This work was supported by grants from the National Natural Science Foundation of China (81570464, 81770271; to Dr. Liao) and the Municipal Planning Projects of Scientific Technology of Guangzhou (201804020083) (to Dr. Liao).

All procedures were performed in accordance with institutional guidelines for animal research, which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). C57BL/6 male mice (8-10 weeks old, weighing 20-25 g) were provided by the Animal Center of South Medical University. After arrival, the mice were housed under a 12/12 h dark/light cycle, with sufficient food and water.
1. Preparation of the surgical instruments and fabrication of the needles
<ol>
	<li>Prepare the sterile surgical instruments (Figure 1A), a 6-0 braided silk suture (Figure 1B[a]) for ligation, and a 5-0 nylon suture for incision closure (Figure 1B[b]).</li>
	<li>Pass the 6-0 braided silk suture (Figure 1B[a]) through a 25 G needle disassembled from a 1 mL injection syringe. Then, curve the needle 90&#176; with hemostatic forceps to make a latch needle (Figure 1C[a]). Curve the 22 G needle 120&#176; (Figure 1C (b)) to make a padding needle.</li>
</ol>
2. Preparation for surgery
<ol>
	<li>Autoclave all surgical instruments before surgery. Adjust the heating pad to 37 &#176;C. Anesthetize the mice by intraperitoneal injection with a mixture of xylazine (5 mg/kg) and ketamine (100 mg/kg) for pain relief. Place the mice in individual boxes to wait for narcotic onset. 
	NOTE: It is also recommended to use 1.5% isoflurane for inhalant anesthesia.</li>
	<li>Monitor the adequacy of anesthesia by the disappearance of the pedal withdrawal reflex. Keep the mice in the supine position on the heating pad by fixing the incisors with a suture and fixing the legs with adhesive tape. Check the reflex again to ensure the depth of anesthesia.</li>
	<li>Apply the depilatory paste on the skin from the neck to the xiphoid process. Disinfect the area with iodine followed by 75% alcohol.</li>
	<li>Perform endotracheal intubation.
	<ol>
		<li>Adjust the animal miniventilator&#39;s (Figure 1D) parameters and set the respiratory rate to 150/min and the tidal volume to 300 &#181;L.</li>
		<li>Pull out the tongue slightly by using pointed pliers, elevate the mandible with a lab made spatula Figure 1C[c]) to expose the glottis, and softly insert a lab made trachea cannula (Figure 1C[d]) through the glottis while a cold light source is directed on the larynx.</li>
		<li>Connect the tube and the ventilator to verify whether the cannula has been inserted into the trachea. Fix the trachea using adhesive tape if the cannula has been inserted correctly.</li>
	</ol>
	</li>
</ol>
3. Surgery
<ol>
	<li>Open the chest.
	<ol>
		<li>Make an incision in the skin parallel to the second rib, about 10 mm in length, with ophthalmic scissors. Ensure that the incision starts from the sternal angle and ends on the left anterior axillary line. Identify the second intercostal space by counting the ribs from the sternal angle.</li>
		<li>Separate and cut the pectoralis major and pectoralis minor muscles with scissors and elbow tweezers above the second intercostal space to expose this space. 
		NOTE: It is also recommended to bluntly separate, mobilize, and move the pectoralis muscles to the right and cranially.</li>
		<li>Bluntly penetrate the second intercostal space with elbow tweezers and open this space. Then, bluntly separate the parenchyma and thymus until the pulmonary trunk is visible.</li>
	</ol>
	</li>
	<li>Constrict the pulmonary artery.
	<ol>
		<li>Bluntly separate the pulmonary trunk and the ascending aorta with elbow tweezers. Pass the suture through the connective tissue between the pulmonary trunk and the ascending aorta with a latch needle.</li>
		<li>Place the padding needle (see step 1.2) on the pulmonary trunk and, then, ligate the pulmonary trunk together with the padding needle, using the 6-0 braided silk suture. Remove the padding needle immediately after the filling of the pulmonary conus is observed and cut the ends of the suture.</li>
		<li>Observe the filling of the pulmonary conus to evaluate whether there is a constriction present. Evaluate the animal&#39;s reflex again to ensure the success of the ligation. 
		NOTE: Perform a sham surgery by following all of the above steps except for the constriction.</li>
	</ol>
	</li>
	<li>Close the chest and the skin with the 5-0 nylon suture. Disinfect the skin again with 75% alcohol.</li>
	<li>Inject 0.5 mL of saline subcutaneously to replace any fluid lost during the surgery. Place the mouse in the cage separately with heating pad to promote recovery. Return the mice to their cages in a 12/12 h light/dark cycle room when consciousness returns. Treat the mice with buprenorphine via their drinking water for the following 3 days.</li>
