Murine (and occasionally rat) models exist for numerous human diseases. These models possess clear, accurately measured endpoints that define disease and enable assessment of an intervening drug/therapeutic approach. Most of the models employed for assessing the effect of therapeutic interventions of heart, vascular, lung, bone, and septic disease are well established in the literature and routinely performed by our team. Less common or tailored protocols for specific investigative needs are also available or can be developed with consultation between your team and ours. In addition to in vivo data generated in a specific disease model, relevant complementary data from tissue- and cell- based assays can be generated.
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Models of heart disease include those for Heart Failure, Myocardial Infarction, Diabetic Cardiomyopathy, and Ischemia/Reperfusion. Drugs/therapeutic strategies for treatment or prophylaxis can be tested for efficacy in each of the models, described below. More reductionist and complementary tissue and cell-based assays for each disease are also described in the associated hyperlink.
In vivo murine models of heart failure include the transverse aortic constriction (TAC) model, which is the most widely used heart pressure overload model. TAC causes an approximately 50% increase in left ventricular mass within two weeks, making this model an excellent choice to examine the utility of pharmacological or molecular interventions that may limit or reverse hypertrophy and pathology associated with heart failure (Rockman et al., 1991; Barrick et al., 2007). Experimental outcomes include, but are not limited to: 1) in vivo cardiac functions, including LV end-diastolic and end-systolic dimensions, heart rate, velocity of circumferential shortening ,and percentage of fractional shortening by transthoracic 2-dimensaional guided M-mode echocardiography (Yan et al., 2015) ; 2) heart rate, aortic pressure, LV systolic and diastolic pressure, all assessed by cardiac catheterization; and 3) immunohistochemical analyses of cardiac structural changes, apoptosis, fibrosis, and inflammation. Additional models of heart failure include those for Dilated Cardiomyopathy (Patten et al., 2009), Diabetic Cardiomyopathy and Ischemia/Reperfusion.
Cellular assays useful for therapeutic heart failure drugs include those employing cardiac myocyte cultures to assess drug effects on hypertrophic stimuli-induced myocyte cell size enlargement, sarcomere reorganization, protein synthesis, expression of hypertrophic marker genes, apoptosis, and activation of transcription factors (Chen et al., 2014 and; Yan et al., 2015).
- Rockman HA, Ross RS, Harris AN, Knowlton KU, Stienhelper ME, Field LJ, Ross J, Chien KR. (1991) Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci USA. 88: 8277–8281.
- Barrick CJ, Rojas M, Schoonhoven R, Smyth SS, Threadgill DW. (2007) Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporal- and background-dependent development of concentric left ventricular hypertrophy. Am J Physiol Heart Circ Physiol. 292: H2119–H2130.
- Patten RD, Hall-Porter MR. (2009) Small animal models of heart failure: development of novel therapies, past and present. Circ Heart Failure. 2:138-144.
- Chen M, Yi B, Sun J. (2014) Inhibition of Cardiomyocyte Hypertrophy by Protein Arginine Methyltransferase 5. J Biol Chem. 289(35):24325-35.
- Yan G, Zhu N, Huang S, Yi B, Zhang G, Shang X, Chen M, Wang N, Talarico JA, Tilley DG, Gao E, Sun J.(2015) Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation. Mol Cell Biol. 35(19):3312-23.
In vivo murine models of myocardial infarction (MI) include permanent occlusion of the left main descending coronary artery occlusion to induce a myocardial infarction (Klocke et al., 2007; Gao et al., 2010; Wang et al., 2015). Experimental outcomes include 1) end-diastolic diameter (EDD), end-systolic diameter (ESD), posterior wall thickness (PWT), and septal wall thickness (SWT), ejection fraction (EF), heart rate, and fractional shortening (FS) determined by echocardiography; 2) area at risk (AAR) and infarct size (IS) determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining; 3) cardiac remodeling including ventricular geometry and wall thickness determined by immunostaining. Another useful and related model is temporary coronary artery occlusion to induce myocardial Ischemia/Reperfusion (I/R) injury.
Cellular assays useful for assessing effects of candidate drugs for MI treatment or prophylaxis include those employing cardiac myocyte cultures to assess drug effects on myocyte survival (Sun et al., 2006), contractile and energetic regulation (Beutner et al., 2005 and; Hom et al., 2010). Tissue-based assays utilize the Langendorff model (Ma et al., 1999) of isolated heart preparation to assess drug regulation of contraction, heart rate, and rhythm.
- Klocke R, Tian W, Kuhlmann MT, Nikol S. (2007) Surgical animal models of heart failure related to coronary heart disease. Cardiovasc Res. 74:29–38.
