Name: ultrasound-based pulse wave velocity evaluation in mice
Uploaded: 2017-02-14T13:00:00
Description: Watch this Scientific Journal Video about Ultrasound-based Pulse Wave Velocity Evaluation in Mice at JoVE.com

Animal experiments were performed in accordance with the European Directive (2010/63/UE) and the Italian law (D.Lvo 26/2014), and it followed principles of laboratory animal care. The Local Ethical Approval Panel approved the study.
1. Imaging Procedure
<ol>
	<li>Place the mouse in an anesthesia induction chamber filled with 2.5% isoflurane in 1 L/min pure oxygen. Verify the depth of anesthesia by unresponsiveness to toe pinch.</li>
	<li>Place the animal on a temperature controlled board in a supine position. Mo...

<ol>
	<li>Zaragoza, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Cardiovascular+Diseases.">Animal Models of Cardiovascular Diseases.</a> J Biomed Biotechnol. 2011, 497-841 (2011).</li><li>Laurent, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+consensus+document+on+arterial+stiffness:+methodological+issues+and+clinical+applications.">Expert consensus document on arterial stiffness: methodological issues and clinical applications.</a> Eur Heart J. 27, 2588-2605 (2006).</li><li>Wang, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+aortic+stiffness+assessed+by+pulse+wave+velocity+in+apolipoprotein+E-deficient+mice.">Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice.</a> Am. J. Physiol. Heart Circ. Physiol. 278, 428-434 (2000).</li><li>Mitchell, G. F., Pfeffer, M. A., Finn, P. V., Pfeffer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+techniques+for+measuring+pulse-wave+velocity+in+the+rat.">Comparison of techniques for measuring pulse-wave velocity in the rat.</a> J Appl Physiol. 82 (1), 203-210 (1997).</li><li>Parczyk, M., Herold, V., Klug, G., Bauer, W. R., Rommel, E., Jakob, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+in+vivo+transit+time+measurements+of+aortic+pulse+wave+velocity+in+mice+with+high-field+CMR+at+17.6+Tesla.">Regional in vivo transit time measurements of aortic pulse wave velocity in mice with high-field CMR at 17.6 Tesla.</a> J Cardiovasc Magn Reson. 12, 72 (2010).</li><li>Hartley, C. J., Taffet, G. E., Michael, L. H., Pham, T. T., Entman, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+determination+of+pulse-wave+velocity+in+mice.">Noninvasive determination of pulse-wave velocity in mice.</a> Am J Physiol. 273 (1), 494-500 (1997).</li><li>Feng, J., Khir, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+wave+speed+and+wave+separation+in+the+arteries+using+diameter+and+velocity.">Determination of wave speed and wave separation in the arteries using diameter and velocity.</a> J Biomech. 43 (3), 455-462 (2010).</li><li>Borlotti, A., Khir, A. W., Rietzschel, E. R., De Buyzere, M. L., Vermeersch, S., Segers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+determination+of+local+pulse+wave+velocity+and+wave+intensity:+changes+with+age+and+gender+in+the+carotid+and+femoral+arteries+of+healthy+human.">Noninvasive determination of local pulse wave velocity and wave intensity: changes with age and gender in the carotid and femoral arteries of healthy human.</a> J Appl Physiol. 113 (5), 727-735 (2012).</li><li>Ch&#233;rin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh+frame+rate+retrospective+ultrasound+microimaging+and+blood+flow+visualization+in+mice+in+vivo.">Ultrahigh frame rate retrospective ultrasound microimaging and blood flow visualization in mice in vivo.</a> Ultrasound Med Biol. 32 (5), 683-691 (2006).</li><li>Gemignani, V., Faita, F., Ghiadoni, L., Poggianti, E., Demi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+real-time+measurement+of+the+brachial+artery+diameter+in+B-mode+ultrasound+images.">A system for real-time measurement of the brachial artery diameter in B-mode ultrasound images.</a> IEEE Trans Med Imaging. 26 (3), 393-404 (2006).</li><li>Di Lascio, N., Stea, F., Kusmic, C., Sicari, R., Faita, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+assessment+of+pulse+wave+velocity+in+mice+by+means+of+ultrasound+images.">Non-invasive assessment of pulse wave velocity in mice by means of ultrasound images.</a> Atherosclerosis. 237 (1), 31-37 (2014).</li><li>Nichols, W. W., O'Rourke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> McDonald's Blood Flow in Arteries: Theoretical, Experimental, and Clinical Principles. , 215-358 (1998).</li><li>Williams, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+ultrasonic+measurement+of+regional+and+local+pulse+wave+velocity+in+mice.">Noninvasive ultrasonic measurement of regional and local pulse wave velocity in mice.</a> Ultrasound Med Biol. 33 (9), 1368-1375 (2007).</li><li>Penny, D. 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In this study, an image processing algorithm based on the lnD-V loop for PWV assessment in mice has been described in detail. The proposed approach is based on the processing of US images only and, thus, could represent a valid alternative to existing techniques6,13 for the evaluation of arterial stiffness in mouse models. In fact, conversely to invasive methods6 which are based on the acquisition of intra-arterial pressure signals and req...

