This work is funded by the National Natural Science Foundation of China (NSFC 81822002). We thank all the members who took part in this work.

The current protocol is approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. This procedure is performed on male adult C57/BL6 mice (&#62;10 weeks of age). All surgical instruments were sterilized by autoclaving before the operation.
1. Preparation of surgical instrument
<ol>
	<li>Prepare a 5 mL syringe and pinch off the tip of the needle with a needle holder to blunt it.&#160;</li>
	<li>Prepare a 27 G needle and blunt it with a needle holder. Cutting the tip of the needle with ophthalmic scissors is an alternative way to blunt the needle.</li>
	<li>Bend the tip of the 27 G needle 90&#176;. 
	NOTE: This needle will be used as a spacer to tie with the aorta for vessel constriction.</li>
	<li>Use a commercially obtained iron wire (medical grade, 0.5 mm in diameter, see Table of Materials) and twine around the needle (Figure 1A). Form a small snare or loop at the blunted tip of the needle (Figure 1B). 
	NOTE: This reusable instrument acts as a special self-made retractor and allows one to pass silk suture under the aorta in the latter steps.</li>
	<li>Autoclave&#160;all the surgical tools before surgery (including the special retractor made above) to sterilize them.&#160;</li>
</ol>
2. Animal preparation
<ol>
	<li>Anesthetize the mouse via intraperitoneal injection of pentobarbital sodium solution (50 mg/kg, see Table of Materials) according to the body weight.&#160;Subcutaneously inject 100 &#956;L of 0.5% lidocaine locally into the surgical area.</li>
	<li>Confirm that the mouse is fully anesthetized by pinching the mouse&#39;s toe&#160;with the tip of the forceps.</li>
	<li>Apply ophthalmic ointment to both the eyes to prevent dryness while under anesthesia.&#160;</li>
	<li>Remove the hair from the throat and upper chest with depilatory cream or a clipper.</li>
	<li>Disinfect the unhaired area with three alternating scrubs of iodine and 70% ethanol in concentric circles moving outward.</li>
	<li>Place the mouse in the supine position on a heating pad covered with a sterile drape with its head pointing to the operator. Fix the extremities with adhesive tape and incisors with a 4-0 suture (Figure 2A).</li>
	<li>Insert a rectal probe (see Table of Materials) to monitor body temperature during the operation. Maintain the body temperature at 37 &#176;C during the whole operation.</li>
	<li>Put on a pair of sterile gloves. 
	NOTE: Once the sterile gloves are put on, do not touch other unsterilized areas or objects.</li>
	<li>Cut a hole (1-1.5 cm in diameter) in a new disposable sterile drape with sterilized ophthalmic scissors. Place the sterile drape over the mouse and ensure the unhaired operating field is visible through the hole. 
	&#8203;NOTE: This step allows the operator to avoid touching other unsterilized areas, which helps maintain sterile conditions during surgery.</li>
</ol>
3. Constriction of transverse aorta
<ol>
	<li>Make a 1.5 cm vertical skin incision at the level of the suprasternal notch (Figure 2B) with scalpel blade. Expand the skin incision down to the level of the sternal angle. 
	NOTE: The suprasternal notch is a notch located on the upper edge of the manubrium sterni. It is a surface symbol that can be detected when mouse hair is removed (Figure 2B). The sternal angle is the joint between manubrium sterni and corpora sterni. When the sternum is exposed, it presents as a horizontal white line on the sternum at the same level of costa secunda (Figure 2C).</li>
	<li>Carefully separate the skin and superficial fascia and expose the trachea and proximal sternal. Carefully make a 3-4 mm longitudinal midline incision to the sternum starting from the upper edge to the sternal angle (Figure 2D). 
	NOTE: The cut must not exceed the sternal angle, for there is a risk of causing acute pneumothorax in the mouse.</li>
	<li>Gently lift the sternum with forceps and the thymus and arcus aortae beneath it. Carefully use straight forceps and bluntly dissect the target vessel and tissues around it.</li>
	<li>Hold the previously self-made retractor (step 1.4) that contained a wire snare on the end of the user&#39;s right hand.</li>
	<li>Carefully pass the snare of the self-made retractor under the aorta between the left common carotid artery and the origin of the right innominate artery (Figure 2E).</li>
