Name: modeling and evaluation of murine diabetic cardiomyopathy model
Uploaded: 2024-11-29T14:10:00.000+00:00
Duration: 6 min 22 s
Description: The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...

This work was supported by the National Natural Science Foundation of China (grant numbers: U23A20395, 81900258, and 82170375); the Key Research and Development Project of Science &#38; Technology Department of Sichuan Province (2022ZDZX0020); Chinese Medical Association Cardiovascular Branch (CSC) Clinical Research Special Fund Project (CSCF2020B04); 1&#183; 3&#183; 5 project of West China Hospital, Sichuan University (ZYGD23021).&#160;Thanks to Qing Yang (Animal Imaging Core Facilities, West China Hospital, Sichuan University) for their help in small animal ultrasonography.

All procedures followed institutional guidelines for animal research in accordance with the Guide for the Care and Use of Laboratory Animals outlined by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). All animal experiments were approved by the Ethics Committee of Animal Care and the Ethics Committee of Sichuan University. Male C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co. (China). Throughout the experimental duration, mice were housed under controlled conditions of 24 &#176;C and a regulated 12 h light/dark cycle, with ad libitum access to food and water.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67189/67189fig01.jpg" /> 
Figure 1: Schematic experiment timeline. The mice used in HFD/STZ and ND groups were both at approximately 7-8 weeks of age. The body weight and random blood glucose level of each mouse were recorded on a weekly basis. At the 12-week and 24-week feeding intervals, the metabolic phenotype of mice was evaluated by an oral glucose tolerance test (OGTT) and serum insulin level measurement. Additionally, echocardiography was assessed at baseline (week 0), 12 weeks, and 24 weeks. Following the 24-week feeding period, the mice were euthanized. <a href="https://www.jove.com/files/ftp_upload/67189/67189fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. HFD feeding
<ol>
	<li>Feed male C57BL/6J mice (7-8 weeks old) either with a normal diet (ND) or HFD (60% kcal fat) for 24 weeks to induce obesity.</li>
	<li>Record the body weight of each mouse every 4 weeks. Ensure the weight curve exhibits a similar upward pattern before 12 weeks, with a steeper slope observed in the HFD group.</li>
</ol>
2. STZ solution preparation and injection
NOTE: Store STZ at -20 &#176;C to prevent degradation due to its high instability at room temperature (RT). Blend STZ with citrate buffer immediately within 5 min before injection.
<ol>
	<li>Prepare citrate buffer. Weigh 2.1 g of citric acid (relative molecular mass: 210.14) and dissolve it thoroughly in 100 mL of distilled water to prepare solution A; weigh 2.94 g of sodium citrate (relative molecular mass: 294.10) and dissolve it thoroughly in 100 mL of distilled water to prepare solution B. Mix solution A and B in a 1: 1.32 ratio to obtain the citric acid working solution. Filter out impurities using a 0.45 &#181;m filter membrane.</li>
	<li>Weigh 1 g of STZ and dissolve it thoroughly in 100 mL of the prepared mixed citric acid working solution. Store the resulting STZ solution at 4 &#176;C. Ensure the injection of the STZ solution occurs within 30 min whenever feasible.</li>
	<li>Using a 1 mL syringe, administer intraperitoneal injections of a solution containing 30 mg/kg of STZ daily for 7 consecutive days to mice in the HFD/STZ group while providing an equal volume of citrate buffer (0.1 mmol/L, pH = 4.5) to mice in the ND group.</li>
	<li>Assay blood glucose levels 1 week after the final injection. Classify mice exhibiting random blood glucose concentrations exceeding 16.7 mmol/L as T2DM mice.
	<ol>
		<li>Carefully position the mouse onto a restraining device, ensuring its tail extends fully outside.</li>
		<li>Prior to blood collection, sanitize the tail&#39;s blood collection site thoroughly with a 70% alcohol swab, maintaining sterility and cleanliness.</li>
		<li>With a sharp-tipped needle, insert it into the tail vein, advancing from the tip towards the base at a depth of 3-4 mm. Once the needle is securely in place, gently squeeze the top of the tail to encourage blood flow.</li>
		<li>Collect approximately 10 &#181;L of blood from a mouse and carefully apply the collected blood onto the dedicated test strip. Once the blood is absorbed, the glucose meter will promptly display the blood glucose reading.</li>
	</ol>
	</li>
</ol>
3. Oral glucose tolerance test (OGTT)14
<ol>
	<li>Before OGTT, fast each mouse overnight for 14 h, while it has unrestricted access to water.</li>