Mouse models are increasingly employed for the investigation of cardiovascular disease (CVD) and particularly used in longitudinal studies which allow the characterization of different phases of disease development1. Elastic properties of large arteries are related to different pathological conditions; from a technical point of view, arterial stiffness can be assessed by measuring pulse wave velocity (PWV), which represents the speed with which the pulse wave travels in a conduit vessel2. Because of its clinical significance, it is increasingly measured even in preclinical small animal models3.
Different techniques are available for assessing PWV in mice. Invasive approaches are based on the use of catheter-tip pressure transducers. PWV is assessed by acquiring pressure signals at two different arterial sites and dividing the distance between the two measurement sites by the time shift between the signals4. The main disadvantage related to these kinds of techniques is that they require animal sacrifice for the evaluation of the distance between the two measurement sites and, thus, cannot be used in longitudinal studies. To overcome this limitation, non-invasive approaches, based on different imaging techniques, have been developed. Previous studies have reported PWV assessments in mice obtained by applying the transit time method on velocity-encoded magnetic resonance imaging data5 and Pulsed-Doppler signals6. However, the PWV value obtained with these methods is a regional evaluation of arterial stiffness. In fact, it represents an average value, accounting for different arteries in terms of size and elastic properties. In addition, these kinds of evaluations require the assessment of the distance between the two measurements sites which is a source of error that could influence the final result.
PWV can be assessed using the diameter-velocity (lnD-V) loop7. This method is based on the simultaneous evaluation of diameter and flow velocity values in a selected vessel. According to this approach, the lnD-V loop is obtained by plotting natural logarithm diameter values vs mean velocity values and PWV is estimated by calculating the slope of the linear part of the obtained loop corresponding to the early systolic phase. With regard to the practical implementation of this method, previous works have already reported results about its application in an in vitro set-up system7 and its use for the assessment of both carotid and femoral PWV in humans8.
The principal aim of the present study is to provide a detailed description of an image processing algorithm that provides a non-invasive arterial PWV measurement in mice using US images only. The proposed approach allows the evaluation of local arterial stiffness by means of the processing of both B-mode and Pulsed-Wave Doppler (PW-Doppler) images and can be applied on arteries of key importance, such as the abdominal aorta.

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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">VEVO2100</td>
			<td style="width:260px;">FUJIFILM VisualSonics Inc, Toronto, Canada</td>
			<td style="width:119px;"></td>
			<td style="width:177px;">micro-ultrasound equipment</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">MS250 Ultrasound Probe</td>
			<td style="width:260px;">FUJIFILM VisualSonics Inc, Toronto, Canada</td>
			<td style="width:119px;"></td>
			<td>micro-ultrasound probe</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:216px;">EKV Software</td>
			<td style="width:260px;">FUJIFILM VisualSonics Inc, Toronto, Canada</td>
			<td style="width:119px;"></td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matlab R2015a&#160;</td>
			<td style="width:260px;">MathWorks Inc, Natick, MA, USA</td>
			<td style="width:119px;"></td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conductive Paste</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petroleum Jelly</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Depilatory Cream</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acoustic Coupling Gel&#160;</td>
			<td style="width:260px;">Chosen by the operator</td>
			<td style="width:119px;"></td>
			<td>Laboratory material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Developed Matlab Software</td>
			<td style="width:260px;"></td>
			<td style="width:119px;"></td>
			<td>The authors are willing to collaborate with those researchers who are interested in the software and to make the software available under their supervision</td>
		</tr>
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ultrasound-based pulse wave velocity evaluation in mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

The proposed approach has been applied to mice abdominal aorta in a previous study11. The following figures show the results of the application of the described approach on real mice images. These data are from a single animal (wild type mice, 13 weeks old, strain: C57BL6, weight: 33 g) In particular, Figure 1 represents the result of the analysis of the US images. Edge detection and contour tracking techniques applied to B-mode images acquired wit...