- Erhe Gao, Yong Hong Lei, Xiying Shang, Z. Maggie Huang, Lin Zuo, Matthieu Boucher, Qian Fan, J. Kurt Chuprun, Xin L. Ma, Walter J. Koch. (2010) A Novel and Efficient Model of Coronary Artery Ligation and Myocardial Infarction in the Mouse. Circulation Res. 107: 1445-1453.
- Wang Y, Gao E, Lau WB, Wang Y, Liu G, Li JJ, Wang X, Yuan Y, Koch WJ, Ma XL. (2015) G-protein-coupled receptor kinase 2-mediated desensitization of adiponectin receptor 1 in failing heart. Circulation.131(16):1392-404.
- Sun J, Yan G, Ren A, You B, Liao JK. (2006) FHL2/SLIM3 decreases cardiomyocyte survival by inhibitory interaction with sphingosine kinase-1. Circulation Research, 99(5):468-76.
- Beutner G, Sharma VK, Lin L, Ryu SY, Dirksen RT, Sheu SS. (2005) Type 1 ryanodine receptor in cardiac mitochondria: transducer of excitation-metabolism coupling. Biochim Biophys Acta. 17(1):1-10.
- Hom J, Yu T, Yoon Y, Porter G, Sheu SS. (2010) Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes. Biochim Biophys Acta. 1797(6-7):913-21.
- Ma XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher TA, Wang C, Lee JC, Feuerstein GZ, Yue TL. (1999) Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation. 99(13):1685-91.
In vivo murine models of Ischemia/Reperfusion (I/R) include temporary coronary artery occlusion to induce I/R injury, and is generally used to examine the short-term consequences of ischemic injury (Guo et al., 1998; Klocke et al., 2007; He et al., 2014). Experimental outcomes include in vivo cardiac function analysis by echocardiography and measurement of the area at risk and infarct size by TTC staining, similar to those outcomes assessed for Heart Failure and Myocardial Infarction.
Assays similar to those performed for heart failure (above) can be conducted.
- Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, Bolli R. (1998) Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol. 275:H1375–H1387.
- Klocke R, Tian W, Kuhlmann MT, Nikol S. (2007) Surgical animal models of heart failure related to coronary heart disease. Cardiovasc Res. 74:29–38.
- He Q, Pu J, Yuan A, Lau WB, Gao E, Koch WJ, Ma XL, He B. (2014) Activation of liver-X-receptor α but not liver-X-receptor β protects against myocardial ischemia/reperfusion injury. Circ Heart Fail. 7(6):1032-41.
In vivo murine models of diabetic cardiomyopathy include type 1 and type 2 diabetes mellitus. Type 1 diabetes is induced after administration of the pancreatic beta-cell toxin streptozotocin. This animal model of type-1 diabetes is the most commonly used and is characterized by consistent hyperglycemia, loss of insulin secretion and signaling, but lacks the immunological components of type-1 diabetes (Hsueh et al., 2007). Control animals will receive intraperitoneal injections of citrate buffer alone. Type-2 diabetes will be induced by feeding C57BL/6 mice with hiegh fat diet (HFD; 60% fat) up to 24 weeks. Controls will be fed standard rodent food (chow) for the same duration as the respective HFD group. This animal model is typified by hyperglycemia, hyperinsulinemia, and obesity, and insulin resistance is a cardinal feature (Witteles et al., 2008). Some of these models are summarized in Table 1. Experimental outcomes include alterations in the circulating levels of glucose and in the lipid profile, cardiac structural abnormalities with diastolic dysfunction, cardiac fibrosis, apoptosis, disruption of intracellular Ca2+ transport, structural changes in extracellular matrix (ECM) regulation, production of oxidative stress and an overwhelming cardiac inflammatory response (Tschope et al., 2004; and Westermann et al., 2007).
Cellular assays useful for assessing effects of candidate drugs for diabetic cardiomyopathy treatment or prophylaxis include those employing cardiac myocyte cultures to assess drug effects on myocyte morphological changes, apoptosis, protein synthesis, activation of transcription factors (Rafiq et al., 2012).
- Hsueh W, Abel ED, Breslow JL, Maeda N, Davis RC, Fisher EA, Dansky H, McClain DA, McIndoe R, Wassef MK, Rabadan-Diehl C, Goldberg IJ. (2007) Recipes for creating animal models of diabetic cardiovasc disease. Circ Res. 100:1415-1427.
- Witteles RM, Fowler MB. (2008) Insulin-resistant cardiomyopathy: Clinical evidence, mechanisms, and treatment options. J Am Coll Cardiol. 51:93-102.
- Tschöpe C, Walther T, Königer J, Spillmann F, Westermann D, Escher F, Pauschinger M, Pesquero JB, Bader M, Schultheiss HP, Noutsias M. (2004) Prevention of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy in rats by transgenic expression of the human tissue kallikrein gene. FASEB J. 18:828–835.