	<li>Prepare approximately 10 mL of a 20% glucose solution. Administer a dose of 1 g/kg glucose via oral gavage to each mouse during the OGTT.</li>
	<li>Monitor blood glucose levels at several time points: immediately (0 min), 15 min, 30 min, 60 min, and 120 min post-administration.</li>
</ol>
4. Echocardiography assessment of cardiac function
<ol>
	<li>Conduct echocardiography on each mouse at baseline (0 weeks), 12 weeks, and 24 weeks after feeding with either HFD or ND.</li>
	<li>Prior to echocardiography, anesthetize the mouse via inhalation of 3% isoflurane which is administered with 100% oxygen using an anesthetic machine. Ensure the mouse shows no reaction to skin pinching using a toothed tweezer or stimulation of its toes and tail. Apply veterinary ointment to the eyes to prevent dryness during anesthesia.</li>
	<li>Position the mouse on a heating pad to maintain body temperature, and secure its claws to an electrode to ensure a stable supine position. Adjust the isoflurane concentration between 1%-2% for maintenance anesthesia to maintain a target heart rate of approximately 450 beats per minute.</li>
	<li>Remove the mouse&#39;s fur using depilatory cream and apply ultrasonic gel to its chest. Remove the depilatory cream and ultrasonic gel with sterile saline following echocardiography.</li>
	<li>Evaluate the cardiac function and structural parameters with a 50 MHz probe.
	<ol>
		<li>To obtain an optimal left ventricle (LV) long-axis view, position the probe on the left side of the animal&#39;s chest.</li>
		<li>Depending on the individual anatomy, rotate the probe counterclockwise between 15&#176; and 45&#176; relative to the left parasternal line, with the notch directed toward its right shoulder. Adjust the x-axis and y-axis under the B-mode15. 
		NOTE: An appropriate LV long-axis view should include (i) the aortic valve and aortic root; (ii) the LV chamber is positioned in the center of the view; (iii) the base-to-apex axis should be parallel to the transducer16.</li>
		<li>Press M-mode 2x to display the measuring line. Position this line at the level of the papillary muscle. Subsequently, take measurements for at least three consecutive heartbeats to ensure accuracy. Tap Save Clip to save the cine loop in the series.</li>
		<li>Identify the end-systolic dimension as the phase that coincided with the ECG T wave, and determine the end-diastolic dimension as the phase corresponding to the ECG R wave.</li>
		<li>Tilt the operating pad, ensuring the upper left corner is lowest, and the lower right corner is highest. Adjust the probe to penetrate along the direction of the mouse&#39;s cardiac apex, parallel to the long axis of the heart, to get a transapical four-chamber view.</li>
		<li>Successively select C (Color Doppler) and PW (PW Doppler) modes, place the sample volume at the highest velocity point, adjust the sampling direction to match the direction of blood flow, and record mitral valve hemodynamic information (E and A wave)16. Tap Save Clip to save the cine loop in the series.</li>
	</ol>
	</li>
	<li>Halt isoflurane inhalation to allow the mice to regain consciousness. Return the animals to their cages, housed in a controlled environment with a 12-h light/dark cycle at both the 0 and 12 week marks. Following the 24-week echocardiographic assessment, humanely euthanize the mice through cervical dislocation following isoflurane inhalation.</li>
	<li>Perform the analysis of the ejection fraction (EF), fraction shortening (FS), left ventricular posterior wall (LVPW), left ventricular internal diameter (LVID), interventricular septum (IVS), and E/A ratio using the workstation dongle.</li>
</ol>
5. Histological staining
<ol>
	<li>After completing the echocardiographic analysis at the 24-week mark, euthanize the mouse by spinal cord dislocation following isoflurane inhalation.</li>
	<li>Dissect the mouse using ophthalmic scissors and tweezers. Sever the ribs carefully to ensure complete exposure of the heart.</li>
	<li>Perfuse the heart through the apex with saline until the liver becomes pallor, indicating successful perfusion. Remove the heart and rinse it thoroughly in saline to ensure complete blood removal.</li>
	<li>Perform paraffin embedding17.
	<ol>
		<li>Fix the heart with 4% formalin at RT for at least 24 h.</li>
		<li>Place the dehydration box containing the heart in a dehydrator to undergo a gradual dehydration process involving gradient alcohol and wax leaching at 65 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare tissue sections17.
	<ol>
		<li>Place the trimmed wax block containing papillary muscle into a microtome for slicing, with a thickness of 4 &#181;m, and store at RT for pathological staining.</li>
	</ol>
	</li>
	<li>Perform stain using Masson&#39;s trichrome18 and wheat germ agglutinin (WGA)19 staining.</li>
</ol>

