Name: implantation of an isoproterenol mini-pump to induce heart failure in mice
Uploaded: 2019-10-03T13:00:00
Description: Watch this Scientific Journal Video about Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice at JoVE.com

The authors acknowledge NIH K08 HL133491 for funding support.

This protocol adheres to the animal care guidelines of the University of California, Los Angeles (ARC protocol #2010-075). Readers are advised to adhere to their own IACUC-approved protocol, as peri-procedure mouse care and analgesia management may be institution-specific.
1. Preparation of isoproterenol osmotic pump
NOTE: This procedure has been applied successfully on 9+ week-old female mice weighing 18+ g from over 100 inbred mouse strains, as well as, in male mice...

<ol>
	<li>Hartupee, J., Mann, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurohormonal+activation+in+heart+failure+with+reduced+ejection+fraction.">Neurohormonal activation in heart failure with reduced ejection fraction.</a> Nature Review Cardiology. 14 (1), 30-38 (2017).</li><li>Wang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Dissection+of+Cardiac+Remodeling+in+an+Isoproterenol-Induced+Heart+Failure+Mouse+Model.">Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model.</a> PLoS Genetics. 12 (7), 1006038 (2016).</li><li>Balakumar, P., Singh, A. 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S., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+expression+of+green+fluorescence+protein+can+cause+dilated+cardiomyopathy.">Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy.</a> Nature Medicine. 6 (5), 482-483 (2000).</li><li>Breckenridge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+failure+and+mouse+models.">Heart failure and mouse models.</a> Disease Model and Mechanism. 3 (3-4), 138-143 (2010).</li><li>Wittstein, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurohumoral+features+of+myocardial+stunning+due+to+sudden+emotional+stress.">Neurohumoral features of myocardial stunning due to sudden emotional stress.</a> New England Journal of Medicine. 352 (6), 539-548 (2005).</li><li>Templin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Features+and+Outcomes+of+Takotsubo+(Stress)+Cardiomyopathy.">Clinical Features and Outcomes of Takotsubo (Stress) Cardiomyopathy.</a> New England Journal of Medicine. 373 (10), 929-938 (2015).</li><li>Shao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+reveals+an+important+role+for+catecholamine-induced+lipotoxicity+in+the+pathogenesis+of+stress-induced+cardiomyopathy.">A mouse model reveals an important role for catecholamine-induced lipotoxicity in the pathogenesis of stress-induced cardiomyopathy.</a> European Journal of Heart Failure. 15 (1), 9-22 (2013).</li><li>Kudej, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+beta-adrenergic+receptor+stimulation+in+mice.">Effects of chronic beta-adrenergic receptor stimulation in mice.</a> Journal of Molecular Cell Cardiology. 29 (10), 2735-2746 (1997).</li><li>Zhuo, X. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol+instigates+cardiomyocyte+apoptosis+and+heart+failure+via+AMPK+inactivation-mediated+endoplasmic+reticulum+stress.">Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress.</a> Apoptosis. 18 (7), 800-810 (2013).</li><li>El-Demerdash, E., Awad, A. S., Taha, R. M., El-Hady, A. M., Sayed-Ahmed, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probucol+attenuates+oxidative+stress+and+energy+decline+in+isoproterenol-induced+heart+failure+in+rat.">Probucol attenuates oxidative stress and energy decline in isoproterenol-induced heart failure in rat.</a> Pharmacology Research. 51 (4), 311-318 (2005).</li><li>Rau, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+genetic+contributions+to+cardiac+pathology+induced+by+Beta-adrenergic+stimulation+in+mice.">Mapping genetic contributions to cardiac pathology induced by Beta-adrenergic stimulation in mice.</a> Circulation Cardiovascular Genetics. 8 (1), 40-49 (2015).</li><li>Berthonneche, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+response+to+beta-adrenergic+blockade+or+activation+in+23+inbred+mouse+strains.">Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.</a> PLoS One. 4 (8), 6610 (2009).</li><li>Chang, S. C., Ren, S., Rau, C. D., Wang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol-Induced+Heart+Failure+Mouse+Model+Using+Osmotic+Pump+Implantation.">Isoproterenol-Induced Heart Failure Mouse Model Using Osmotic Pump Implantation.</a> Methods Molecular Biology. 1816, 207-220 (2018).</li><li>Shusterman, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+patterns+of+autonomic+nervous+system+activity+and+heart+failure+susceptibility+in+mice.">Strain-specific patterns of autonomic nervous system activity and heart failure susceptibility in mice.</a> American Journal of Physiology-Heart Circulation Physiology. 282 (6), 2076-2083 (2002).</li><li>Faulx, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+beta-adrenergic+receptor+function+influences+myocardial+responses+to+isoproterenol+stimulation+in+mice.">Strain-dependent beta-adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice.</a> American Journal of Physiology-Heart Circulation Physiology. 289 (1), 30-36 (2005).</li><li>Zhang, G. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+oxidative+stress+in+acute+and+chronic+isoproterenol-infused+rats.">Cardiac oxidative stress in acute and chronic isoproterenol-infused rats.</a> Cardiovascular Research. 65 (1), 230-238 (2005).</li><li>Boluyt, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol+infusion+induces+alterations+in+expression+of+hypertrophy-associated+genes+in+rat+heart.">Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart.</a> American Journal of Physiology. 269 (2), 638-647 (1995).</li><li>Hohimer, A. R., Davis, L. E., Hatton, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+daily+injections+and+osmotic+pump+infusion+of+isoproterenol+cause+similar+increases+in+cardiac+mass+but+have+different+effects+on+blood+pressure.">Repeated daily injections and osmotic pump infusion of isoproterenol cause similar increases in cardiac mass but have different effects on blood pressure.</a> Canadian Journal of Physiology and Pharmacology. 83 (2), 191-197 (2005).</li><li>Cross, H. R., Murphy, E., Koch, W. J., Steenbergen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+mice+overexpressing+the+beta(2)-adrenergic+receptor+exhibit+differences+in+ischemia/reperfusion+injury:+role+of+nitric+oxide.">Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide.</a> Cardiovascular Research. 53 (2), 662-671 (2002).</li><li>Klingman, G. I., McKay, G., Ward, A., Morse, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+isoproterenol+treatment+of+mice:+effects+on+catecholamines+and+rectal+temperature.">Chronic isoproterenol treatment of mice: effects on catecholamines and rectal temperature.</a> Journal of Pharmaceutical Sciences. 62 (5), 798-801 (1973).</li></ol>