- Tschöpe C, Spillmann F, Rehfeld U, Koch M, Westermann D, Altmann C, Dendorfer A, Walther T, Bader M, Paul M, Schultheiss HP, Vetter R. (2004) Improvement of defective sarcoplasmic reticulum Ca2+ transport in diabetic heart of transgenic rats expressing the human kallikrein-1 gene. FASEB J. 18:1967–1969.
- Westermann D, Rutschow S, Jäger S, Linderer A, Anker S, Riad A, Unger T, Schultheiss HP, Pauschinger M, Tschöpe C. (2007) Contributions of inflammation and cardiac matrix metalloproteinase activity to cardiac failure in diabetic cardiomyopathy: the role of angiotensin type 1 receptor antagonism. Diabetes 56:641–646.
- Rafiq K, Guo J, Vlasenko L, Guo X, Kolpakov MA, Sanjay A, Houser SR, Sabri A. (2012) c-Cbl ubiquitin ligase regulates focal adhesion protein turnover and myofibril degeneration induced by neutrophil protease cathepsin G. J. Biol. Chem 287(8):5327-39.
Models of vascular disease include those for Hypertension, Peripheral Ischemia, and Vascular Injury/Remodeling. Drugs/therapeutic strategies for treatment or prophylaxis can be tested for efficacy in each of the models, described below. More reductionist and complementary tissue and cell-based assays for each disease are also described in the associated hyperlink.
In vivo murine models of peripheral ischemia include the hindlimb ischemia model, in which the entire left femoral artery and vein are excised surgically (Brenes et al., 2012). Experimental outcomes include, but are not limited to: Functional scoring using the tarlov scale, ischemia scale, and modified ischemia scale; Doppler measurement of blood flow; and histological analysis of fiber area and numbers and capillary density (Most et al., 2013). With the use of laser Doppler imaging analysis, this is an excellent system for studying postnatal arteriogenesis and angiogenesis and effects of therapeutic candidates (Limbourg et al., 2009).
Cellular assays useful for assessing effects of candidate drugs for treatment or prophylaxis of peripheral ischemia include those employing endothelial cell cultures to assess drug effects on endothelial cell proliferation, migration, Matrigel tube formation, and ex vivo aortic ring sprouting assays (Sun et al., 2004; and Chen et al., 2016).
- Most P, Lerchenmüller C, Rengo G, Mahlmann A, Ritterhoff J, Rohde D, Goodman C, Busch CJ, Laube F, Heissenberg J, Pleger ST, Weiss N, Katus HA, Koch WJ, Peppel K. (2013) S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation. Circ Res. 112(1):66-78.
- Brenes RA, Jadlowiec CC, Bear M, Hashim P, Protack CD, Li X, Lv W, Collins MJ, Dardik A. (2012) Toward a mouse model of hind limb ischemia to test therapeutic angiogenesis. J Vasc Surg. 56(6):1669-79.
- Limbourg A, Korff T, Napp LC, Schaper W, Drexler H, Limbourg FP. (2009) Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat Protoc. 4(12):1737-46.
- Sun J, Liao JK. (2004) Induction of angiogenesis by heat shock protein 90 mediated by protein kinase Akt and endothelial nitric oxide synthase. Arterioscler Thromb Vasc Biol. 24(12): 2238-44.
- Chen M, Yi B, Zhu N, Wei X, Zhang GX, Huang S, Sun J. (2016) Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633. Cardiovasc Res. 109(1):141-50.
In vivo rodent models of vascular injury/remodeling include the rat carotid artery balloon injury model, a well-established model for studying mechanisms involved and therapies affecting vascular smooth muscle dedifferentiation, neointima formation, and vascular remodeling. This model is also useful for the study of vascular injury-induced restenosis (Lamfers et al., 2001). Experimental outcomes include, but are not limited to: neointimal area and neointima/media (N/M) ratio and in vivo smooth muscle cell proliferation and migration (Huo et al., 2014). Specific atherosclerosis models including the ApoE−/− model and the LDLR−/− model fed with a chow or high fat diet. Experimental outcomes in this model include aortic root lesion size, en face staining, plasma lipid levels, plasma cytokine levels, and macrophage infiltration (Hartmann et al., 2016; and Fang et al., 2014).
Cellular assays useful for assessing effects of candidate drugs for treatment or prophylaxis of Vascular Injury/Atherosclerosis/Remodeling include those cell-based assays of inflammatory cytokine-induced endothelial cell activation, adhesion of monocytes to endothelial cells, and FITC-dextran-based permeability assays (You at al., 2009); aortic ring contraction and relaxation assays (Yi et al., 2015); and smooth muscle cell proliferation and migration assays (Sun et al., 2002; and Li et al., 2013).
- Lamfers ML, Lardenoye JH, de Vries MR, Aalders MC, Engelse MA, Grimbergen JM, van Hinsbergh VW, Quax PH. (2001) In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI. Gene Ther. 8(7):534-41.
