This work is supported by the Goethe University Hospital Frankfurt and a grant of the German Research Foundation to U.C.

1. Intravenous injection of Ad-2D6 into ophthalmic sinus
<ol>
	<li>Under sterile conditions, dilute the Ad-2D6 virus stock solution to a concentration of 5 x 109 pfu/ml in RPMI and keep the Ad-2D6 virus solution on ice. Injection of two doses of 5 x 108 pfu (intravenous and intraperitoneal) has proven to result in the most reliable outcome of autoimmune hepatitis in mice of the FVB strain.</li>
	<li>Anesthetize mouse with 4% isoflurane in an anesthesia chamber. Pinch the hind leg to make sure the animal is anesthetized.</li>
	<li>Hold the mouse by the back of the neck and tighten the loose skin of the head with thumb and middle finger. Like this, the eye protrudes slightly by the traction to the skin adjacent to the eye.</li>
	<li>Place the needle vertically into the medial canthus (junction of eyelids closest to the animal's nose) and slowly inject 100 &mu;l Ad-2D6 virus solution. It is important not to apply too much pressure, to avoid damage of the vessels and trachea.</li>
	<li>Slowly withdraw the needle to avoid spillage and close the eyelids.</li>
	<li>Injection worked well if the virus solution does not leak out trough the nose and the eye slides back to its initial position.</li>
	<li>Immediately following the intravenous injection, perform a standard intraperitoneal injection of the second 100 &mu;l of the Ad-2D6 virus solution.</li>
</ol>
Alternatively, the intravenous injection can be executed via the tail vein. However, injection via the ophthalmic sinus is much easier to perform and minimizes the loss of virus solution. It has been demonstrated that these two techniques can be used interchangeably and that both routes are equally effective 10.
Infected mice are monitored for adverse effects and obvious signs of suffering and pain, such as loss of appetite, loss of weight, loss of mobility, failure to groom, rough hair coat, hunched appearance, as well as licking, biting, scratching, or shaking a particular area (i.e. site of injection). Although the mice display a massive damage to the liver in the CYP2D6 model, the mice seem healthy and show no signs of suffering, pain or stress. Nevertheless, indicators of severe liver failure, such as serum aminotransferases are determined on a regular basis or are part of the performed experiments. Serum aminotransferase levels of &gt;1000 U/l should only appear as a direct result of the virus infection, but not chronically. If the serum aminotransferase levels are chronically above 1000 U/l (severity limit) the mice are sacrificed. 
2. Mouse eye bleeding
<ol>
	<li>Anesthetize mouse with 4% isoflurane in an anesthesia chamber. Pinch the hind leg to make sure the animal is anesthetized.</li>
	<li>Hold the mouse by the back of the neck and tighten the loose skin of the head with thumb and middle finger. Like this, the eye protrudes slightly by the traction to the skin adjacent to the eye.</li>
	<li>Place the tip of the capillary tube at the lower or inner corner of the eye and gently but firmly slid alongside the eyeball to the ophthalmic venous sinus. The venous capillaries rupture and the resulting hemorrhage fills the orbital cavity.</li>
	<li>Slightly withdraw the capillary tube to free the tip so that the accumulated blood is drawn into the tube.</li>
	<li>When enough blood is collected, the tube is withdrawn and the eyelids closed. Bleeding stops upon withdrawal of the tube and reestablishment of normal ocular pressure upon the venous complex.</li>
	<li>Blood in the capillary tube is transferred to a Microtainer tube and incubated for 30 min on ice.</li>
	<li>The Microtainer tube is centrifuged at 7500 x g for 2 min at 4 &deg;C.</li>
	<li>The supernatant corresponds to the serum which is transferred to a fresh tube and stored at -80 &deg;C.</li>
	<li>Serum can be used to determine antibody titer by ELISA. Indicators of liver damage such as the serum levels of the hepatic enzymes alanine aminotransferase (ALT/GPT) and aspartate aminotransferase (AST/GOT) can be assessed using the according test strips from Roche Diagnostics (Mannheim, Germany) and a Reflotron Plus analyzer (Roche Diagnostics, Mannheim, Germany).</li>
</ol>
Alternatively, blood sampling can be done via the tail vein or via the superficial temporal vein11.
3. Mouse liver perfusion
Remark: This step is important to avoid a contamination of isolated liver lymphocytes by blood lymphocytes
<ol>
	<li>Euthanize the mouse by lethal doses of CO2 followed by cervical dislocation.</li>
	<li>Mount the animal laying on its back on the styrofoam board. Use 23 G needles to pin extremities.</li>