We have applied this method to over 100+ strains of inbred mice to assess cardiac outcomes due to chronic beta-adrenergic stimulation2,13. Significant differences in susceptibility to isoproterenol are known to exist among mouse strains and may be customized to the strain of interest as needed16. This may be due to the variation in beta-adrenergic receptor function between mouse strains17, strain-dependent differenc...

Heart failure with reduced ejection fraction (HFrEF) is accompanied by a well-recognized compensatory response of the sympathetic nervous system to maintain cardiovascular homeostasis1. Hemodynamic stress and deleterious effects on the heart and circulation were observed with chronic activation. These have become the cornerstone of contemporary pharmacotherapy for heart failure and are important mechanisms in the progression of heart failure and therapeutic antagonism of neurohormonal systems1.
Several mouse models are available for basic investigations of heart failure. Genetic models are attractive for exploring molecular therapies and investigating signaling pathways. However, these models may not be relevant to common forms of heart failure. Other common models include left anterior descending (LAD) artery ligation, transaortic constriction (TAC), and isoproterenol (ISO), each aiming at a different pathological etiology2,3,4,5,6. LAD artery ligation induces an anterior wall myocardial infarction thus creating a model specific for ischemic cardiomyopathy. TAC induces acute pressure overload to create a hypertensive model of heart failure. Although the pressure gradient can be measured, allowing for the stratification of hypertrophy, the acute onset of hypertension lacks direct clinical relevance4. Both LAD and TAC models require a high level of surgical expertise to execute. Acute ISO model of heart failure mimics stress-induced cardiomyopathy, also known as Takotsubo disease, which is characterized by a marked increase in catecholamines and activity in the left ventricle that mimics acute myocardial infarction7,8. In contrast, chronic ISO models of heart failure present symptoms characteristics of advanced heart failure, with chronically elevated levels of catecholamines1. The advantages of the chronic ISO model are that it provides chronic adrenergic stimulation that mimics advanced heart failure and that it is relatively easy to create. The investigator should choose a model that best recapitulates their pathology of interest.
The overall goal of this method is to induce heart failure in mice using an implanted mini pump that releases ISO continuously to mimic chronic sympathetic activation found in heart failure patients1. The method is simple and reproducible. Although there is clear variation among mouse strains, ISO administered over 3 to 4 weeks at 30 mg/kg/day is sufficient to induce cardiac remodeling in most mice. Specifically, ISO leads to a pro-hypertrophic compensatory phase during week 1 followed by wall thinning, ventricular dilation and decrease in systolic function by week 2 and 32. Success for the ISO model creation can be assessed in vivo by serial echocardiography demonstrating hypertrophy, dilation, and ventricular dysfunction, as well as ex vivo via histological and molecular assessment of harvested cardiac tissue for intramyocardial lipid accumulation, fibrosis, ER stress, apoptosis, and gene expression9,10,11,12.