- Hartmann P, Zhou Z, Natarelli L, Wei Y, Nazari-Jahantigh M, Zhu M, Grommes J, Steffens S, Weber C, Schober A. (2016) Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4. Nat Commun. 7:10521.
- Fang P, Zhang D, Cheng Z, Yan C, Jiang X, Kruger WD, Meng S, Arning E, Bottiglieri T, Choi ET, Han Y, Yang XF, Wang H. (2014) Hyperhomocysteinemia potentiates hyperglycemia-induced inflammatory monocyte differentiation and atherosclerosis. Diabetes. 63(12):4275-90.
- You B, Jiang Y, Chen S, Yan G, Sun J. (2009) The nuclear orphan receptor Nur77 suppresses endothelial cell activation through induction of IkappaB expression. Circulation Research. 104(6):742-9.
- Sun J, Sui X, Bradbury JA, Zeldin DC, Conte MS, Liao JK. (2002) Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenase-derived eicosanoids. Circulation Research. 90(9): 1020-7.
- Yi B, Ozerova M, Chen M, Yan G, Huang S, Sun J. (2015) Posttranscriptional regulation of endothelial nitric oxide synthase expression by polypyrimidine tract binding protein 1 (PTB1). Arterioscler Thromb Vasc Biol. 35(10):2153-60.
- Huo Y, Yi B, Chen M, Wang, N, Chen P, Guo C, Sun J. (2014) Induction of Nur77 by Hyperoside Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Formation. Biochem Pharmacol. 92(4):590-8.
- Li P, Zhu N, Yi B, Wang N, Chen M, You X, Zhao X, Slolomides CC, Qin Y, Sun J. (2013) MicroRNA-663 Regulates Human Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Neointimal Formation. Circulation Research. 113(10):1117-27.
In vivo rodent models of asthma include the well-established models of allergen-induced allergic lung inflammation using ovalbumin (OVA) or house dust mite (HDM) or its components Dermatophagoides pteronissinus or Dermatophagoides farinae, in sensitization/challenge or challenge protocols. The models, which can be performed in either mice or rats, elicit a predominately eosinophilic, type 2 cytokine dominant allergic lung inflammation accompanied by changes in airway responsiveness to methacholine or other contractile stimuli. Protocols of longer duration also elicit multiple features of airway remodeling. Drugs/strategies can be tested for their ability to:
- when administered immediately prior to acute challenge with a contractile agent (e.g., methacholine), inhibit acute bronchoconstriction (i.e., inhibit increases in lung resistance); or
- when administered prior to or after the allergen treatment protocol, inhibit or reverse the various pathological features of allergic lung inflammation.
Experimental outcomes include, but are not limited to: multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, multiple indices of airway remodeling including airway smooth muscle mass, mucous cell metaplasia, and matrix abundance/remodeling. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice) whereas rat models are slightly preferable for assessing regulation of remodeling or age-dependent effects.
Cellular assays assessing drug/therapy effects in either human or rodent airway smooth muscle include assays of cellular stiffness/contraction, cytokine production, and matrix production (Ammit et al., 2001; Misior et al., 2008; Misior et al., 2009; Morgan et al., 2014). Assays of human airway epithelia include assays of cytokine production or other induced gene products (e.g., Muc5A), and of cell permeability/barrier function (Park et al., 2008). Additional assays of inflammatory cell (t-cell, mast cell, etc.) function are also possible (Loza et al., 2008). Tissue-based assays include analysis of smooth muscle contractile regulation using precision cut lung slices, or isolated airways using classical organ bath systems, obtained from human or murine lung (Balenga et al., 2014; Morgan et al., 2014).
- Ammit, A. J., A. T. Hastie, L. C. Edsall, R. K. Hoffman, Y. Amrani, V. P. Krymskaya, S. A. Kane, S. P. Peters, R. B. Penn, S. Spiegel and R. A. Panettieri, Jr. (2001) Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. Faseb J. 15(7): 1212-1214.
- Balenga, N. A., W. Jester, M. Jiang, R. A. Panettieri, Jr. and K. M. Druey (2014) Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation. J Allergy Clin Immunol. 134(2): 451-459.
- Deshpande, D. A., R. M. Pascual, S. W. Wang, D. M. Eckman, E. C. Riemer, C. D. Funk and R. B. Penn (2007) PKC-dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation. FASEB J. 21(10): 2335-2342.
- Forkuo, G. S., H. Kim, V. J. Thanawala, N. Al-Sawalha, D. Valdez, R. Joshi, S. Parra, T. Pera, P. A. Gonnella, B. J. Knoll, J. K. Walker, R. B. Penn and R. A. Bond (2016) PDE4 Inhibitors Attenuate the Asthma Phenotype Produced by beta-adrenoceptor Agonists in PNMT-KO Mice. Am J Respir Cell Mol Biol. 55(2):234-242.