	<li>Moisturize and disinfect the mouse with 70 % EtOH</li>
	<li>About 1 cm away from hind legs, make an incision through the skin and the muscle layer. Cut on both sides of the animal working up to the diaphragm.</li>
	<li>When the diaphragm is reached the skin can be flipped backwards.</li>
	<li>Cut the diaphragm away from the rips then cut the vena cava.</li>
	<li>Move the stomach and the intestines off to the side, exposing the portal vein.</li>
	<li>In the portal vein insert a 27 G needle connected to a 5 ml syringe filled with cold PBS. Let the PBS slowly wash the blood out of the liver.</li>
	<li>Dissect the liver by grabbing onto the diaphragm and cutting the diaphragm away from the body.</li>
	<li>Transfer the liver to a petri dish and remove the diaphragm, the gallbladder and any other tissue still connected to the liver</li>
	<li>Transfer the liver to 10 ml PBS in a 50 ml tube on ice or embed in OCT compound.</li>
</ol>
4. Isolation of liver lymphocytes
<ol>
	<li>Prepare the following stock solutions:
	<ul>
		<li>Collagenase IV stock (100 mg/ml) in PBS. (Make small aliquots and store at -20 &deg;C).</li>
		<li>DNase stock (25 mg/ml) in PBS. (Make small aliquots and store at -20 &deg;C).</li>
		<li>Collagenase buffer: PBS containing 0.2 mg/ml collagenase IV, 0.02 mg/ml DNase and 5% FCS; for 1 liver, mix 9.5 ml ice-cold PBS, 20 &mu;l collagenase IV (100 mg/ml stock), 8 &mu;l DNase (25 mg/ml stock), and 500 &mu;l heat-inactivated FCS.</li>
		<li>35 % Percoll; mix 175 ml Percoll, 20 ml 10 x PBS stock; 305 ml PBS.</li>
		<li>RPMIcomplete = RPMI containing 10% heat-inactivated FCS, 100 &mu;/ml penicillin, 100 &mu;g/ml streptomycin, 2 mM L-glutamine.</li>
	</ul>
	</li>
	<li>Transfer the perfused liver (see section 3) to 10 ml of fresh PBS in a petri dish on ice and cut into small pieces using scissors.</li>
	<li>Transfer into a 70 &mu;m cell strainer and squeeze liver junks through with a glass pestle or a pestle from a 2 ml syringe. Carefully add 10 ml cold collagenase buffer and press through strainer. Collect suspension and filter through strainer two times more.</li>
	<li>Transfer suspension to fresh 50 ml tube on ice. Process next liver.</li>
	<li>Incubate liver cell suspension at 37 &deg;C for 60 min, mix gently every 15 min.</li>
	<li>Centrifuge at 30 x g for 3 min at 4 &deg;C.</li>
	<li>Transfer supernatant to a fresh tube, leaving 5 mm fluid above pellet</li>
	<li>Centrifuge at 650 x g for 10 min at 4 &deg;C.</li>
	<li>Discard supernatant, leaving 3 mm above pellet</li>
	<li>Resuspend pellet in 20 ml Percoll buffer</li>
	<li>Centrifuge at 600 x g for 20 min at 4 &deg;C.</li>
	<li>Discard supernatant and resuspend pellet by flicking the tube.</li>
	<li>Wash pellet 1 x with PBS.</li>
	<li>Wash pellet 1 x with RPMIcomplete</li>
	<li>Resuspend pellet in 3 ml RPMIcomplete and count cells in a 1:10 dilution.</li>
	<li>Resuspend cells at ~107 cells/ml in RPMIcomplete and transfer tube to ice.</li>
</ol>
5. Intracellular cytokine staining (ICCS)
5.1 Stimulation:
<ol>
	<li>Prepare liver lymphocytes at ~107 cells/ml in RPMIcomplete as described. Plate 100 &mu;l (106 cells)/well into a flat 96-well plate which is not tissue-culture treated.</li>
	<li>Add 50&mu;l RPMIcomplete containing 2&mu;g/ml Brefeldin A and then add 50&mu;l RPMIcomplete containing 2 &mu;g/ml stimulating CYP2D6 peptide (i.e. the immunodominant CD4 epitope CYP2D641-60 PGLGNLLHVDFQNTPYCFDQ12 or the immunodominant CD8 epitope CYP2D6193-212 RRFEYDDPRFLRLLDLAQEG12). Mix by pipetting.</li>
	<li>Incubate for 5 hrs at 37&deg;C (optimal stimulation time but overnight incubation works as well).</li>
</ol>
5.2. Staining:
<ol>
	<li>Prepare the following stock solutions:
	<ul type="disc">
		<li>FACS buffer: PBS containing 1% fetal calf serum (FCS).</li>
		<li>Fixation/Permeabilization buffer: FACS buffer containing 0.1% saponin and 4% paraformaldehyde</li>
		<li>FACS/saponin wash buffer: FACS buffer containing 0.1% saponin</li>
		<li>FACS/PFA buffer: FACS buffer containing 1% paraformaldehyde (PFA)</li>
	</ul>
	</li>
	<li>Transfer cells into V-bottom microtiter plate (96-well) and spin at 460 x g for 3 min at 4 &deg;C. Discard medium and vortex plate</li>
	<li>Add 150 &mu;l FACS buffer and centrifuge at 460 x g for 3 min at 4 &deg;C. Discard medium and vortex plate. Repeat wash step.</li>
	<li>Block surface FcR if necessary (when using secondary antibodies) with 1 &mu;g/ml &alpha;CD16/32 cocktail (FcR block) in FACS buffer for 15 min at 4 &deg;C and wash 2 x with 150 &mu;l staining buffer (460 x g for 2 min at 4 &deg;C).</li>
	<li>Stain for surface molecules: i.e. Anti-CD8a-FITC mAb 10 &mu;g/ml in 50 &mu;l FACS buffer for 30 min at 4 &deg;C in the dark.</li>
