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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

461 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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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 ...

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

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

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

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

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

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

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

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...