Watch this Scientific Journal Video about Ultrasound-based Pulse Wave Velocity Evaluation in Mice at JoVE.com

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

The overall goal of this procedure is to provide an image processing algorithm for assessing arterial stiffness in mice based on the ultrasound imaging of different arterial sites. Arterial stiffness is associated with cardiovascular disease and is increasing used as therapeutic target due to its prognostic value. After confirming the appropriate levels of sedation by toe pinch, place the animal on a 40 degree Celsius temperature controlled board in the supine position and moisten the animals eyes with ointment.Coat the four limbs of the animal with conductive paste and tape the limbs onto the ECG electrodes embedded in the board. Measure the body temperature with a rectal probe lubricated with petroleum jelly and confirm that all of the physiological measurements are correctly displayed. Next, use depilatory cream to chemically remove the hair from the abdomen and coat the exposed skin with acoustic coupling gel.Place a 13 to 24 mega hertz ultrasound probe in the mechanical arm and fix the probe parallel to the animal. Adjust the probe so that the desired longitudinal images of the abdominal aorta located in the focal zone can be obtained and select high frame rate ECG gate at acquisition with a frame rate acquisition of 700 frames per second. Start the acquisition, then using the same scan projection select pulse wave doppler and move the sample volume to the center of the vessel.Obtain images with the cineloop of at least three seconds, maintaining a small an angle correction as possible. After all of the images have been acquired, remove the animal from the board with monitoring until full recovery. For post processing analysis of the ultrasound data, first export the B mode and post wave doppler images as dicom files.After saving the files, convert the post wave doppler dicom files to tif images Next using the dedicated graphical user interface, initialize the contours by drawing a line close to the far wall of the vessel and double click close to the near wall. A line parallel to the drawn line will automatically appear. Click analyze to apply the algorithm on a single frame.If the edges have been correctly identified click go"to apply the algorithm to the whole cineloop. Then click record"and save the corresponding mat file containing the instantaneous diameter values related to a single cardiac cycle. For the diameter velocity loop implementation, open the dedicated graphical user interface.To begin the pulse wave doppler image processing, click velocity"generating a single beat mean velocity curve. After identifying the post wave doppler trace, select white line"to locate the line corresponding to a velocity value equal to zero. Then, in the calibration panel, click velocity"to draw lines with lengths that correspond to the calibration factor.Repeat the operation for the time calibration clicking time. Using the region of interest physio button, manually select a region of interest containing the physiological signals, then click region of interest signal to manually select a region of interest containing the post wave doppler trace. Select analyze"to check if the envelope is identified.If the result is not satisfactory, enter the new value in the velocity threshold editable text field and analyze the envelope again. Next, click elaboration"and check for errors in our peaks detection of the ECG signal. Change the minimum peaks distance value, and then click update"for a correct identification of our peaks.Click choose beats, select all of the beats that are uncorrupted by noise or located in the inspiration phase. Press the return button on the keyboard to re-sample selected beats and to obtain a single beat velocity wave form, then select the mean velocity check box to achieve a single beat mean velocity signal and click okay. Now click diameter"and press the interpolate button on the keyboard to obtain a single beat diameter signal with the same sample frequency as the single beat velocity signal.Click okay, then select the second derivative approach as the alignment method and click update. The two curves will be automatically time aligned using the second derivative method. The natural logarithm of the single beat diameter values are automatically plotted against the single beat mean velocity values.And the corresponding pulse wave value is assessed. In this image, edge detection and contour tracking techniques were applied to B mode images acquired with a high frame rate, ECG gated modality to provide the diameter wave form. Identification of the post wave doppler signal envelope and the average of data from different cardiac cycle leads to the single beat mean velocity curve assessment.Single beat diameter and mean velocity wave forms are interpolated in both the frequency and time domain and then time aligned. A diameter velocity loop is obtained by plotting the natural logarithm diameter values against the mean velocity measurements, allowing assessment of the pulse wave velocity value. This is possible by calculating the slope of the linear part of the loop, which is known to correspond to the early systolic phase as it happens in humans using a similar approach.to assess local arterial stiffness, noninvasively.

ultrasound-based-pulse-wave-velocity-evaluation-in-mice

Research

JoVE Journal

Medicine

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1.&#160;Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.

Pulse wave velocity (PWV) measurement assesses arterial stiffness by a tonometry-based system that measures the speed with which arterial pressure wave travels along the arterial tree, typically approximating the time that it takes this wave to travel from the descending aorta (using the carotid artery as a surrogate) to the femoral artery. Carotid-femoral PWV increases two- to threefold across the adult lifespan.