<table>
	<tbody>
		<tr>
			<td>Micro-Osmotic Pump System with Flow Moderator in Place</td>
			<td>Alzet</td>
			<td>Model 1004</td>
			<td>Includes filling tube, flow moderator and pump body</td>
		</tr>
		<tr>
			<td>(-)-Isoproterenol hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>16504-1G</td>
			<td>(-)-Isoproterenol hydrochloride is a powder that needs to be stored at -20&#176;C.</td>
		</tr>
		<tr>
			<td>1 ml sterile syringe</td>
			<td>VWR</td>
			<td>BD309602</td>
			<td></td>
		</tr>
		<tr>
			<td>30 W LED Fiber optic O-ring light microscope illuminator</td>
			<td>AmScope</td>
			<td>SKU: LED-30WR</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 COATED VICRYL (polyglactin 910) Suture</td>
			<td>Ethicon</td>
			<td>J303H</td>
			<td>5-0, absorbable</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>FST</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>FST</td>
			<td>18000-46</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizers</td>
			<td>FST</td>
			<td>18000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris forceps</td>
			<td>WPI</td>
			<td>15915</td>
			<td></td>
		</tr>
		<tr>
			<td>Look Sharpoint 6-0, 18&#34; Black Nylon Monofilament Suture</td>
			<td>LOOK</td>
			<td>AA-2176</td>
			<td>6-0, non-absorbable</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>WPI</td>
			<td>15926</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline, 0.9% NaCl</td>
			<td>Fisher</td>
			<td>89167-772</td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation of an isoproterenol mini-pump to induce heart failure in mice

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics stress-induced cardiomyopathy, the chronic model, administered through an osmotic pump, mimics advanced heart failure in humans. The purpose of the described protocol is to create the chronic ISO-induced heart failure model in mice using an implanted mini-pump. This protocol has been used to induce heart failure in 100+ strains of inbred mice. Techniques on surgical pump implantation are described in detail and may be relevant to anyone interested in creating a heart failure model in mice. In addition, the weekly cardiac remodeling changes based on echocardiographic parameters for each strain and expected time to model development are presented. In summary, the method is simple and reproducible. Continuous ISO administered via the implanted mini-pump over 3 to 4 weeks is sufficient to induce cardiac remodeling. Finally, the success for ISO model creation may be assessed in vivo by serial echocardiography demonstrating hypertrophy, ventricular dilation, and dysfunction.

In our previously published study, we administered an ISO dosage of 30 mg/kg/d over 21 days via the osmotic pump across 105 Hybrid Mouse Diversity Panel (HMDP) strains2,13. We assessed outcomes using echocardiogram performed at baseline, week 1, 2 and 3 of ISO treatment (Figure 1). Similar to a prior study where the authors studied the impact of isoproterenol among 23 mouse strains14, we observed striking inte...

Watch this Scientific Journal Video about Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice at JoVE.com

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics ...