	<li>Pay special attention to the healing of the thoracotomy wound by monitoring the mice 2x a day during the first week to detect any signs of insufficient healing, impaired mobility, or weight loss.</li>
</ol>
4. Echocardiographic assessment of the RV function after surgery
NOTE: Echocardiographic changes can be detected 3 days after surgery.
<ol>
	<li>Anesthetize the mice with 3% isoflurane through inhalation and maintain the depth of anesthesia with 1.5% isoflurane. Fix a mouse on the platform, tape its claws to the electrode, and maintain the animal in a supine position. Maintain the mouse&#39;s heart rate between 450-550 beats/min by adjusting the concentration of isoflurane between 1.5% and 2.5%.</li>
	<li>Remove the hair on the mouse&#39;s chest with depilatory cream and apply ultrasound gel to the skin of the chest.</li>
	<li>Assess the pulmonary trunk constriction with a 30 MHz probe.
	<ol>
		<li>Keep the probe at 30&#176; counterclockwise relative to the left parasternal line, while orienting the notch in the caudal direction. Regulate the y-axis and x-axis under the B-mode until the mitral valve, aorta, and LV chamber are clearly visible.</li>
		<li>Rotate the probe 30&#176;-40&#176; on its y-axis toward the chest. Regulate the y-axis and x-axis until the pulmonary conus is clearly visible.</li>
		<li>Place the cursor at the tip of the pulmonary valve leaflets to measure the peak flow velocity. Use the Color Doppler mode by pressing Color, followed by PW, and then moving the cursor to place the PW-dashed line parallel to the direction of the blood flow.</li>
		<li>Measure the pulmonary artery peak velocity. Save the data and image with Cine Store and Frame Store.</li>
	</ol>
	</li>
	<li>Assess the RV parameters with a 30 MHz probe.
	<ol>
		<li>Adjust the left side of the pad so that it is lower than the right side. Keep the probe oriented at 30&#176; to the horizon along the right anterior axillary line with the notch pointed in the caudal direction. Regulate the y-axis and x-axis until the RV is clearly shown.</li>
		<li>Press M-Mode 2x to show the indicator line. Measure the RV chamber dimension, fractional shortening, and RV wall thickness. Save the data and image with Cine Store and Frame Store.</li>
	</ol>
	</li>
	<li>Stop isoflurane inhalation to allow the mice to recover consciousness and then return the animals to their cages in a 12 h light/dark cycle room.</li>
</ol>
5. Right heart catheterization for assessing the RV function
NOTE: Right heart catheterization was performed 8 weeks after surgery to assess the RV function, using a 1.0 F catheter and a monitoring system.
<ol>
	<li>Autoclave all surgical instruments. Anesthetize the animal via intraperitoneal injection with a mixture of xylazine (5 mg/kg) and ketamine (100 mg/kg).</li>
	<li>After the pedal withdrawal reflex disappears, fix the mouse on the platform, tape its claws to the electrode, and maintain the mouse in supine position. Remove the hair in the surgical area with depilatory cream.</li>
	<li>Disinfect the skin of the surgical area with 75% alcohol. Using pointed pliers,pull the tongue out slightly, elevate the mandible with a spatula made inhouse to expose the glottis, and softly insert the trachea cannula made inhouse through the glottis while a cold light source is directed on the larynx. Use a ventilator (Figure 1E) to assist with ventilation.</li>
	<li>Open the chest cavity by means of a 1.5 cm bilateral incision below the xiphoid process through the diaphragm with ophthalmic scissors and forceps. Cut through the diaphragm and ribs with ophthalmic scissors to expose the heart. Penetrate the RV free wall with a 23 G needle and remove the needle; press the puncture point gently with a cotton swab to stop any bleeding. Puncture the ventricle with the catheter tip through the wound. 
	NOTE: It is also recommended to perform right heart catheterization via the right jugular vein6. When the catheter tip is in the ventricle, the monitor will display the RV pressure curve.</li>
	<li>Record the RV systolic blood pressure, the RV end-diastolic pressure, the RV dP/dt, the mouse&#39;s heart rate, and the RV exponential time constant of relaxation (Tau) for 10 min when the curve is stable. Using the software, click Select and then click Analyze.</li>
	<li>Regulate the tip of the catheter toward the RV outflow tract. Pull the catheter out after the recording is complete. Place the catheter in saline when the measurements are finished.</li>
	<li>Euthanize the mice by intraperitoneal injections of pentobarbital sodium 150 mg/kg, followed by cervical dislocation. Then, harvest the heart, lungs, and tibia for histomorphological and molecular biological analyses.</li>
</ol>

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The authors have nothing to disclose.