	<li>Add 100 &mu;l FACS buffer and spin at 460 x g for 3 min at 4 &deg;C.</li>
	<li>Wash cells 2 x with 150 &mu;l FACS buffer (460 x g; 3 min; 4 &deg;C). Vortex plate.</li>
	<li>Fix/permeabilize cells with 100 &mu;l fixation/permeabilization buffer for 10 min at RT.</li>
	<li>Spin at 460 x g for 7 min at 4 &deg;C. Note that it is important to extend the centrifugation time to 7 min, since the fixation/permeabilzation steps changes the overall density of the cells. Vortex plate.</li>
	<li>Wash 2 x with 150 &mu;l FACS/saponin wash buffer (460 x g; 7 min; 4 &deg;C). Vortex plate.</li>
	<li>Stain for intracellular molecules: i.e. Anti-IFN&gamma;-PE mAb 10 &mu;g/ml in 50 &mu;l FACS/saponin wash buffer for 30 min at 4 &deg;C.</li>
	<li>Add 100 &mu;l FACS/saponin wash buffer and spin at 460 x g for 7 min at 4 &deg;C.</li>
	<li>Wash cells 2 x with 150 &mu;l FACS/saponin wash buffer (460 x g; 7 min; 4 &deg;C). Vortex plate.</li>
	<li>Wash cells 1 x with FACS buffer (460 x g; 7 min; 4 &deg;C). Vortex plate.</li>
	<li>Resuspend cells in 200 &mu;l FACS/PFA buffer, transfer into tubes, and store at 4 &deg;C in the dark until acquisition of data by flow cytometry. We normally use a FACSCanto II or a FACSCalibur (BD Biosciences, Heidelberg, Germany).</li>
</ol>
6. Immunohistochemistry
6.1. H&amp;E staining for the assessment of general liver pathology:
<ol>
	<li>Harvest liver, immerse in Tissue-Tek O.C.T. in a disposable base mold and quick-freeze on dry ice.</li>
	<li>Cut 7 &mu;m liver tissue sections using a Leica cryostat set at -17 &deg;C and mount the tissue sections on Superfrost Plus microscope slides. Store slides at -20 &deg;C until further use.</li>
	<li>Fix cryosections in pre-cooled EtOH for 15 min at -20 &deg;C. Let dry for 10 min.</li>
	<li>Wash 2 x in distilled water for 2 min at room temperature (the following steps, 6.1.5 - 6.1.13, are performed at room temperature).</li>
	<li>Stain in Meyer's Hematoxylin solution for 8 min.</li>
	<li>Wash in warm (30 &deg;C) tap water for 10 min. Change water several times.</li>
	<li>Rinse in distilled water.</li>
	<li>Rinse in 95% EtOH for 20 sec.</li>
	<li>Counterstain in Eosin G/Y solution for 40 sec.</li>
	<li>Dehydrate 2 x in 95% EtOH for 5 min per incubation.</li>
	<li>Dehydrate 2 x in 100% EtOH for 5 min per incubation.</li>
	<li>Clear in xylene 2 x for 5 min per clearing.</li>
	<li>Mount in Roti-Histokitt.</li>
</ol>
6.2. Immunohistochemistry for collagen I deposition:
<ol>
	<li>Harvest liver, immerse in Tissue-Tek OCT. in a disposable base mold and quick-freeze on dry ice.</li>
	<li>Cut 7 &mu;m liver tissue sections using a Leica cryostat set at -17 &deg;C and mount the tissue sections on Superfrost Plus microscope slides. Store slides at -20 &deg;C until further use.</li>
	<li>Fix cryosections in cold EtOH for 15 min at -20 &deg;C. Let dry for 10 min.</li>
	<li>Wash 2 x in PBS for 2 min at room temperature.</li>
	<li>Incubate in PBS containing 0.3% H2O2 and 0.1% Na-azide for 10 min at room temperature.</li>
	<li>Wash 2 x in PBS for 2 min at room temperature.</li>
	<li>Block with an avidin-biotin blocking kit according the manufacturer's guidelines.</li>
	<li>Block with 10 % FCS in PBS (FCS/PBS) for 30 min at room temperature.</li>
	<li>The primary antibody is a rabbit anti-mouse collagen I antibody which is diluted 1:200 in 10 % FCS/PBS. Incubate sections with primary antibody for 2 hrs at room temperature.</li>
	<li>Wash sections 3 x in PBS for 4 min at room temperature.</li>
	<li>Dilute biotinylated anti-rabbit antibody to 1:500 and incubate with the sections for 1.5 hr at room temperature.</li>
	<li>Wash sections 3 x in PBS for 4 min at room temperature.</li>
	<li>Color reaction is obtained by sequential incubation with avidin-peroxidase conjugate and diaminobenzidine-hydrogen peroxide according the manufacturer's guidelines.</li>
	<li>Counterstain in Meyer's Hematoxylin solution for 5 min, wash 2 x in PBS for 2 min at room temperature and mount in Aquatex.</li>
</ol>
7. Representative Results
Infection of mice with Ad-2D6 induced two distinct stages of liver damage. In the first days after infection, virus infection of the liver causes an acute increase of serum aminotransferase levels, an indicator of hepatocyte death6. This first, acute stage occurs independent of the expression of hCYP2D6 and can also be observed after infection with an empty control virus (Ad-Control) or a virus expressing green fluorescence protein (Ad-GFP). In contrast, the second stage is autoimmune-mediated and dependent on the expression of the triggering molecule hCYP2D6. This autoimmune stage occurs within 2-4 weeks post-infection and persists for several months6.
 