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Measuring the Carotid to Femoral Pulse Wave Velocity (Cf-PWV) to Evaluate Arterial Stiffness

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively by the measurement of carotid to femoral pulse wave velocity (cf-PWV) (Class I; Level of Evidence A). In literature, numerous community-based or disease-specific studies have reported that higher cf-PWV is associated with increased cardiovascular risk. Here, we discuss strategies to evaluate arterial stiffness with cf-PWV. Following the well-defined steps detailed here, e.g., proper position operator, distance measurement, and tonometer position, we will obtain a standard cf-PWV value to evaluate arterial stiffness. In this paper, a detailed stepwise method to record a good quality PWV and pulse wave analysis (PWA) using a non-invasive tonometry-based device will be discussed.

Measuring the Carotid to Femoral Pulse Wave Velocity Cf-PWV to Evaluate Arterial Stiffness

This protocol describes a method to standardize the measurements of carotid-femoral pulse wave velocity to evaluate arterial stiffness.

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Author Spotlight: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Liujunzi Decoction: An Effective Treatment for UC-CRC Prevention

Establishment of Coloproctitis Cancer Model in Mice and Evaluation of Therapeutic Effect of Chinese Medicine

Colorectal cancer (CRC) is a common malignancy of the digestive system and has become the third most common malignancy worldwide and the second leading cause of malignancy-related death. Ulcerative coloproctitis (UC) is a precancerous lesion, and UC-associated CRC (UC-CRC) is the most common subtype of CRC. Therefore, a reasonable UC-CRC model is the cornerstone and guarantee of new drug development. Traditional Chinese medicine (TCM) has been widely used in the treatment of UC-CRC due to its good efficacy. As a classic tonic prescription of TCM, Liujunzi decoction (LJZD) has been widely used in the treatment of UC-CRC. In this study, a UC-CRC model was established by combining azomethane and dextran sulfate sodium, and the LJZD was administered. The data confirmed that LJZD can effectively inhibit cancer transition in UC-CRC by using mouse body weight, colorectal length, pathological and inflammatory factors, colorectal barrier function, and cancer markers. This protocol provides a system for evaluating the efficacy of TCM in the prevention and treatment of UC-CRC.

Author Spotlight: Liujunzi Decoction as a Traditional Chinese Treatment for Coloproctitis Cancer

This protocol provides a mouse model of ulcerative coloproctitis-associated colorectal cancer induced by azomethane combined with dextran sulfate sodium. The model was used to evaluate the efficacy of traditional Chinese medicine compounds in the prevention and treatment of colorectal cancer.

Colorectal cancer (CRC) is a common malignancy of the digestive system and has become the third most common malignancy worldwide and the second leading ...

Assessing Leg Blood Flow in Training Workloads

Doppler Ultrasound-Based Leg Blood Flow Assessment During Single-Leg Knee-Extensor Exercise in an Uncontrolled Setting

Doppler ultrasound has revolutionized the assessment of organ blood flow and is widely used in research and clinical settings. While Doppler ultrasound-based assessment of contracting leg muscle blood flow is common in human studies, the reliability of this method requires further investigation. Therefore, this study aimed to investigate the within-day test-retest, between-day test-retest, and inter-rater reliability of Doppler ultrasound for assessing leg blood flow during rest and graded single-leg knee-extensions (0 W, 6 W, 12 W, and 18 W), with the ultrasound probe being removed between measurements. The study included thirty healthy subjects (age: 33 &#177; 9.3, male/female: 14/16) who visited the laboratory on two different experimental days separated by 10 days. The study did not control for major confounders such as nutritional state, time of day, or hormonal status. Across different exercise intensities, the results demonstrated high within-day reliability with a coefficient of variation (CV) ranging from 4.0% to 4.3%, acceptable between-day reliability with a CV ranging from 10.1% to 20.2%, and inter-rater reliability with a CV ranging from 17.9% to 26.8%. Therefore, in a real-life clinical scenario where controlling various environmental factors is challenging, Doppler ultrasound can be used to determine leg blood flow during submaximal single-leg knee-extensor exercise with high within-day reliability and acceptable between-day reliability when performed by the same sonographer.

Author Spotlight: Assessing the Reliability of Doppler Ultrasound in Measuring Leg Blood Flow 

This test-retest study evaluated leg blood flow measured by the Doppler ultrasound technique during single-leg knee-extensor exercise. The within-day, between-day, and inter-rater reliability of the method was investigated. The approach demonstrated high within-day and acceptable between-day reliability. However, the inter-rater reliability was unacceptably low during rest and at low workloads.