	<li>Pass the self-made retractor to the left hand to spare the right hand for other instruments.</li>
	<li>With microscopic tweezers in the right hand, pass a 7-0 silk suture through the loop of the snare at the end and pull out the retractor. This will allow to leave the silk suture around the artery for further ligation (Figure 2F). 
	NOTE: Be gentle and do not drag the aortic arch too much from its original position.</li>
	<li>Place a 27 G needle parallel to the aorta and tie the suture around the needle and the aorta (Figure 2G,H). 
	NOTE: The sham procedure should be the same, except that there is no aorta ligation.</li>
	<li>Hold on to the 27 G needle with ophthalmic forceps and another forceps holding the knot tied to the 27 G needle and aorta. Quickly but gently pull out the 27 G needle from the ligation (Figure 2H). 
	&#8203;NOTE: The knot must not be too tight, or it won&#39;t be easy to pull out the needle. Be careful when pulling out the needle in case of rupturing the aorta.</li>
	<li>Suture the skin with 4-0 synthetic monofilament&#160;suture in an interrupted suture pattern (Figure 2I).&#160;</li>
</ol>
4. Postoperative care
<ol>
	<li>Place the mouse on a warm pad (see Table of Materials) and wait for it to revive. Generally, the mouse revives in about 60 min. 
	NOTE: The animal must not be left unattended until it regains sufficient consciousness to the main sternal recumbency.</li>
	<li>Subcutaneously inject 0.5 mL of warm saline and monitor the animals&#39; fluid homeostasis. 
	NOTE: This step prevents animals from dehydration and hypovolemic circulation status.</li>
	<li>Apply erythromycin ointment to the wound surface every day for 3 days.</li>
	<li>For post-surgical pain treatment, administer 1 mg/L of buprenorphine local anesthetic&#160;parenterally to the incision site&#160;(see Table of Materials).</li>
	<li>Postoperatively, monitor the mouse for signs of infection and impairment (malnourishment, hunched posture, and ruffled fur). 
	&#8203;NOTE: Animals that underwent surgery are not returned to the company of other animals until fully recovered.</li>
</ol>
5. Ultrasound imaging
<ol>
	<li>Confirm successful ligation of the transverse aorta by calculating blood flow velocity via an ultra-sound imaging system12,13 (24 MHz, see Table of Materials) 28 days after surgery.
	<ol>
		<li>Anesthetize the animal with 4.5% isoflurane in 20% oxygen-enriched air and maintain with 0.5% isoflurane in 20% oxygen-enriched air.</li>
		<li>Place the mouse in a supine position and fix its extremities to the electrode with adhesive tape.</li>
		<li>Using depilatory cream, remove the chest hair and apply an ultrasonic coupling agent to the chest.</li>
		<li>Tilt the platform to the left as far as possible. While keeping the probe in the vertical position, gradually lower it along the right parasternal line on the chest with the notch pointing to the animal&#39;s chin.</li>
		<li>Under the B mode of the instrument, adjust the X and Y-axis until the aortic arch and constriction site are clearly visible.</li>
		<li>Click on the Pulse Wave Doppler button to switch to pulse wave mode.</li>
		<li>Adjust the dashed cursor sample volume box distal to the constricted site and find the stenotic jet with the highest velocity.</li>
		<li>Click on the Pulse Wave Doppler button again to get the waveform of aortic flow and measure the peak velocity. Calculate the blood flow velocity according to the peak of the wave patterns (Figure 3A,B).</li>
		<li>Set the imager to B mode. Rest the platform in the horizontal position. Rotate the probe 30&#176; to the left parasternal line.</li>
		<li>Adjust X and Y-axis to obtain a short axis view. Press M mode to show the indicator line and click on Cine Store to save the image for later measurement of cardiac wall thickness, chamber dimension, and ejection fraction. 
		NOTE: The formula for calculating ejection fraction: 100% * ((LV Vol;d - LV Vol;s) / LV Vol;d). The formula for calculating cardiac output: Stroke Volume * Heart rate (at the first frame drawn) / 1000. Most indexes such as ejection fraction and cardiac output can be measured automatically via the ultrasonic imaging system&#39;s software. LV Vol;d: diastolic volume of the left ventricle. LV Vol;s: systolic volume of the left ventricle.</li>
	</ol>
	</li>
</ol>