<img alt="Figure 1" src="/files/ftp_upload/3644/3644fig1.jpg" />
Figure 1. The CYP2D6 model. Wildtype FVB or C57BL/6 mice are injected with two doses of Ad-2D6 (intravenous and intraperitoneal). At a time of interest the liver and the serum are collected and assessed for liver damage and formation of hCYP2D6-specific antibodies and T cells.
The following features are characteristic for the persistent autoimmune hepatitis developing after Ad-2D6-infection of wildtype FVB or C57BL/6 mice in the CYP2D6 model: First, the liver shows an aberrantly changed morphology. In particular, the liver lobes are fused, hyperplasic nodules appear scattered over the entire liver and a massive capsular fibrosis is visible (figure 2).
 
<img alt="Figure 2" src="/files/ftp_upload/3644/3644fig2.jpg" />
Figure 2. Liver morphology. Typical Ad-2D6-induced liver damage as detected at week 4 post-infection. Note that the individual liver lobes are fused (arrowheads). Hyperplasic nodules appear scattered over the entire liver (arrows). Infection with a control adenovirus not expressing hCYP2D6 has no effect on the liver morphology.
Second, extensive and persistent cellular infiltrations appear in the peri-portal and parenchymal regions of the livers of Ad-2D6-infected but not Ad-Control-infected mice (figure 3A). Fibrosis predominantly develops in the subcapsular region with some collagen bundles protruding into the parenchyma (figure 3B).
 