- Liu, T., T. M. Laidlaw, H. R. Katz and J. A. Boyce (2013) Prostaglandin E2 deficiency causes a phenotype of aspirin sensitivity that depends on platelets and cysteinyl leukotrienes. Proc Natl Acad Sci U S A 110(42): 16987-16992.
- Loza, M. J., S. Foster, S. P. Peters and R. B. Penn (2008) Interactive effects of steroids and beta-agonists on accumulation of type 2 T cells. J Allergy Clin.Immunol. 121(3): 750-755.
- Misior, A. M., D. A. Deshpande, M. J. Loza, R. M. Pascual, J. D. Hipp and R. B. Penn (2009) Glucocorticoid- and protein kinase A-dependent transcriptome regulation in airway smooth muscle. Am J Respir Cell Mol Biol. 41(1): 24-39.
- Misior, A. M., H. Yan, R. M. Pascual, D. A. Deshpande, R. A. Panettieri Jr and R. B. Penn (2008) Mitogenic effects of cytokines on smooth muscle are critically dependent on protein kinase A and are unmasked by steroids and cyclooxygenase inhibitors. Mol Pharmacol. 73(2): 566-574.
- Morgan, S. J., D. A. Deshpande, B. C. Tiegs, A. M. Misior, H. Yan, A. V. Hershfeld, T. C. Rich, R. A. Panettieri, S. S. An and R. B. Penn (2014) beta-Agonist-mediated relaxation of airway smooth muscle is protein kinase A-dependent. J Biol Chem. 289(33): 23065-23074.
- Park, J., S. Fang, A. L. Crews, K. W. Lin and K. B. Adler (2008) MARCKS regulation of mucin secretion by airway epithelium in vitro: interaction with chaperones. Am J Respir Cell Mol Biol. 39(1): 68-76.
- Wagner, E. M., J. Jenkins, A. Schmieder, L. Eldridge, Q. Zhang, A. Moldobaeva, H. Zhang, J. S. Allen, X. Yang, W. Mitzner, J. Keupp, S. D. Caruthers, S. A. Wickline and G. M. Lanza (2015) Angiogenesis and airway reactivity in asthmatic Brown Norway rats. Angiogenesis 18(1): 1-11.
Chronic Obstructive Pulmonary Disease (COPD), Smoking/Emphysema
In vivo murine models of COPD include two well-established models, the 1) LPS (Pera et al., 2014; Pera et al., 2011) and 2) “smoking” (Kistemaker et al., 2013) mouse models. In the LPS model, animals are subject to repeated intranasal instillations of LPS (up to 12 weeks). In the smoking mouse model, mice are exposed to cigarette smoke for 4 days. Experimental outcomes for each model include indices of pulmonary inflammation (inflammatory cell counts and cytokines) as well as mucus production, airway fibrosis and emphysema.
Cellular assays assessing drug/therapy effects of LPS, cigarette smoke, or, cigarette smoke extract (CSE) in human airway smooth muscle include assays of cellular stiffness/contraction, cytokine production, and matrix production that are dysregulated in COPD/emphysema (Pera et al., 2010; Pera et al., 2011; Pera et al., 2012). Assays of human airway epithelia include assays of cytokine production or other induced gene products, and of cell permeability/barrier function (Oldenburger et al., 2014).
- Pera T, Zuidhof AB, Smit M, Menzen MH, Klein T, Flik G, Zaagsma J, Meurs H, Maarsingh H. (2014) Arginase inhibition prevents inflammation and remodeling in a guinea pig model of chronic obstructive pulmonary disease. J Pharmacol Exp Ther. 349(2):229-38.
- Pera T, Zuidhof A, Valadas J, Smit M, Schoemaker RG, Gosens R, Maarsingh H, Zaagsma J, Meurs H. (2011)Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD. Eur Respir J. 38(4):789-96.
- Kistemaker LE, Bos IS, Hylkema MN, Nawijn MC, Hiemstra PS, Wess J, Meurs H, Kerstjens HA, Gosens R. (2013) Muscarinic receptor subtype-specific effects on cigarette smoke-induced inflammation in mice. Eur Respir J. 42(6):1677-88.
- Pera T, Gosens R, Lesterhuis AH, Sami R, van der Toorn M, Zaagsma J, Meurs H. (2010) Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype. Respir Res. 11:48.
- Pera T, Sami R, Zaagsma J, Meurs H. (2011) TAK1 plays a major role in growth factor-induced phenotypic modulation of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 301(5):L822-8.
- Pera T, Atmaj C, van der Vegt M, Halayko AJ, Zaagsma J, Meurs H. (2012) Role for TAK1 in cigarette smoke-induced proinflammatory signaling and IL-8 release by human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 303(3):L272-8.
