This research was supported by the Intramural Research Program of the NIH, National Institute on Aging and the Baltimore Longitudinal Study on Aging. A portion of that support was through an R&amp;D contract with MedStar Research Institute. Standard protocol and technical expertise was provided by AtCor.

1. Procedure
<ol>
	<li>Open the software via the shortcut on your Windows desktop by double-clicking on the icon.</li>
	<li>Click Patient to activate the Patient Screen.</li>
	<li>To enter a new patient into the database, click the New button in the patient search area, and then click on the Yes button to confirm you would like to enter a new patient.</li>
	<li>Enter patient details such as last name, first name, date of birth, and gender. Click on the Save button to advance to the next step.</li>
	<li>Click the PWA or PWV button to perform measurement. If only one of these modes is available, there is no need to click on the button, simply proceed to the study screen.</li>
</ol>
2. Central Blood Pressure Measurement Using Pulse Wave Analysis (PWA)
<ol>
	<li>Click on the Study button to enter study parameters. Enter the brachial pressure taken from a calibrated sphygmomanometer and any other details you wish to include.</li>
	<li>Click on the Capture Data button to proceed with a measurement. Attach the three ECG electrodes and leads to the patient in a modified Lead II configuration or using the patient&#39;s limbs as indicated on the cables.</li>
	<li>Palpate the patient&#39;s radial artery. This is located on the lateral of the wrist, immediately above the wrist joint near the base of the thumb. Gently press the radial artery against the underlying bone with the middle and index fingers.</li>
	<li>Place the tonometer over the strongest pulse point. Gently press the tonometer down until you see a consistent pressure waveform displayed on the data capture screen.</li>
	<li>If Auto Capture is enabled (see Note below), the system will automatically save the measurement. To manually save the measurement for analysis, press the spacebar on your keyboard (or step on the footswitch, if used).</li>
	<li>If a repeat measurement is required with the same study parameters, click on the Repeat button to return to the Capture Data screen and repeat the measurement as outlined in steps 2.4-2.5 above.</li>
	<li>To perform a measurement on a new patient, click on the Patient button and return to step 1.3 above.</li>
</ol>
3. Arterial Stiffness Measurement Using Pulse Wave Velocity (PWV)
<ol>
	<li>Click on the Study button to enter study parameters.</li>
	<li>Enter brachial pressure taken from a calibrated sphygmomanometer 
	Enter the distance measured for the distal (supra-sternal notch to femoral pulse) and proximal (supra-sternal notch to carotid pulse) sites. Medication, Notes, and Operator fields may be entered, if desired</li>
	<li>Click on the Capture button to proceed with the measurement.</li>
	<li>Ensure the ECG signal is of acceptable quality.</li>
	<li>Palpate the carotid artery pulse. This is located between the larynx and the sternocleidomastoid muscle in the neck. It can be felt by pushing the muscle to the side and pressing the artery against the larynx.</li>
	<li>Place the tonometer on the carotid artery and gently press the tonometer down until you receive a consistent pressure waveform displayed on the data capture screen.</li>
	<li>When you have a minimum of 13 sec of consistent waveforms, click the OK button or (press on the footswitch, if used).</li>
	<li>Palpate the femoral artery pulse. This is located over the ventral thigh halfway between the pubic symphysis and anterior superior iliac spine.</li>
	<li>Place the tonometer on the femoral artery and gently press the tonometer down until you receive a consistent pressure waveform displayed on the capture screen. When you have a minimum of 13 sec of consistent waveforms, click the OK button (or press the on the footswitch, if used).</li>
	<li>The report(s) can now be reviewed. These measurements are typically conducted in triplicate. If the user wishes to measure the PWV between carotid and radial arteries, these steps just shown would be repeated except that instead of measuring at the femoral artery, the user would place the tonometer at the radial artery. To perform a measurement on a new patient, click on the Study button and repeat this procedure or use the Search feature to search for an existing patient.</li>
</ol>
4. Shut Down
<ol>
	<li>The software automatically saves all reports. To close the software, click on System from the main menu, and then click on Exit. Place the tonometer in the module tray for storage.</li>
</ol>