<img alt="Figure 3" src="/files/ftp_upload/3644/3644fig3.jpg" /> 
Figure 3. Liver histology. A: H&amp;E staining of liver tissue section of FVB mice infected with either Ad-Control or Ad-2D6 at week 4 post-infection. Note that significant infiltrations of mononuclear cells are only present in Ad-2D6 infected mice (single arrows). Triple arrows indicate larger cellular infiltrations in the liver parenchyma bridging between neighboring portal tracts. B: Immunohistochemical representation of liver fibrosis at week 4 after infection with Ad-2D6 or Ad-control. The liver sections have been stained for collagen using an anti-collagen I antibody. Note multiple layer of collagen disposition under the liver capsule (triple arrows) with some bundles protruding into the parenchyma (single arrows) and the presence of large clusters of infiltrating cells (arrowheads). Two representative liver sections are displayed for each condition. Size bars: 100&mu;m.
A third feature is the generation of high titers of anti-CYP2D6 antibodies, which have a similar epitope specificity to human LKM-1 antibodies 6,13. Last, hCYP2D6-specific CD4 and CD8 T cells are generated that predominantly home to the liver. Such hCYP2D6-specific T cells can be detected by stimulation with immunodominant hCYP2D6 peptides and subsequent measurement of IFN&gamma; expression by an intracellular cytokine staining (ICCS) (figure 4). hCYP2D6-specific CD8 T cells kill Ad-2D6-infected target cells in vivo 12.
 
<img alt="Figure 4" src="/files/ftp_upload/3644/3644fig4.jpg" />
Figure 4. hCYP2D6-specific T cells. Flow cytometric analysis of hCYP2D6-specific T cells. Liver lymphocytes have been isolated at week 4 after Ad-2D6-infection and were stimulated with either the immunodominant CD4 or CD8 hCYP2D6 epitope overnight. Generation of IFN&gamma; was detected by intracellular cytokine staining and analyzed by flow cytometry using a FACS Canto II (BD Biosciences, Heidelberg, Germany). The percentage of hCYP2D6-specific CD4 and CD8 cells is indicated.

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Immunol. 169, 4889-4896 (2002).</li><li>Christen, U., Hintermann, E., Holdener, M., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+triggers+for+autoimmunity:+is+the+'glass+of+molecular+mimicry'+half+full+or+half+empty.">Viral triggers for autoimmunity: is the 'glass of molecular mimicry' half full or half empty.</a> J. Autoimmun. 34, 38-44 (2010).</li><li>Krebs, P., Scandella, E., Odermatt, B., Ludewig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+exhaustion+and+deletion+of+CTL+following+immunization+with+recombinant+adenovirus.">Rapid functional exhaustion and deletion of CTL following immunization with recombinant adenovirus.</a> J. Immunol. 174, 4559-4566 (2005).</li><li>Hintermann, E., Bayer, M., Pfeilschifter, J., Christen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+delivery+of+the+liver+autoantigen+cytochrome+P450+2D6+chronically+activates+hepatic+stellate+cells+and+induces+fibrosis.">Adenoviral delivery of the liver autoantigen cytochrome P450 2D6 chronically activates hepatic stellate cells and induces fibrosis.</a> Manuscript Submitted. , (2011).</li></ol>

The authors have nothing to disclose.