Murine Model for Diabetic Cardiomyopathy via High-Fat Diet and STZ Injection

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+type+ii+diabetes+in+c57bl/6j+mice.">Diet-induced type ii diabetes in c57bl/6j mice.</a> Diabetes. 37 (9), 1163-1167 (1988).</li><li>Clee, S. M., Attie, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+landscape+of+type+2+diabetes+in+mice.">The genetic landscape of type 2 diabetes in mice.</a> Endocr Rev. 28 (1), 48-83 (2007).</li><li>Lee, J. Y., Kim, M. J., Moon, C. K., Chung, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+products+of+streptozotocin+do+not+induce+hyperglycemia+in+rats.">Degradation products of streptozotocin do not induce hyperglycemia in rats.</a> Biochem Pharmacol. 46 (11), 2111-2113 (1993).</li><li>Ozturk, N., Uslu, S., Ozdemir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes-induced+changes+in+cardiac+voltage-gated+ion+channels.">Diabetes-induced changes in cardiac voltage-gated ion channels.</a> World J Diabetes. 12 (1), 1-18 (2021).</li><li>Bohne, L. J., Johnson, D., Rose, R. A., Wilton, S. B., Gillis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+diabetes+mellitus+and+atrial+fibrillation:+Clinical+and+mechanistic+insights.">The association between diabetes mellitus and atrial fibrillation: Clinical and mechanistic insights.</a> Front Physiol. 10, 135 (2019).</li></ol>

The authors have nothing to disclose.

Given the widespread prevalence of diabetes mellitus and its associated cardiovascular complications globally, there is an urgent need to uncover the underlying molecular mechanisms and develop preventative and therapeutic strategies for this condition20. The pathogenesis of DbCM, one of the cardiovascular complications for patients with T2DM, remains unclear, with no effective approaches to prevent and treat21. The absence of reliable preclinical models that accurately mim...

Type 2 diabetes mellitus (T2DM) is a progressively escalating global health concern, standing as a leading cause of morbidity and mortality among affected individuals. The prevalence of T2DM is closely related to the rising epidemic of obesity1,2. More than one-third of patients with T2DM exhibit a distinct cardiovascular phenotype termed diabetic cardiomyopathy (DbCM), characterized by myocardial dysfunction that occurs independently of coronary artery disease, hypertension, and valvular heart disease3. Emerging evidence suggests that approximately 20% of diabetic patients are predisposed to developing heart failure3, a condition closely associated with their prognosis4. Several pathophysiological mechanisms of DbCM have been proposed, including inflammation5, cardiac remodeling and dysfunction6, mitochondrial dysfunction7, oxidative stress8, and metabolic disturbances9. Despite extensive research, the complete spectrum of mechanisms and their individual contributions to DbCM remain incompletely understood10. Therefore, well-established preclinical animal models are essential for advancing the study of this condition.
T2DM is characterized by insulin resistance and progressive insulin insufficiency11, with most being overweight or obese12. In this study, we establish a stable and modified murine model of DbCM based on previous studies, combining high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In T2DM, insulin resistance and pancreatic &#946;-cell destruction contribute to the disease&#39;s pathophysiology12. This model leverages two prominent risk factors of T2DM. Specifically, mice fed with HFD develop insulin resistance, and subsequent STZ injections further impair pancreatic islet &#946;-cell function, significantly leading to the progression of T2DM-like pathophysiology. STZ, isolated from Streptomyces achromogenes, was first described in 1963 for its selective destruction of pancreatic islet &#946;-cells and its diabetogenic properties13. There are two STZ treatment models:&#160;administering a relatively high dose of STZ results in a short-term cardiomyopathy model, while repeated low-dose STZ injections induce a T2DM model that naturally progresses to DbCM. As a result of the latter method, animals develop hyperglycemia, polydipsia, and polyuria, all of which are characteristics of human T2DM and develop DbCM naturally.
This study found that wild-type C57BL/6J mice fed with HFD followed by intraperitoneal STZ injections (30 mg/kg) exhibited cardiac function, morphology, and histology consistent with the characteristics of DbCM by the end of the experiment. This approach provides an effective method for establishing a murine DbCM model.

<table><tbody><tr><td>Animal ultrosound system</td><td>Fujifilm Visual Sonics</td><td>VEVO 3100</td><td>Echocardiography</td></tr><tr><td>Blood glucometer</td><td>Yuwell</td><td>GU100</td><td>Assess blood glucose level</td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>251275</td><td></td></tr><tr><td>Isoflurane</td><td>RWD life science</td><td>R510-22</td><td>Anesthesia</td></tr><tr><td>Isoflurane vaporizer</td><td>RWD life science</td><td>R500</td><td>Anesthesia</td></tr><tr><td>Mouse insulin (INS) ELISA Kit</td><td>Wuhan Feiyue Biotechnology Co.,Ltd</td><td>FY-EM14029</td><td>Assess serum insulin level</td></tr><tr><td>Nair hair removal cream</td><td>Nair</td><td>255g</td><td>Remove the fur of mouse</td></tr><tr><td>Rodent diet with 60% kcal fat</td><td>Research Diets Inc</td><td>D12492</td><td>High fat diet feeding</td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>S4641</td><td></td></tr><tr><td>Sterile filter</td><td>Merck Millipore</td><td>SLHV033N</td><td></td></tr><tr><td>Streptozocin</td><td>Solarbio</td><td>S8050</td><td></td></tr><tr><td>Ultrasound gel</td><td>Keppler</td><td>KL-250</td><td>Echocardiography</td></tr><tr><td>Workstation Dongle</td><td>Fujifilm Visual Sonics</td><td>Vevo LAB</td><td>Echocardiographic data analysis</td></tr></tbody></table>

modeling and evaluation of murine diabetic cardiomyopathy model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

This study involved random allocation of mice into two groups: the ND group and the HFD/STZ group, with 6 mice in each group. Subsequent to the final echocardiography and OGTT tests 24 weeks after feeding, the mice were euthanatized to harvest their heart tissues for a histological assessment.
HFD/STZ caused an obvious body weight gain, reaching its peak in 12 weeks, and was significantly higher compared with the ND group. Following the administration of STZ, a notable decrease in body weight ...

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...

modeling-and-evaluation-of-murine-diabetic-cardiomyopathy-model

Research

JoVE Journal

Medicine

477 Views.  Sichuan University. This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

Video: Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Immunology and Infection

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are limited for investigating the fundamental mechanisms of HFpEF and identifying potential therapeutic targets. This work provides a detailed description of the surgical procedure of descending aortic constriction (DAC) in Tibetan minipigs to establish a large animal model of HFpEF. This model used a precisely controlled constriction of the descending aorta to induce chronic pressure overload in the left ventricle. Echocardiography was used to evaluate the morphological and functional changes in the heart. After 12 weeks of DAC stress, the ventricular septum was hypertrophic, but the thickness of the posterior wall was significantly reduced, accompanied by dilation of the left ventricle. However, the LV ejection fraction of the model hearts was maintained at &gt;50% during the 12-week period. Furthermore, the DAC model displayed cardiac damage, including fibrosis, inflammation, and cardiomyocyte hypertrophy. Heart failure marker levels were significantly elevated in the DAC group. This DAC-induced HFpEF in minipigs is a powerful tool for investigating molecular mechanisms of this disease and for preclinical testing.

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are ...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

Exploring HR-Dependent Cardiac Performance Using an Atrial-Pacing Protocol

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While exercise-stressed hemodynamic tests or MRI can be used to detect diastolic dysfunction and diagnose HFpEF in humans, such modalities are limited in basic research using mouse models. A treadmill exercise test is commonly used for this purpose in mice, but its results can be influenced by body weight, skeletal muscle strength, and mental state. Here, we describe an atrial-pacing protocol to detect heart rate (HR)-dependent changes in diastolic performance and validate its usefulness in a mouse model of HFpEF. The method involves anesthetizing, intubating, and performing pressure-volume (PV) loop analysis concomitant with atrial pacing. In this work, a conductance catheter was inserted via a left ventricular apical approach, and an atrial pacing catheter was placed in the esophagus. Baseline PV loops were collected before the HR was slowed with ivabradine. PV loops were collected and analyzed at HR increments ranging from 400 bpm to 700 bpm via atrial pacing. Using this protocol, we clearly demonstrated HR-dependent diastolic impairment in a metabolically induced HFpEF model. Both the relaxation time constant (Tau) and the end-diastolic pressure-volume relationship (EDPVR) worsened as the HR increased compared to control mice. In conclusion, this atrial pacing-controlled protocol is useful for detecting HR-dependent cardiac dysfunctions. It provides a new way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and may help develop new treatments for this condition.

Author Spotlight: Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach 

The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function&#160;in mouse models of heart failure.