The author would like to acknowledge funding from NHLBI (K08 HL102066 and R01 HL114832).

<ol>
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The author has nothing to disclose.

There are three critical steps in this procedure: 1) the placement of the endotracheal tube and appropriate ventilation, 2) placement of the jugular IV catheter, and 3) the proper placement of the PV catheter in the left ventricle. Determining the appropriate respiratory rate is an important part of providing ventilatory support. Conscious mice generally maintain alveolar ventilation with rapid shallow breaths. In general, ventilated mice will have much larger tidal volumes. Thus a slower respiratory rate is required. This is important as too little ventilation will result in respiratory acidosis and too much ventilation will lead to respiratory alkalosis, both conditions that will alter cardiac function. A simple way of optimizing the respiratory rate is to take cues from the mouse&#39;s respiratory effort and use the lowest respiratory rate that eliminates respiratory effort from the anesthetized mouse.
A critical feature of PV loop analysis is to maintain a sufficient level of anesthesia without significant depression of cardiac function. There are many anesthetic regimes that can be successfully applied to perform PV loop analysis. These can be divided into two broad categories: inhalant and injectable anesthetics. Within this latter group are a variety of mixtures that can be used, however, many of these mixtures have potential cardio-depressive effects 19. Of the injectable cocktails, urethane based anesthetics have the least cardio-depressive effects 4,19. Take care to limit exposure of personnel to urethane as it is a suspected carcinogen 28. Isoflurane and sevoflurane are the currently available inhalant anesthetic agents. Both of these agents have cardio-depressive effects at doses sufficient for surgical manipulation. Importantly, inhaled anesthetic agents can be titrated to effect. This allows a higher analgesic dose during the surgical preparation and then a lower sedative dose for the measuring of cardiac function, thus minimizing the cardio-depressive effects of these compounds.
If the hemodynamic parameters are below the normal levels, there are several common causes. First is low blood volume, secondary to blood loss or evaporation. This complication is generally addressed with the fluid administration outlined in section 2.7. As noted in section 1.5, maintenance of core body temperature is also very important for the assessment of hemodynamic function. Thus careful monitoring is essential, a digital feedback system can be useful for maintaining core body temperature during recording. Ensuring that the anesthetic has been properly reduced during the measurement phase is also an important aspect of improving cardiac performance.
Measuring load-independent parameters of contractility is one of the primary advantages of PV loop analysis. The real-time simultaneous collection of pressure and volume data provides the unique capacity to measure the hemodynamic changes in response to varying loading conditions on the heart. This analysis allows the contractile function of the heart to be isolated from the actions of the vessels. Reductions in preload through occlusion of venous return have been used to assess load-independent measures of contractility in many animal models and man 4,7,12-16,18,29-32. In small rodents, this procedure results in a curvilinear end-systolic pressure-volume relationship 4,31,33. This likely results from the reduction in coronary perfusion that dramatically accelerates the decline in systolic function 34. In the mouse, 2 - 3 sec of gentle abdominal compression results in a rightward shift of the PV loops. This results from both an increase in afterload and preload 12. Prolonged increases in afterload result in an increase in contractile function, a phenomena called the Anrep effect 35. However the short duration of the abdominal compression used in these studies indicates that the Anrep effect does not affect cardiac function with this procedure. In other studies, acute aortic constriction in dogs was demonstrated to increase contractile function, but this was hypothesized to result from the effects of reduced systemic perfusion 36. Again the duration and severity of the disruption of aortic flow resulting from abdominal compression, as described here, is not sufficient to significantly increase contractile function secondary to hypo-perfusion of systemic tissues. Analysis of PV loops obtained by abdominal compression align well with PV loops obtained shortly after caudal vena cava occlusion. Together, these observations demonstrate that the transient abdominal compression described here does not significantly alter the contractility of the heart. Furthermore, this procedure provides an important method to assess the passive properties of the heart over a broader range of end-diastolic volumes.
The analysis of load-independent measures requires the selection of specific PV loops to be included in the analysis. It is critically important that this be done consistently across an experimental data set. Positive pressure ventilation creates a hemodynamic artifact through respiratory dependent alterations of preload to the left ventricle 37. To avoid this artifact, PV loops should be collected during short periods of apnea (3 - 4&#160;sec). Pauses in respiration are particularly useful as they provide better control of experimental changes in loading on the heart. It is important to keep these periods of apnea short to avoid hypoventilation. The procedures described in this manuscript collect load-independent data from two distinct procedures, caudal vena cava occlusion and abdominal compression, collected at roughly the same time. The loops isolated from these two procedures should be combined and analyzed together as they both measure the function of the same heart under, roughly, the same conditions. There are several principles to consider when selecting loops for analysis. Avoid arrhythmic beats. The beat following a dropped beat is always abnormally large and premature beats are abnormally small; both will disrupt the analysis of the data. Avoid beats where the pressure is declining but volume is constant. These are common in the mouse following the occlusion of the caudal vena cava and are likely secondary to poor perfusion of the myocardium. Finally, include only data from beats directly after the change in loading; the beats during the recovery period are likely affected by alterations in sympathetic nervous activity secondary to changes in systemic blood pressure resulting from manipulation of the loading conditions of the heart.
PV loop analysis provides an extremely detailed assessment of cardiac function. When applied in conjunction with the genetic flexibility and low housing costs of the mouse it can provide a practical means of assessing cardiac physiology on the molecular level. There are several important limitations that must be considered when deciding to perform these assays. First, this is an invasive procedure in which mice are anesthetized, which may impact important aspects of cardiac function. Furthermore, interpretation of PV loop data requires a detailed understanding of cardiac physiology both to identify patterns within the data and potential confounding variables. Additionally, because these assays are terminal they cannot be used to assess the same mouse repeatedly. The ventricular volumes derived from PV catheters tend to be less accurate than the anatomical ventricular volumes provided by MRI. This is not surprising as PV catheters model the ventricle as a cylinder, which is clearly an estimate of the overall volume of the ventricle. The real strength is the ability to collect this volume information at a high frequency, thus allowing beat-to-beat analysis of changes in ventricular volume.
The collection and analysis of PV data from the mouse can be challenging, but the method provides information regarding cardiac function that is not available through any other methodology. This procedure provides the most complete picture of cardiac function available and its utilization in the murine model will provide an important platform for the determination of the molecular underpinnings of complex cardiac disease states such as heart failure and inherited cardiomyopathies. This manuscript provides detailed information on the most critical aspects of performing this procedure. However, like all complicated procedures, this requires practice to build the microsurgical skills that are necessary to successfully perform these experiments.