Pathological increases in RV filling pressures result in a leftward shift of the septum, which can alter the LV geometry21. These changes contribute to reduced cardiac output and LV ejection fraction (LVEF), which can cause a hemodynamic disorder of the circulatory system22. Therefore, an efficient, stable, and economical model for studying the mechanism of RVF is valuable.
We developed a more effective and highly reproducible approach to PAC usi...

RV dysfunction (RVD), defined here as evidence of an abnormal RV structure or function, is associated with poor clinical outcomes. RVF, as the end stage of RV function, is a clinical syndrome with signs and symptoms of heart failure that result from progressive RVD1. With differences in structure and physiological function, left ventricular (LV) failure and RVF have different pathophysiological mechanisms. A few independent pathophysiological mechanisms in RVF have been reported, including overexpression of &#946;2-adrenergic receptor signaling2, inflammation3, transverse tubule remodeling, and Ca2+ handling dysfunction4.
RVF can be caused by volume or pressure overload of the RV. Previous animal models have used SU5416 (a potent and selective inhibitor of the vascular endothelial growth factor receptor) combined with hypoxia (SuHx)5,6 or monocrotaline7 to induce pulmonary hypertension, which results in RVF secondary to pulmonary vascular disease2. The researchers conducting these studies focused on the vasculature instead of the pathological progression of RVF. Moreover, monocrotaline has extra-cardiac effects that cannot precisely represent cardiogenic disease. Other models have used arteriovenous shunts to induce volume overload and RVF8. However, this surgery is difficult to perform and inappropriate for mice, who require long induction periods for the production of RVF.
Rat PAC models using banding clips also exist9,10. Compared with rats, mice have many advantages as animal models of cardiac diseases, such as easier reproduction, more widespread use, reduced costs, and access to gene modification11. However, the diameters of the banding clips usually range from 0.5 mm to 1.0 mm, which are too large for mice9. In addition, the banding clip is hard to produce, imitate, and popularize in other labs.
We provide a protocol to develop a modified reproductive RVF mouse model based on reported studies, which uses PAC to mimic the tetralogy of Fallot and Noonan syndrome or other pulmonary arterial hypertensive diseases12,13,14,15,16,17,18,19. This PAC approach is created by ligating the pulmonary trunk of mice using a latch and padding needle made inhouse to control the degree of constriction. The latch needle is made of a 90&#176; curved injection syringe with a braided silk suture passed through the syringe. The needle is made from common materials using a process that is easy to master. The padding needle is curved 120&#176; from the gauge needle. Padding needles with different diameters (0.6-0.8 mm) are used, depending on the mice&#39;s weight (20-35 g). Additionally, we establish an evaluation criterion to determine the stability and quality of the RVF model by echocardiography and right heart catheterization. We use mice as the model animal because of their widespread use in other experiments. The needles made in the lab are easy to reproduce and can be widely used in other labs. This study provides a good approach for researchers to investigate the mechanism of RVF.

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			<td style="width:327px;">For right heart catheterization</td>
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induction of right ventricular failure by pulmonary artery constriction and evaluation of right ventricular function in mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

In this study, mice were randomly assigned to the PAC group (n = 9) or the sham operation group (n = 10). Echocardiography was performed at 1, 4, and 8 weeks after the surgery. Eight weeks after surgery, following the last echocardiography and catheterization assessments, the mice were euthanized, and their hearts were harvested for a morphological and histological assessment.
Pulmonary trunk constriction cause...

Watch this Scientific Journal Video about Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice at JoVE.com

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

induction-right-ventricular-failure-pulmonary-artery-constriction

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10.4K Views.  Southern Medical University. Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

Video: Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6 However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.

This video describes a right hemihepatectomy with intrahepatic transection of the right hemipedicle leaving the hepatoduodenal ligament completely untouched.

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...