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The authors declared that there are no conflicts of interest.

The induction of sustained pressure overload can gradually cause cardiac hypertrophy and heart failure. This model has been used in numerous laboratories around the world14,15,16. The protocol provided an improved TAC method that does not need microsurgical skills or mechanical ventilation.
The most important step in this protocol is passing silk suture under the aortic arch. When the snare has hooked...

Heart failure is a complex clinical symptom that results from impaired structure and function of ventricular filling or ejecting blood1. The disease stage is mainly defined via the New York Heart Association function classification based on the severity of symptoms and physical activity2. For those patients with an ejection fraction of over 50%, structural and/or functional abnormalities raised natriuretic peptides to support the diagnosis of heart failure with preserved ejection fraction (HFpEF)2. Ischemic heart disease is a leading cause among multiple etiologies of heart failure. Thus, the myocardial infarction model (such as permanent coronary ligation) is often used to study pathophysiology after cardiac hypoperfusion or ischemia-reperfusion injury3,4. Besides acute myocardial injury, other risk factors such as hypertension, diabetes, obesity, and a family history of cardiomyopathy also contribute to the development of heart failure. After patients pass Stage A (at risk for heart failure) and enter Stage B (pre-heart failure), structural modification occurs1. For example, hypertensive patients first go through adaptive left ventricle hypertrophy, and then gradually develop into maladaptive cardiac hypertrophy and transit to heart failure through pathological remodeling5.
As the terminal stage of various cardiovascular diseases, chronic heart failure has been studied for decades6. Multiple mouse models have been widely used in heart failure research, including drug infusion (angiotensin II), metabolic disorders (diabetes or high caloric diet), and aortic constriction7. Among these models, angiotensin II perfusion is accompanied by various organ side effects, such as kidney7. Inducing metabolic disorders usually require a rather long period of time. Ascending aortic constriction has been considered to have limited relevance to human disease7.
TAC is a reliable model that increases afterload and induces cardiac hypertrophy as well as heart failure8. Open-chest TAC model was first described by Rockman et al. and was used in numerous laboratories around the world9. However, this classical TAC procedure causes a rather large trauma to mice and changes their normal behavior, which may take a long recovery time and disturb further treatment10. Other modified closed-chest TAC procedures did reduce some invasive steps but required microsurgical skills or mechanical ventilation10,11.
The present protocol details a step-by-step method with a minimally invasive approach to the aortic arch using a self-made retractor via a 3 mm midline incision of the upper edge of the sternum. This model does not need microsurgical skill, mechanical ventilation, or cutting through the ribs, thereby providing a rapid, surgical trauma-limited, uncomplicated, inexpensive way to perform TAC surgery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-0 nonabsorbable suture</td>
			<td>Jinhuan</td>
			<td>HM403</td>
			<td>Used for suturing the skin</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Haifuda Technology Co., Ltd.</td>
			<td>BD-309628</td>
			<td>Used for making snare containing retractor</td>
		</tr>
		<tr>
			<td>7-0 nonabsorbable suture</td>
			<td>Jinhuan</td>
			<td>HM701</td>
			<td>Used for aorta ligation</td>
		</tr>
		<tr>
			<td>Animal temperature monitor</td>
			<td>Kaerwen</td>
			<td>FT3400</td>
			<td>Used for monitoring body temperature</td>
		</tr>
		<tr>
			<td>Buprenorphine&#160;</td>
			<td>Sigma</td>
			<td>B-044</td>
			<td>Used for post-surgical pain treatment</td>
		</tr>
		<tr>
			<td>Depilatory cream&#160;</td>
			<td>Veet</td>
			<td>N/A</td>
			<td>Used for remove body hair from the surgical area</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Xiaochuangxin</td>
			<td>N/A</td>
			<td>Used for maintaining body temperature</td>
		</tr>
		<tr>
			<td>Ibuprofen</td>
			<td>MCE</td>
			<td>HY-78131</td>
			<td>Used for post-surgical pain treatment</td>
		</tr>
		<tr>
			<td>Iron wire (0.5 mm)</td>
			<td>Qing Yuan</td>
			<td>Iron wire #26</td>
			<td>Used for making snare containing retractor</td>
		</tr>
		<tr>
			<td>Microscopic tweezers</td>
			<td>RWD</td>
			<td>F12006-10</td>
			<td>Used for penetrating and separating the tissue to open operation space</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>RWD</td>
			<td>F12005-10</td>
			<td>Used for pinching off the tip of gauge needle and blunting it</td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>RWD</td>
			<td>F14012-10&#160;</td>
			<td>Used for holding skin and other tissues</td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>RWD</td>
			<td>S11001-08</td>
			<td>Used for making sking incision of mouse</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td>Used for mouse anesthesia</td>
		</tr>
		<tr>
			<td>Sterile operating mat</td>
			<td>Hale &#38; hearty</td>
			<td>211002</td>
			<td>Used for placing animal during surgery</td>
		</tr>
		<tr>
			<td>Ultra-sound imaging system</td>
			<td>Fujifilm visualsonics</td>
			<td>vevo1100</td>
			<td>Used for measure the blood flow velocity, left ventricular wall thickness and ejection fraction, https://www.visualsonics.com/product/imaging-systems/vevo-1100</td>
		</tr>
	</tbody>
</table>