The chronic administration of isoproterenol via an osmotic pump has been widely used to recapitulate changes in advanced heart failure in mice. This method is simple and requires less surgical expertise than other models, such as LAD and TAC. Demonstrating the procedure will be Dr.Vincent Ren, a research scientist from Dr.Yibin Wang's laboratory.After weighing and recording the body weight of each mouse, calculate the appropriate amount and concentration of isoproterenol for each animal, and dissolve the isoproterenol in 120 microliters of sterile 0.9%sodium chloride. Mix thoroughly for one minute to completely solubilize the isoproterenol, and weigh and record the weight of one empty osmotic pump, together with and without its flow moderator, per mouse. Attach a 27-gauge, blunt-tipped filling tube provided with the osmotic pumps, and load one, one-milliliter syringe with 120 microliters of the isoproterenol solution per mouse.Holding a pump in the upright position, insert the filling tube through the pump opening at the top until the tip of the filling tube sits near the bottom of the pump reservoir, and slowly depress the syringe plunger until the isoproterenol solution fills to the pump opening. When the pump has been loaded, carefully remove the filling tube, and wipe off the excess solution. Insert back the flow moderator to close the pump, and wipe off any excess solution.Then reweigh the pump to confirm that over 90%of the reservoir volume has been filled. Place the mouse in the supine position on a heated pad, and remove the air from the lower abdomen. For surgical osmotic pump implantation, confirm a lack of response to toe pinch in an anesthetized mouse, and apply ointment to the animal's eyes.Clean the exposed skin with a suitable disinfectant, and make a one-centimeter midline skin incision. Use a pair of blunt-ended scissors to carefully dissect the skin from the underlying peritoneal walls, and use forceps to pull the peritoneal walls away from the underlying bowel. Use fine scissors to cut a 0.8-centimeter hole in the peritoneal wall, and insert the osmotic pump flow moderator end first into the peritoneal cavity.When the pump is in place, use 5-0 absorbable sutures in an interrupted fashion to close the hole in the peritoneal wall, and use 6-0 nonabsorbable sutures to close the skin. Then place the mouse in a dedicated incubator with monitoring until full recumbency before returning the animal to its home cage. Here, a summary of the weekly changes based on the selected echocardiographic parameters and the expected time to model development are presented.Although variations are observed between mouse strains, on average, interventricular septal wall thickness at end diastole and fractional shortening increase in the first week but decrease by later time points. The left ventricular internal diameter at end diastole and the left ventricular mass increase over a period of three weeks. Further, mouse hearts demonstrate visibly larger left ventricular chamber dimensions under isoproterenol, as well as greater amounts of fibrosis relative to saline-treated controls.It is critical to maintain a sterile surgical environment during the survival surgery to minimize post-operative infection and morbidity. Successful model creation can be assessed in vivo by serial echocardiography for hypertrophy, dilation, and dysfunction, as well as ex vivo via histological and molecular assessment of harvested cardiac tissue. We have applied this method to over 100 strains of inbred mice to assess cardiac remodeling traits due to chronic beta-adrenergic stimulation.The results are published and readily available.

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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 ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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 ...

Implantation of the Syncardia Total Artificial Heart

With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of such patients are generally well served by left ventricular assist devices (LVADs). However, a subset of patients with late stage biventricular failure or other significant anatomic lesions are not adequately treated by isolated left ventricular mechanical support. Examples of concomitant cardiac pathology that may be better treated by resection and TAH replacement includes: post infarction ventricular septal defect,&#160;aortic root aneurysm / dissection, cardiac allograft failure, massive ventricular thrombus, refractory malignant arrhythmias (independent of filling pressures), hypertrophic / restrictive cardiomyopathy, and complex congenital heart disease. Patients often present with cardiogenic shock and multi system organ dysfunction. Excision of both ventricles and orthotopic replacement with a total artificial heart (TAH) is an effective, albeit extreme, therapy for rapid restoration of blood flow and resuscitation. Perioperative management is focused on end organ resuscitation and physical rehabilitation. In addition to the usual concerns of infection, bleeding, and thromboembolism common to all mechanically supported patients, TAH patients face unique risks with regard to renal failure and anemia. Supplementation of the abrupt decrease in brain natriuretic peptide following ventriculectomy appears to have protective renal effects. Anemia following TAH implantation can be profound and persistent. Nonetheless, the anemia is generally well tolerated and transfusion are limited to avoid HLA sensitization. Until recently, TAH patients were confined as inpatients tethered to a 500 lb pneumatic console driver. Recent introduction of a backpack sized portable driver (currently under clinical trial) has enabled patients to be discharged home and even return to work. Despite the profound presentation of these sick patients, there is a 79-87% success in bridge to transplantation.

The purpose of this manuscript is to briefly review indications, management, and outcomes for the total artificial heart. Video operative techniques for device implantation are presented.

With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of ...

Implantation of Total Artificial Heart in Congenital Heart Disease

In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital heart disease (CHD), the malformed heart presents a challenge to TAH implantation. 
In the case presented here, a 17 year-old patient with congenital transposition of the great arteries (CCTGA) experienced progressively worsening HF due to his congenital condition. He was hospitalized multiple times and received an implantable cardioverter defibrillator (ICD). However, his condition soon deteriorated to end-stage HF with multisystem organ failure. 
Due to the patient's grave clinical condition and the presence of complex cardiac lesions, the decision was made to proceed with a TAH. The abnormal arrangement of the patient's ventricles and great arteries required modifications to the TAH during implantation.
With the TAH in place, the patient was able to return home and regain strength and physical well-being while awaiting a donor heart. He was successfully bridged to heart transplantation 5 months after receiving the device. This report highlights the TAH is feasible even in patients with structurally abnormal hearts, with technical modification.

This is a case report of a patient with congenitally corrected transposition of the great arteries (CCTGA) who received a total artificial heart (TAH) as a bridge to heart transplant. The TAH was successfully implanted with modifications to accommodate the patient's congenitally malformed heart.

In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

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 ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...