Cardiovascular disease continues to be a significant cause of mortality and morbidity throughout the world 1. Diseases of the heart present particularly difficult challenges in developing new therapies. Advances in genetics provide for the possibility to identify a multitude of potential genetic contributors to the development of heart disease. The integrative nature of the cardiovascular system requires that these genetic targets be validated in intact animal models. The genetic flexibility and low housing costs of the mouse have brought it to the forefront for the assessment of the physiological role of a given gene. The small size of the mouse presents some unique challenges for the assessment of cardiac function. There are several modalities that can provide information regarding cardiac function, but only the simultaneous measurement of ventricular pressure and volume allows pressure-volume (PV) loop analysis of ventricular function. PV loops allow cardiac function to be analyzed independent of its connection to the vasculature; an important factor in determining the functional role of a particular genetic element.
The assessment of pressure-volume loops has been used both experimentally and clinically for many years and extensive literature exists regarding the analysis of these data sets 2,3. The adaptation of PV loop technology to the mouse has been an important advancement for the understanding of murine cardiac physiology 4-6. Catheter based PV loop technologies couple a pressure transducer and the use of conductance to estimate ventricular volume. The ventricular volume is determined by examining changes in an electrical field generated by the catheter. This method models the ventricle as a cylinder, the height of which is defined by the distance between the electrodes on the catheter and the radius is calculated from conduction of an electrical field through the blood in the ventricle 7-9. The conductance signal measured by the catheter has two components. The first is the conduction through the blood; this varies with the volume of the ventricle and constitutes the primary signal used to determine ventricular volume. The second component results from conduction through and along the wall of the ventricle. This is called parallel conductance and must be removed in order to determine the absolute ventricular volume. There are two commercially available systems for the collection of pressure-volume data in the research laboratory and the method used to calculate and remove the parallel conductance is the primary difference between them 6,10,11. Conductance catheters require the injection of hypertonic saline for the calculation of parallel conductance. This injection transiently changes the conductivity of the blood in the ventricle, while the conductivity of the wall remains constant. From this data it is possible to determine the component of the conductance signal that originates from the blood and what comes from the ventricular wall. This approach assumes that parallel conductance does not vary during the cardiac cycle. The admittance method relies on phase changes in the electrical field to assess the contribution of the ventricular wall to the overall volume signal. This method relies on a variety of predetermined constants for the conductivity of the blood and myocardium to determine the final volume, but makes continuous measures of parallel conductance during the cardiac cycle. Both of these systems provide good estimates of left ventricular volume and the differences between them are not likely to be physiologically significant. The cylindrical model of the ventricle and other assumptions render these catheter-based approaches not as accurate as other modalities, but this data is provided on a beat-by-beat basis that is essential for the assessment of load independent measures of cardiac function.
The procedure outlined here is used in my laboratory and has provided data for a large number of studies examining the basic pathophysiological mechanisms of dystrophic cardiomyopathy 12-18. The procedure outlined below is one of two that can be used to obtain PV loop data. While many of the principles are applicable for either approach, this protocol will focus on an open-chest apical approach; a closed chest protocol has been detailed elsewhere 19,20. While the procedure will be described in detail, the important overarching principles are to expose the heart with minimal damage to either the heart or the lungs. Throughout the protocol it is important to remember that this is a non-survival procedure and that having a good exposure of the heart is critically important for the proper placement of the catheter.