In previous studies, experimental hepatitis was often reported to be only transient and many current models for autoimmune liver disease depend on a rather complex disease induction protocols (for reviews see 3,5). For example, several models use transgenic mice expressing specific target antigens and adoptively transferred target antigen-specific, mostly TcR-transgenic, T cells14,15. Often an additional infection with livertropic viruses, bacteria or parasites is necessary to induce disease16-18. Alternatively, DNA-vaccination with plasmids encoding for target antigens, including CYP2D619, is used to induce hepatitis. However, an additional vaccination with plasmids encoding for pro-inflammatory cytokines, such as IL-12, is required20. Unfortunately, with a few exceptions15,20 hepatitis is only transient.
The CYP2D6 model6,21 uses a straightforward method to induce autoimmune hepatitis by simply infecting mice with an Adenovirus expressing hCYP2D6, the major autoantigen in AIH-27,8. Delivery of CYP2D6 by an adenovirus construct guarantees both a direct targeting of the liver as well as a local inflammation that promotes the breakdown of tolerance. It is important to note however that both the virus titer as well as the route of administration is crucial to this model. On the one hand, Ad-2D6 is a replication deficient virus, thus, a sufficient high titer of virus is required to generate a critical amount of antigen within the liver. On the other hand, too high a titer might result in a fatal acute liver failure. In addition, it has been demonstrated previously that infection of mice by high titers of adenovirus leads to a functional exhaustion of the immune response22. In our model, we used a combination of intravenous and intraperitoneal infection in order to obtain both chronic cellular infiltration as well as extensive fibrosis. We have observed that intravenous infection alone still causes massive cellular infiltration, but no fibrosis of the subcapsular region, indicating that peritoneal inflammation due to the intraperitoneal injection might be involved in the continuous subcapsular accumulation of collagen (Hintermann &amp; Christen, manuscript in preparation). However, it is important to note that the observed hepatic fibrosis is antigen-specific, since we did not detect activation of hepatic stellate cells and collagen deposition after infection with equal virus-titers of Ad-GFP or Ad-Control23.
The CYP2D6 model reflects many aspects of human AIH1,2, such as chronic hepatitis characterized by persistent cellular infiltration and liver fibrosis6. In addition, the presence of high-titer anti-CYP2D6 antibodies with similar epitope specificity and spreading13 to LKM-1 antibodies found in AIH-2 patients indicates the presence of a common chronic autoimmune reactivity. CYP2D6 is the major antigen in AIH-2, but it is not recognized by patients diagnosed with the more frequent type 1 AIH. In addition, patients suffering from other liver diseases with autoimmune etiology, such as primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC), generate hallmark autoantibodies with specificity to distinct liver autoantigens and their livers show different pathological features centering on small (PBC) or large (PSC) bile ducts. Nevertheless, the CYP2D6 models offers the opportunity to analyze immunopathogenic mechanisms involved in chronic hepatic inflammatory processes as occurring during autoimmune liver diseases, to identify key players that drive the autoimmune destruction of hepatocytes and to evaluate possible therapeutic interventions to cure the disease.