<ol>
	<li>Najjar, S. S., Scuteri, A., 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=Pulse+wave+velocity+is+an+independent+predictor+of+the+longitudinal+increase+in+systolic+blood+pressure+and+of+incident+hypertension+in+the+Baltimore+Longitudinal+Study+of+Aging.">Pulse wave velocity is an independent predictor of the longitudinal increase in systolic blood pressure and of incident hypertension in the Baltimore Longitudinal Study of Aging.</a> J. Am. Coll. Cardiol. 51 (14), 1377-1383 (2008).</li><li>Doonan, R. J., Scheffler, P., 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=Altered+arterial+stiffness+and+subendocardial+viability+ratio+in+young+healthy+light+smokers+after+acute+exercise.">Altered arterial stiffness and subendocardial viability ratio in young healthy light smokers after acute exercise.</a> PloS one. 6 (10), (2011).</li><li>Laurent, S., Cockcroft, J., 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 (21), 2588-2605 (2006).</li><li>Mitchell, G. F., Hwang, S. -. J., 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=Arterial+stiffness+and+cardiovascular+events:+the+Framingham+Heart+Study.">Arterial stiffness and cardiovascular events: the Framingham Heart Study.</a> Circulation. 121 (4), 505-511 (2010).</li><li>Abbatecola, A. M., Chiodini, P., 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=Pulse+wave+velocity+is+associated+with+muscle+mass+decline:+Health+ABC+study.">Pulse wave velocity is associated with muscle mass decline: Health ABC study.</a> Age. , (2011).</li><li>Watson, N. L., Sutton-Tyrrell, K., 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=Arterial+stiffness+and+gait+speed+in+older+adults+with+and+without+peripheral+arterial+disease.">Arterial stiffness and gait speed in older adults with and without peripheral arterial disease.</a> Am. J. Hypertens. 24 (1), 90-95 (2011).</li><li>Mackenzie, I. S., Wilkinson, I. B., Cockcroft, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+arterial+stiffness+in+clinical+practice.">Assessment of arterial stiffness in clinical practice.</a> QJM. 95 (2), 67-74 (2002).</li><li>O'Rourke, M. F., Pauca, A., Jiang, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse+wave+analysis.">Pulse wave analysis.</a> Br. J. Clin. Pharmacol. 51 (6), 507-522 (2001).</li><li>Stoner, L., Young, J. M., Fryer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessments+of+arterial+stiffness+and+endothelial+function+using+pulse+wave+analysis.">Assessments of arterial stiffness and endothelial function using pulse wave analysis.</a> Int. J. Vasc. Med. 2012, 903107 (2012).</li><li>Farro, I., Bia, D., 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=Pulse+wave+velocity+as+marker+of+preclinical+arterial+disease:+reference+levels+in+a+uruguayan+population+considering+wave+detection+algorithms,+path+lengths,+aging,+and+blood+pressure.">Pulse wave velocity as marker of preclinical arterial disease: reference levels in a uruguayan population considering wave detection algorithms, path lengths, aging, and blood pressure.</a> Int. J. Hypertens. 2012, (2012).</li><li>Lakatta, E. G., Wang, M., Najjar, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+aging+and+subclinical+arterial+disease+are+fundamentally+intertwined+at+macroscopic+and+molecular+levels.">Arterial aging and subclinical arterial disease are fundamentally intertwined at macroscopic and molecular levels.</a> Med. Clin. N. Am. 93 (3), 583-604 (2009).</li><li>Ambrose, 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> Clinical Manual (CVMS) Rev. 2, 1-51 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+Values+for+Arterial+Stiffness'+Collaboration+Determinants+of+pulse+wave+velocity+in+healthy+people+and+in+the+presence+of+cardiovascular+risk+factors:+'establishing+normal+and+reference+values.">Reference Values for Arterial Stiffness' Collaboration Determinants of pulse wave velocity in healthy people and in the presence of cardiovascular risk factors: 'establishing normal and reference values.</a> Eur. Heart J. 31 (19), 2338-2350 (2010).</li><li>van Bortel, L. M., 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+the+measurement+of+aortic+stiffness+in+daily+practice+using+carotid-femoral+pulse+wave+velocity.">Expert consensus document on the measurement of aortic stiffness in daily practice using carotid-femoral pulse wave velocity.</a> J. Hypertens. 30 (3), 445-448 (2012).</li><li>Scuteri, A., Brancati, A., Gianni, W., Assisi, A., Volpe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+is+an+independent+risk+factor+for+cognitive+impairment+in+the+elderly:+a+pilot+study.">Arterial stiffness is an independent risk factor for cognitive impairment in the elderly: a pilot study.</a> J. Hypertens. 23 (6), 1211 (2005).</li><li>Khoshdel, A. R., Carney, S. L., Nair, B. R., Gillies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+management+of+cardiovascular+diseases+by+pulse+wave+velocity:+combining+clinical+practice+with+clinical+research+using+evidence-based+medicine.">Better management of cardiovascular diseases by pulse wave velocity: combining clinical practice with clinical research using evidence-based medicine.</a> Clin. Med. Res. 5 (1), 45-52 (2007).</li></ol>

The authors declare that they have no competing financial interests. The authors received assistance with protocol description and performance of study from SphygmoCor which manufactures this product described.