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			<td height="42" style="height:42px;width:216px;">Dumont 5/45 (2)</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Castroviejo Micro Dissecting Spring Scissor</td>
			<td style="width:127px;">Roboz Instruments</td>
			<td style="width:119px;">RS-5668</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Octogon Forceps - Serrated/Curved</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">11041-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Octogon Forceps - Serrated/Straight</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">11040-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissector Scissors- Heavy Blade</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">14082-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transpore Surgical Tape</td>
			<td style="width:127px;">3M</td>
			<td style="width:119px;">1527-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3-0 Silk Suture</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">18020-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TOPO Ventilator</td>
			<td style="width:127px;">Kent Scientific</td>
			<td style="width:119px;">TOPO</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Martin ME 102 Electrosurgical Unit</td>
			<td style="width:127px;">Harvard Apparatus</td>
			<td style="width:119px;">PY2 72-2484</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:127px;">Lucca Technologies</td>
			<td style="width:119px;">GenieTouch</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stereomicroscope with boom stand</td>
			<td style="width:127px;">Nikon</td>
			<td style="width:119px;">SMZ-800N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermocouple Thermometer</td>
			<td style="width:127px;">Cole Parmer</td>
			<td style="width:119px;">EW-91100-40</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T/Pump Warm Water Recirculator</td>
			<td style="width:127px;">Kent Scientific</td>
			<td style="width:119px;">TP-700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ADVantage Pressure-Volume System</td>
			<td style="width:127px;">Transonic</td>
			<td style="width:119px;">ADV500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Data Acquision and Analysis</td>
			<td style="width:127px;">DSI</td>
			<td style="width:119px;">Ponemah ACQ-16</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring pressure volume loops in the mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