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.

After successful TAC surgery, pressure overload was detected using an ultrasound imaging system. Four weeks after surgery, mice develop decreased heart function. In the present study, the efficacy of TAC surgery was validated via ejection fraction (EF), fractional shortening (FS), left ventricular mass (LV mass) and left ventricular internal diameter (LVID) of mice who underwent TAC surgery after 4 weeks. EF was significantly reduced in TAC mice after 4 weeks compared to sham mice (47% &#177; 10% vs. 78% &#177; ...

Watch this Scientific Journal Video about A Modified Technique for Transverse Aortic Constriction in Mice at JoVE.com

A Modified Technique for Transverse Aortic Constriction in Mice

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 ...

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4.1K Views.  Huazhong University of Science and Technology. 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.

Video: A Modified Technique for Transverse Aortic Constriction in Mice

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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 ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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.

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 ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...

Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate time of day. Studies over the last few decades have shown that rhythmic food intake (i.e., the time at which organisms eat food during the 24 h day), significantly contributes to the rhythmic regulation of gene expression in various organs and tissues throughout the body. The effects of rhythmic food intake on health and physiology have been widely studied ever since and have revealed that restricting food intake for 8 h during the active phase attenuates metabolic diseases arising from a variety of obesogenic diets. These studies often require the use of controlled methods for timing the delivery of food to animals. This manuscript describes the design and use of a low-cost and efficient system, built in-house for measuring daily food consumption as well as manipulating rhythmic food intake in mice. This system entails the use of affordable raw materials to build cages suited for food delivery, following a user-friendly handling procedure. This system can be used efficiently to feed mice on different feeding regimens such as ad libitum, time-restricted, or arrhythmic schedules, and can incorporate a high-fat diet to study its effect on behavior, physiology, and obesity. A description of how wild-type (WT) mice adapt to the different feeding regimens is provided.

Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice.

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate ...