 70% ethanol.</li>
	<li>Stain the slides with Masson&#39;s trichrome staining as described18.</li>
	<li>Dehydrate stained slides in 100% ethanol 2 times for 10 min each. Clear with xylene 2 times for 10 min each and mount in mounting medium.</li>
	<li>View the slides with a bright field microscope. Use a lens with 5x magnification and a numerical aperture of 0.12 for an overview picture or a lens with 20x magnifications with a numerical aperture of 0.35 for detailed observation.</li>
</ol>
4. Immunofluorescence Microscopy
<ol>
	<li>Rehydrate tissue slides using a decreasing series of ethanol. Start with 100% ethanol 2 times for 5 min, followed by 3 min of 95%, 80% and 70% ethanol.</li>
	<li>Perform antigen-retrieval by heating the slides in antigen-retrieval solution in a steamer for 20 min.</li>
	<li>Let the slides cool down to room temperature.</li>
	<li>After washing the slides for three times with phosphate buffered saline (PBS), apply antigen blocking solution on sections for 30 min.</li>
	<li>Wash slides three times for 5 min with PBS.</li>
	<li>Incubate sections with primary antibody diluted in primary antibody diluent. 
	NOTE: The right concentration and incubation time should be chosen separately for each antibody.</li>
	<li>Wash slides three times for 5 min with PBS to remove unbound antibody.</li>
	<li>Incubate sections with a pre-conjugated secondary antibody diluted in secondary antibody diluent. 
	NOTE: The right concentration and incubation time should be chosen separately for each antibody.</li>
	<li>Remove unbound antibodies by washing the slides for 5 min three times.</li>
	<li>Counterstain the cell nuclei using 4&#39;,6-diamidino-2-phenylindole (DAPI) for 15 min; the final DAPI concentration should be 350 nM.</li>
	<li>Mount slides in immunofluorescence compatible mounting solution. 
	NOTE: Using the wrong mounting solution can obscure the fluorescence signal.</li>
	<li>View slides with a fluorescence microscope. Use a 40x magnification lens with a numerical aperture of 1.3.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+differentiation+of+vascular+smooth+muscle+cells.">Regulation of differentiation of vascular smooth muscle cells.</a> Physiol Rev. 75 (3), 487-517 (1995).</li><li>Owens, G. K., Kumar, M. S., Wamhoff, B. 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The authors have nothing to disclose.

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>
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			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12075-14</td>
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			<td>Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
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			<td>Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
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			<td>Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
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		<tr>
			<td>PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
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		<tr>
			<td>PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td></td>
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		<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>
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			<td>Replaceable Fine Tip</td>
			<td>Bovie</td>
			<td>H101</td>
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			<td>Resorcin-Fuchsin Weigert</td>
			<td>Waldeck</td>
			<td>2E-30</td>
			<td>Trichrome staining</td>
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			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>400684.00.00</td>
			<td>Carprofen</td>
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			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
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			<td>Scissors Vannas-style</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
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			<td>Secondary antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
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			<td>Fast acting Adhesive MINIS 3x1g</td>
			<td>UHU</td>
			<td>45370</td>
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			<td>Slide Rack</td>
			<td>Ted Pella</td>
			<td>21057</td>
			<td></td>
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			<td>SM22</td>
			<td>abcam</td>
			<td>ab10135</td>
			<td>Antibody</td>
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			<td>SMA</td>
			<td>abcam</td>
			<td>ab21027</td>
			<td>Antibody</td>
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			<td>Staining dish</td>
			<td>Ted Pella</td>
			<td>21075</td>
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			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>M651</td>
			<td></td>
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		<tr>
			<td>Tabotamp fibrillar</td>
			<td>Ethicon</td>
			<td>431962</td>
			<td>Absorbable hemostat</td>
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		<tr>
			<td>Transpore Surgical Tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
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		<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>
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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 ...

balloon-based-injury-to-induce-myointimal-hyperplasia-mouse-abdominal

Research

JoVE Journal

Medicine

9.5K Views.  University Heart Center. This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

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

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 ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

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 ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

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 ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...