<table border="1">
<tbody>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>23G needle</td>
 <td>BD Biosciences</td>
 <td>300800</td>
 <td></td>
 </tr>
 <tr>
 <td>27&frac12;G needle</td>
 <td>VWR</td>
 <td>612-0151</td>
 <td></td>
 </tr>
 <tr>
 <td>30&frac12; G needle</td>
 <td>BD Biosciences</td>
 <td>304000</td>
 <td></td>
 </tr>
 <tr>
 <td>Alanine aminotransferase test strips (GPT/ALT)</td>
 <td>Roche Diagnostics</td>
 <td>10 745 138 202</td>
 <td></td>
 </tr>
 <tr>
 <td>Aspartate aminotransferase test strips (GOT/AST)</td>
 <td>Roche Diagnostics</td>
 <td>10 745 120 202</td>
 <td></td>
 </tr>
 <tr>
 <td>Anaesthesia Unit Univentor 400</td>
 <td><a href="http://www.agnthos.se" target="_blank" >AgnTho's</a></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Base molds, disposable 37x24x10 mm </td>
 <td>VWR</td>
 <td>720-0208</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell strainer 70mm</td>
 <td>VWR</td>
 <td>734-0003</td>
 <td></td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Leica</td>
 <td>CM1850 UV </td>
 <td></td>
 </tr>
 <tr>
 <td>Heparinized capillary tubes</td>
 <td>Fisher</td>
 <td>3123987</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope slides Superfrost Plus </td>
 <td>Menzel Gl&auml;ser </td>
 <td>J1800AMNZ </td>
 <td></td>
 </tr>
 <tr>
 <td>Microtainer SST tubes</td>
 <td>BD Biosciences</td>
 <td>365951</td>
 <td></td>
 </tr>
 <tr>
 <td>Microtiter plates, V-bottom</td>
 <td>VWR</td>
 <td>391-1924</td>
 <td></td>
 </tr>
 <tr>
 <td>Reflotron Plus</td>
 <td>Roche Diagnostics</td>
 <td></td>
 <td>Determiniation of serum AST / ALT</td>
 </tr>
 <tr>
 <td colspan="4">&nbsp;</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Rabbit anti-mouse anti-Collagen type I IgG,</td>
 <td>Chemicon</td>
 <td>AB765P</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotinylated goat anti-rabbit IgG</td>
 <td>Vector (AXXORA)</td>
 <td>VC-BA-1000-MC15 </td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated anti mouse CD8a antibody</td>
 <td>Southern Biotech</td>
 <td>1550-02</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-conjugated anti mouse IFN&gamma; antibody</td>
 <td>BD Biosciences</td>
 <td>554412</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-conjugated anti-mouse CD16/CD32 antibody (FcR block)</td>
 <td>BD Biosciences</td>
 <td>553141</td>
 <td></td>
 </tr>
 <tr>
 <td>Aquatex </td>
 <td>VWR </td>
 <td>1.08562.0050</td>
 <td></td>
 </tr>
 <tr>
 <td>Avidin/Biotin Blocking kit </td>
 <td>Vector (AXXORA) </td>
 <td>VC-SP-2001-KI01 </td>
 <td></td>
 </tr>
 <tr>
 <td>Brefeldin A</td>
 <td>Sigma</td>
 <td>B6542</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase IV</td>
 <td>Sigma</td>
 <td>C5138-100MG</td>
 <td></td>
 </tr>
 <tr>
 <td>DAB substrate kit 3'3'diaminobenzidine </td>
 <td>Vector (AXXORA) </td>
 <td>VC-SK-4100-KI01 </td>
 <td></td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma</td>
 <td>DN-25</td>
 <td></td>
 </tr>
 <tr>
 <td>Elite ABC Reagent </td>
 <td>Vector (AXXORA) </td>
 <td>VC-PK-7100-L050</td>
 <td></td>
 </tr>
 <tr>
 <td>Eosin G/Y solution </td>
 <td>Roth</td>
 <td>X883.1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum (FCS)</td>
 <td>Biochrom AG</td>
 <td>S 0115</td>
 <td></td>
 </tr>
 <tr>
 <td>Isofluran</td>
 <td>Forene</td>
 <td>B506</td>
 <td></td>
 </tr>
 <tr>
 <td>Meyer's Hematoxylin solution </td>
 <td>AppliChem </td>
 <td>A4840,1000</td>
 <td></td>
 </tr>
 <tr>
 <td>OCT Compound</td>
 <td>Sakura Finetek Europe</td>
 <td>4583</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS-buffered formaldehyde 10% </td>
 <td>Roth </td>
 <td>A146.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Percoll </td>
 <td>Amersham </td>
 <td>17-0891-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Roti-Histokit</td>
 <td>Roth</td>
 <td>6638.1</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Invitrogen</td>
 <td>61870</td>
 <td></td>
 </tr>
 <tr>
 <td>Saponin from quillaja bark </td>
 <td>Sigma</td>
 <td>S7900</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice at JoVE.com

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

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 ...

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18.9K Views.  Goethe University Hospital Frankfurt. 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.

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

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 ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

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.

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 ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...