The measurement of pulse wave velocity (PWV) is a noninvasive method for analyzing central arterial stiffness in a relatively simple and reproducible manner3. To assess PWV, the carotid (as a surrogate for the descending aorta) and femoral pulse waveforms are acquired using a high-fidelity tonometer; the time delay in pulse wave arrival at the between the two sites is estimated referencing to a concurrently recorded EKG waves by custom designed computer software that conforms to the propagative model of the ar...

A correction was made to <a href="http://www.jove.com/video/50817">Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging</a>. There was an error with the author, Majd AlGhatrif's, name. The author's name has been corrected to:
Majd AlGhatrif
instead of:
Majd al Ghatrif

A correction to the author list has been made for article <a href="http://www.jove.com/video/50817">Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging</a>.

Erratum: Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Pulse Wave Velocity (PWV) is regarded as the gold standard in the assessment of arterial stiffness2. While not used clinically in the United States, it has been validated and widely used in clinical settings throughout Europe3. Changes in PWV have been correlated with cardiovascular risk factors such as hypertension, obesity, impaired glucose tolerance, and dyslipidemia. Higher aortic stiffness has been associated with increased risk for a first cardiovascular event4, muscle mass decline5, and slower gait speed in subjects with peripheral arterial disease6. Assessment of PWV is accomplished by analyzing the speed with which a pulse of blood travels from the carotid artery to the femoral artery7. It is a relatively simple method with highly reproducible results. Advances in technology continue to simplify the procedure, reducing operator dependency and decreasing the amount of time required for the exam. The system used in our laboratory is produced by SphygmoCorand utilizes an EKG and a high-fidelity tonometer to acquire waveforms from the carotid and femoral pulses. The software then analyzes these variables to determine the velocity by 1) estimating the time that the pulse wave takes to travel between the carotid and femoral sites by referencing the timing of the pressure wave arrival at each arterial site relative to the electrocardiographic waveforms 2) dividing the measured distance between the two sites by the estimated time. The closer together the sampling sites, the less accurate the PWV, limiting the use of this method to larger vessels such as the femoral and carotid arteries. In addition to determining PWV, the system can calculate the pressure in the ascending aorta by applying a transfer function to the waveform produced by the radial pulse8 which is the &#34;central pressure&#34; and the pressure that most directly impacts the heart at time of ejection. This information can aid in the assessment of the arterial system&#39;s effect on the left ventricle in a reliable and noninvasive manner9. Alternative techniques used for the examination of vascular stiffness, such as carotid distensibility via echo tracking, require a great deal of technical expertise and are more time consuming than the PWV. Echo tracking also generally utilizes video-image analysis and therefore the accuracy of these measurements are limited3. Other techniques for examining vascular changes include assessment of carotid-intima medial thickness (IMT) which provides results more closely related to atherosclerotic progression although the cross-sectional and transverse images obtained can provide a crude approximation of carotid elastance. This method, however, is plagued by some of the same constraints as the distensibility studies described above. As researchers continue to look at the role arterial stiffness plays in various disease states, PWV measurement will likely continue to provide a quantitative and reproducible surrogate endpoint.

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			<td height="61" style="height:61px;width:64px;">SphygmoCor System</td>
			<td style="width:64px;">AtCor&#160;</td>
			<td style="width:64px;">SPT-304, Module EM3</td>
			<td style="width:64px;"></td>
		</tr>
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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.

<img alt="Table 1" fo:content-width="5in" fo:src="/files/ftp_upload/50817/50817table1highres.jpg" src="/files/ftp_upload/50817/50817table1.jpg" /> 
Table 1.&#160;PWV results are produced in meters per second. In healthy subjects, these speeds can range from an average 6.2 m/sec (in subjects under 30) to 10.9 m/sec (in subjects 70 and older). These values were established in the article &#34;Determinants of pulse wave velocity in healthy People and in the presenc...

Watch this Scientific Journal Video about Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging at JoVE.com

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

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 ...

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17.0K Views.  National Institute of Aging. 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.

Video: Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

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.

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 Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...