- Oldenburger A, Poppinga WJ, Kos F, de Bruin HG, Rijks WF, Heijink IH, Timens W, Meurs H, Maarsingh H, Schmidt M. (2014) A-kinase anchoring proteins contribute to loss of E-cadherin and bronchial epithelial barrier by cigarette smoke. Am J Physiol Cell Physiol. 306(6):C585-97.
In vivo murine models of lung fibrosis includes four different well-established models:
- The intratracheal bleomycin instillation model. This model is generated by delivering a one-time dose of bleomycin (0.075 units) into posterior oropharyngeal space of anesthetized rodents (Romero et al., 2014). The reversibility of this model makes it an excellent choice for studying both injury and repair mechanisms.
- The systemic bleomycin delivery model. This model is generated by intravenously administering a single dose of bleomycin (80 mg/kg) or by administering bleomycin via continuous subcutaneous infusion for 7 days using an osmotic minipump (Moore et al., 2013). The reversibility of the single intravenous dose model makes it an excellent choice for studying both injury and repair mechanisms while the subcutaneous model is better for modeling chronic lung disease because fibrosis tends to persist.
- The silicosis model. This model is generated by instilling a one-time dose of silica particles (20 mg of sterile silica crystal, median diameter 1-5 µm) into the posterior pharynx of anesthetized mice as described previously (Romero et al., 2014). This model is an excellent model of occupational/environmental-induced chronic fibrotic lung disease.
- The radiation-induced lung injury. This model is generated by exposing anesthetized mice to a one-time dose of 16 Gy delivered to the thorax while shielding the rest of the body (Romero et al., 2014). This model is an excellent model of radiation-induced lung fibrosis. The disadvantages of this model is the long time between injury and fibrosis (6 months).
Experimental outcomes for each model include, but are not limited to multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, multiple indices of extracellular matrix abundance and tissue remodeling. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice).
Cellular assays of fibroblast activation employ human and rodent lung fibroblast cell lines to assess effects on cell differentiation, proliferation, and survival and extracellular matrix production. Cellular assays of epithelial cells employ human and rodent alveolar epithelial cells to provide insight into drug effects on growth, survival, proliferation and senescence.
- Romero F, Shah D, Duong M, Penn RB, Fessler MB, Madenspacher J, Stafstrom W, Kavuru M, Lu B, Kallen CB, Walsh K, Summer R. (2014) A Pneumocyte-macrophage paracrine lipid axis drives the lung toward fibrosis. Am J Respir Cell Mol Biol. 53(1):74-86.5
- Moore B, Lawson WE, Oury TD, Sisson TH, Raghavendran K, Hogaboam CM. (2013) Animal models of fibrotic lung disease. Am J Respir Cell Mol Biol. 49(2):167-79.
Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS)
In vivo murine models of Acute Lung Injury including systems providing insight into human ARDS pathology. 5 different models are available:
1) The HCl instillation model. This direct lung injury model is generated by instilling a one-time dose of 50 μl of HCl (0.1N) into posterior oropharyngeal space of anesthetized rodents (Shah et al., 2015).
2) The LPS instillation model – This direct lung injury model is generated by instilling a one-time dose of 100 μl of lipopolysaccride (LPS, 100 mcg) into posterior oropharyngeal space of anesthetized rodents (Shah et al., 2015). This model mimics many features of the ARDS, including massive immune cell infiltrations and vascular permeability.
3) The bleomycin model – This direct lung injury model is generated by instilling a one-time dose of 100 μl of bleomycin (bleomycin, 0.05-0.075 units) into posterior oropharyngeal space of anesthetized rodents (Shah et al., 2014). The disadvantages of this model are that fibrotic reactions develops late after instillation and that inflammation tends to predominantly mononuclear at late time points.
4) The oleic acid model – This indirect lung injury model is generated by infusing 70 mg/kg of oleic acid in a bolus form through the left femoral vein of anesthetized rodents (more info ... ). This model has the advantage of injuring the endothelium; however, this model is less clinically relevant.
5) The cecal-ligation and puncture model – This indirect lung injury model is generated by ligating and then puncturing the cecum 3 to 5 times with a needle to induce intra-abdominal sepsis in anesthetized rodents. This is a better model to mimic ARDS caused by systemic sepsis.
Experimental outcomes for each model include, but are not limited to multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, indices of barrier function including BAL protein concentration, lung wet:dry ratio and junctional protein expression as well as markers of apoptotic and necrotic cell death. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice).
Cellular assays assessing drug/therapy effects on endothelial or epithelial function to LPS, bacteria, drugs (bleomycin), chemicals (HCl) or fatty acids (palmitate) include assays of barrier function, inflammation and cell death using a variety of approaches.
- Shah, D., Romero, R., Duong, M., Ying, J., Walsh, K., Summer, R. (2015) C1q Deficiency Promotes Lung Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury. J Biol Chem. 290(49):29642-51.