By convention, volume is plotted on the X-axis and pressure on the Y-axis as in Figure 1. The pressure-volume loops resulting from plotting pressure against volume should resemble a rectangle, the vertical edges representing isovolumic changes in pressure (i.e., when both mitral and aortic valves are closed). The bottom horizontal represents ventricular filling through the mitral valve and the upper horizontal portion represents ventricular emptying through the aortic valve. In a healthy wild type mouse left ventricular pressures of 90 - 110 mmHg are expected with maximal dP/dt of 8,000 - 12,000 mmHg/sec (see Table 1 for ranges of normal hemodynamic parameters). The normal ranges provided in the table are based on values obtained from wild type C57BL/10 and C57BL/6 mice; however, it is important to note that there are significant differences between strains 21. The procedure outlined here focuses on the use of an apical stab incision to position the catheter within the left ventricle. Another popular approach is to insert the catheter retrograde through the aortic valve following catheter insertion in the right carotid artery. The retrograde approach as the advantage in that it can be performed with a closed chest, resulting in the maintenance of normal intra-thoracic pressures; however, animals are frequently ventilated during this procedure which limits this advantage. The closed chest approach limits control over the catheter orientation within the ventricle, while the open chest approach provides greater ability to maneuver the catheter within the ventricle. A potential problem with retrograde insertion of the catheter is the potential for outflow track obstruction. The diameter of the mouse aorta varies from 0.8 to 1.2 mm 22,23, the diameter of commercially available pressure-volume catheters ranges from 0.33 (1.0 French) to 0.47 mm (1.4 French). The relative sizes of these catheters in the context of a large and small aorta are depicted in Figure 2. The fraction of the outflow tract blocked by the catheter crossing the aorta can become a significant issue in smaller hearts and should be taken into account when performing PV loop studies in small hearts. There are several artifacts of measurement that can complicate the analysis of PV data, one of the most common is catheter entrapment. This is evident as a spike in pressure at the end of systole likely resulting from direct compression of the pressure transducer by a papillary muscle or other dynamic structure within the ventricle (Figure 3). This is problematic, because most methods for determining systolic function use the maximum pressure. The systolic pressure and maximum derivative of pressure (dP/dt) from data sets with catheter entrapment need to be examined closely and the analysis may need to be altered to obtain meaningful data.
Pressure-Volume loops can be used in a wide variety of protocols to assess cardiac function. These include assessments of cardiac reserve via &#946;-adrenergic stimulation 12,14,16,17. A wide variety of drugs may be infused to assess any acute effects on cardiac physiology. Control of the airway also allows for the administration of altered gas mixtures allowing the effects of hypoxia and/or acidosis on cardiac function to be addressed directly 24-27. Furthermore, analysis of this PV data can also provide a detailed assessment of the passive properties of the left ventricle, which can be altered significantly in various disease states 12.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Normal Range</td>
		</tr>
		<tr>
			<td>Systolic Pressure</td>
			<td>90 &#8211; 110 mmHg</td>
		</tr>
		<tr>
			<td>Diastolic Pressure</td>
			<td>4 &#8211; 10 mmHg</td>
		</tr>
		<tr>
			<td>Maximum dP/dt</td>
			<td>8,000 &#8211; 12,000 mmHg/sec</td>
		</tr>
		<tr>
			<td>Minimum dP/dt</td>
			<td>-8,500 &#8211; -12,000 mmHg/sec</td>
		</tr>
		<tr>
			<td>Tau</td>
			<td>5 &#8211; 6 sec</td>
		</tr>
		<tr>
			<td>Heart Rate</td>
			<td>550 &#8211; 600 bpm</td>
		</tr>
		<tr>
			<td>Cardiac Output</td>
			<td>10 &#8211; 13 ml/min</td>
		</tr>
		<tr>
			<td>Ejection Fraction</td>
			<td>40 &#8211; 60%</td>
		</tr>
	</tbody>
</table>
Table 1. Normal Values for Selected Hemodynamic Parameters.
<img alt="Figure 1" src="/files/ftp_upload/53810/53810fig1.jpg" /> 
Figure 1: Representative Pressure-volume Loops from a Wild Type C57BL/10 Mouse. Representative data that was collected using the procedures described in this manuscript. <a href="https://www.jove.com/files/ftp_upload/53810/53810fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53810/53810fig2.jpg" /> 
Figure 2: Schematic Representation of the Importance of Aortic Diameter and Potential Outflow Track Obstruction. Schematic drawing of the relative sizes of the mouse aorta and the commercially available pressure-volume catheters for mice. These data underscore the importance of considering outflow track obstruction when assessing smaller mice using a retrograde catheter insertion approach. <a href="https://www.jove.com/files/ftp_upload/53810/53810fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53810/53810fig3.jpg" /> 
Figure 3: Pressure-volume Loops with Catheter Entrapment. These data represent an example of a pressure spike in late systole, likely resulting from direct compression of the catheter by an internal cardiac structure. Declines in end-diastolic volume (A) result in an increase in the pressure peak as the ventricle gets smaller, however the systolic pressure prior to the peak declines as expected, depicted as a dashed line in B. (C and D) represent the pressure-volume loops (C) and pressure-time (D) curves following abdominal compression. As the left ventricular volume increases, the pressure spike gets smaller and the underlying increases in systolic function becomes apparent; dashed line in D. <a href="https://www.jove.com/files/ftp_upload/53810/53810fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring Pressure Volume Loops in the Mouse at JoVE.com

Measuring Pressure Volume Loops in the Mouse

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

measuring-pressure-volume-loops-in-the-mouse

Nanyang Technological University

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JoVE Journal

Biology

16.2K Views.  University of Minnesota. This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Video: Measuring Pressure Volume Loops in the Mouse

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...