Center for Translational Medicine Preclinical Core

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.

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.

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 and toxicology/PD/PK in each of the models, described below.

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).

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).

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.

We can collect organs (heart, lung, bone or bladder etc.), blood and cells per experimental design. The tissue can be fixed for histopathology and immunohistology analysis, or snap-frozen tissue can be used for omic (mRNA, miRNA, or protein/phoshphoprotein) analyses. Standard analyses can be performed on whole blood or its components. Specific cells (see below) can be isolated and cultures generated for various signaling or functional assays. All procedures are performed according to the Institutional Animal Care & Use Committee (IACUC) approved Animal Use Protocol (AUP) Protocol.

Among the many cell cultures that can be generated from murine tissue are:

• Adult cardiomyocytes
• ‎Neonatal cardiomyocytes‎
• Airway, alveolar epithelial cells
• Airway, aortic smooth muscle cells
• Endothelial cells
• Osteoblasts, osteoclasts

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).

• 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.
• 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.
• 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. This model has the advantage of injuring the endothelium; however, this model is less clinically relevant.
• 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).

In vivo murine models of lung fibrosis includes four different well-established models:

1. 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).

COPD Models in Mice

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.

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:

1. 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.

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.

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 assessed by transthoracic 2-dimensaional guided M-mode echocardiography (Yan et al., 2015) ; 2) heart rate, aortic pressure, LV systolic and diastolic pressure, following bolus doses of a catecholamine, 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 Diabetic Cardiomyopathy and Ischemia/Reperfusion. When requesting a quote, please note desired strain of mice desired, # of mice per experimental condition, mode of drug delivery, and duration of protocol. When designing your desired protocol, assume an ~30% mortality within first few weeks after TAC (control group) which varies depending on strain of mouse used.

Ischemia/ Reperfusion

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.

Peripheral Ischemia

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 (Tschope et al., 2004; and Westermann et al., 2007).

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).

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 (Tschope et al., 2004; and Westermann et al., 2007).