- Shah, D., Romero, R., Duong, M., Wang, N., Paudyal, B., Suratt, B., Kallen, C.B., Sun, J., Walsh, K., Summer., R. (2015) Obesity-induced adipokine imbalance impairs mouse pulmonary vascular endothelial function and primes the lung for injury. Sci Rep. 5:11362.
- Shah, D., Romero, F., Stafstrom, W., Duong, M., Summer, R. (2014) Extracellular ATP mediates the late phase of neutrophil recruitment to the lung after LPS induced injury. Am J Respir Cell Mol Biol. 306(2):L152-61.
Alcoholic Fatty Lung
In vivo murine and rat models of Alcoholic Fatty Lung include acute and chronic alcohol feeding models, in which rodents are typically fed for 3-4 months to induce the “fatty lung” phenotype (Romero et al., 2014). Measurements include lung and BAL fluid lipid levels and analysis of tissues for changes in cellular metabolism. This model can also be used to study foamy macrophage formation and the effects of alcohol on immune homeostasis in the lung.
Cellular assays assessing the effects of alcohol on lung metabolic and immune homeostasis employ in mouse, rat and human airway epithelial cells or alveolar macrophages exposed to clinically relevant doses of alcohol followed by assessment of metabolic and immune responses. Additional assays of cellular responses to inflammatory agonists such as LPS, live/dead bacteria can also be tested.
- Romero F, Shah D, Duong M, Stafstrom W, Hoek JB, Kallen CB, Lang CH, Summer R. (2014) Chronic alcohol ingestion in rats alters lung metabolism, promotes lipid accumulation, and impairs alveolar macrophage functions. Am J Respir Cell Mol Biol. 51(6):840-9.
Models of Sepsis include systemic sepsis induced by either intraperitoneal injection of LPS or Cecal Ligation and Puncture. Both of these models induce many features of the sepsis syndrome including end-organ dysfunction, endothelial activation and barrier dysfunction and cytokine storm. Drugs/therapeutic strategies for treatment or prophylaxis can be tested for efficacy in each of the models, described below. More reductionist and complementary tissue and cell-based assays for each disease are also described in the associated hyperlink.
LPS Model of Sepsis
LPS model of Sepsis entails instilling a one-time dose of 100 μl of lipopolysaccride (LPS, 100 mcg) into the peritoneal cavity of rodents (Doi et al., 2009). Outcomes include, but are not limited to measurement of serum cytokines, vascular leak, vascular inflammation and organ-specific injury (elevated Cr, hypoxia, liver dysfunction)
Experimental outcomes for each model include, but are not limited to multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, multiple indices of epithelial and endothelial barrier function. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice).
Cellular assays assessing drug/therapy effects of LPS, bacterial products, high levels of fatty acids and other agents on mouse and human endothelial cell function employ assays to measure endothelial activation, permeability and cytokine production.
- Doi, K., A. Leelahavanichkul, P. S. Yuen and R. A. Star (2009) Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119(10): 2868-2878.
Cecal Ligation & Puncture
Cecal Ligation and Puncture involve ligating and then puncturing the cecum 3 to 5 times with a needle to induce intraabdominal sepsis (Dejager et al., 2011; Pan et al., 2015). Outcomes include, but are not limited to measurement of serum cytokines, vascular leak, vascular inflammation and organ-specific injury (elevated Cr, hypoxia, liver dysfunction).
Cellular assays useful for assessing effects of candidate drugs for treatment of sepsis include blood count, profiling of plasma cytokines, adhesion molecule expression and apoptosis (Weaver et al., 2004; Zhang et al., 2015).
- Pan S, Wang N, Bisetto S, Yi B, Sheu SS. (2015) Downregulation of adenine nucleotide translocator 1 exacerbates tumor necrosis factor-α-mediated cardiac inflammatory responses. Am J Physiol Heart Circ Physiol. 308:H39-48.
- Dejager L, Pinheiro I, Dejonckheere E, Libert C. (2011) Cecal ligation and puncture: the gold standard model for polymicrobial sepsis. Trends Microbiol. 4:198-208.
- Zhang J, Yang GM, Zhu Y, Peng XY, Li T, Liu LM (2015) Role of connexin 43 in vascular hyperpermeability and relationship to Rock1-MLC20 pathway in septic rats. Am J Physiol Lung Cell Mol Physiol 309:L1323-L1332.
- Weaver JG, Rouse MS, Steckelberg JM and Badley AD (2004) Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis. FASEB J. 18(11):1185-91.
Models of bone disease include those for Osteoporosis and Osteoarthritis, each described in detail below. Drugs/therapeutic strategies for treatment or prophylaxis can be tested for efficacy in each of the models, described below. More reductionist and complementary tissue and cell-based assays for each disease are also described in the associated hyperlink.
In vivo models of osteoporosis include mouse or rat estrogen-deficient bone loss generated by bilateral ovariectomy (OVX), which are the most common osteoporotic models (Bouxsein et al., 2005; Masiukiewicz et al., 2000). The OVX mouse model is suitable for the study of pathogenesis of postmenopausal osteoporosis, and various gene knockout mice are available for the experiments. In addition, continuous parathyroid hormone (PTH) administration by infusion pump can cause bone loss and hypercalcemia (Lida-Klein et al., 2005) in mouse or rats. Experimental outcomes include, but are not limited to analyses of bone formation and resorption, and osteoblastogenesis and osteoclastogenesis (Fig.1).
Cellular assays assess drug/therapy effects on primary osteoblast mineralization (Wang et al., 2013), osteoclast formation and resorptive activity (Yang et al., 2015) (Fig.2).
- Bouxsein, M. L., Myers, K. S., Shultz, K. L., Donahue, L. R., Rosen, C. J., and Beamer, W. G. (2005) Ovariectomy-induced bone loss varies among inbred strains of mice. J Bone and Mineral Res. 20: 1085-1092.
- Masiukiewicz, U. S., Mitnick, M., Grey, A. B., and Insogna, K. L. (2000) Estrogen modulates parathyroid hormone-induced interleukin-6 production in vivo and in vitro. Endocrinology 141: 2526-2531.
- Iida-Klein A, Lu SS, Kapadia R, Burkhart M, Moreno A, Dempster DW, Lindsay R. Short-term continuous infusion of human parathyroid hormone 1-34 fragment is catabolic with decreased trabecular connectivity density accompanied by hypercalcemia in C57BL/J6 mice. J Endocrinol. 186: 549-57.
- Wang B, Yang Y, Liu L, Blair HC, Friedman PA. NHERF1 regulation of PTH-dependent bimodal Pi transport in osteoblasts. Bone 52: 268-77.
- Yang Y, Blair HC, Shapiro IM, Wang B. The Proteasome Inhibitor Carfilzomib Suppresses Parathyroid Hormone-induced Osteoclastogenesis through a RANKL-mediated Signaling Pathway. J Biol Chem. 290: 16918-28.
Models of arthritis include those for osteoarthritis and rheumatoid arthritis.
- Osteorthritis. In vivo models of osteoarthritis include two separate surgical models, anterior cruciate ligament transection (ACLT; performed in the mouse (Culley et al., 2015)) and destabilization of the medial meniscus (DMM), commonly used to study the onset and progression of posttraumatic osteoarthritis (OA) (Glasson et al., 2007). The DMM model mimics clinical meniscal injury and permits the study of structural and biological changes over the course of the disease. Experimental outcomes for both models include, but are not limited to, analyses of cartilage damage and cartilage hypertrophic markers.
- Rheumatoid arthritis. In vivo models of rheumatoid arthritis include rat adjuvant-induced arthritis (AIA) (Wang et al., 2014), and rat collagen-induced arthritis (CIA) (Godler et al., 2005). AIA is induced by intradermal injection of inactive Bacillus Calmette-Guérin suspended in paraffin oil into the hind food pat. The hallmarks of AIA are reliable onset of robust polyarticular inflammation and marked bone resorption. CIA is induced by intradermal injection of type II collagen emulsified in incomplete Freund's adjuvant at the base of the tail. CIA is polyarthritis characterized by marked cartilage destruction associated with immune complex deposition on articular surfaces and bone resorption.
Relevant cellular assays include assessment of chondrogenic differentiation using a chondrogenic cell line (ATDC5 cells)(Fig. 3) or chondrocytes derived from mesenchymal stem cells, and assays of cartilage proteoglycan synthesis. Cell based assays of rheumatoid arthritis include macrophages isolated from peritoneal lavage, synoviocytes prepared from synovial membranes of knee joints, and lymphocytes isolated from spleens for studies of drug efficacy on inflammatory cytokine production and immune response (Wang et al., 2014).
- Culley, K. L., Dragomir, C. L., Chang, J., Wondimu, E. B., Coico, J., Plumb, D. A., Otero, M., and Goldring, M. B. (2015) Mouse models of osteoarthritis: surgical model of posttraumatic osteoarthritis induced by destabilization of the medial meniscus. Methods in molecular biology 1226: 143-173.
- Glasson, S. S., Blanchet, T. J., and Morris, E. A. (2007) The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage 15: 1061-1069.
- Godler DE, Stein AN, Bakharevski O, Lindsay MM, Ryan PF (2005) Parathyroid hormone-related peptide expression in rat collagen-induced arthritis. Rheumatology (Oxford) 44: 1122-1131.
- Wang, B., and Chen, M. Z. (2014) Astragaloside IV possesses antiarthritic effect by preventing interleukin 1beta-induced joint inflammation and cartilage damage. Arch Pharm Res. 37: 793-802.