המחבר מבקש להודות במימון NHLBI (K08 HL102066 ו R01 HL114832).

<ol>
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The author has nothing to disclose.

ישנם שלושה שלבים קריטיים בהליך זה: 1) את המיקום של הטובוס ואוורור המתאים, 2) המיקום של קטטר IV הצוואר, ו -3) את המיקום הנכון של הקטטר PV בחדר השמאלי. קביעת קצב הנשימה המתאימה היא חלק חשוב של מתן תמיכה נשימתית. עכברים מודעים בדרך כלל לשמור על אוורור המכתשית עם נשימות שטחיות מה...

מחלות לב וכלי דם ממשיכה להיות גורם משמעותי לתמותה ותחלואה בעולם 1. מחלות של הלב להציב אתגרים קשים במיוחד בפיתוח טיפולים חדשים. התקדמות בגנטיקה לספק לאפשרות לזהות מספר רב של תורמים גנטיים פוטנציאל להתפתחות של מחלות לב. האופי האינטגרטיבי של מערכת הלב וכלי הדם מחייב המטרות הגנטיות הללו להיות מאומתים במודלים של בעלי חיים ללא פגע. הגמישות הגנטית והעלויות נמוכים דיור של העכבר הביאו אותו לקדמה עבור הערכת התפקיד הפיזיולוגי של גן מסוים. גודלו הקטן של העכבר מציבה מספר אתגרים ייחודיים להערכת תפקוד הלב. ישנן שיטות שיכולות לספק מידע לגבי תפקוד הלב, אבל רק מדידה בו זמנית של הלחץ חדרית ונפח מאפשר בנפח הלחץ (PV) ניתוח הלולאה של פונקציה חדרית. PV לולאות כלתפקוד לב ow להיות מנותח עצמאי של הקשר בינו לבין כלי הדם; גורם חשוב בקביעת התפקיד הפונקציונלי של מרכיב גנטי מסוים. הערכת לולאות לחצו-נפח נעשתה שימוש הוא בניסוי קליני במשך שנים רבות ספרות ענפה שקיימת לגבי הניתוח של נתונים אלה קובע 2,3. התאמת טכנולוגית לולאת PV העכבר כבר התקדמות חשובה להבנת פיזיולוגית murine לב 4-6. טכנולוגיות PV לולאת צנתר מבוסס זוג מתמר לחץ והשימוש מוליך להעריך נפח חדרית. היקף חדרית נקבע על ידי בחינת שינויי שדה חשמלי שנוצר על ידי קטטר. מודלי שיטה זו החדר כמו גליל, הגובה אשר מוגדר על ידי המרחק בין האלקטרודות על הקטטר ואת הרדיוס מחושבים הולכה של שדה חשמלי דרך הדםהחדר 7-9. אות מוליכות נמדדת הקטטר יש שני מרכיבים. הראשונה היא ההולכה דרך הדם; זה משתנה עם הנפח של החדר ומהווה האות העיקרית המשמשת לקביעת נפח חדרית. המרכיב השני נובע הולכה דרך ולאורך הקיר של החדר. זה נקרא מוליכות במקביל וצריך להסיר על מנת לקבוע את עוצמת קול חדרית המוחלטת. ישנן שתי מערכות זמינות מסחרית לאיסוף נתונים לחצו-נפח ששמש למחקר במעבדה לבין השיטה הנהוגה לחישוב ולהסיר מוליכות המקבילי ההבדל העיקרי ביניהם 6,10,11. צנתרים מוליכים דרש הזרקת סליין היפרטוני לחישוב מוליכות במקביל. הזרקה זה זמני משנה את המוליכות של דם החדר, בעוד המוליכות של הקיר נשארה קבועות. מנתונים אלה ניתן לקבוע אתרכיב של אות מוליכות שמקורה בדם ומה מגיע מקיר חדרית. גישה זו מניחה כי מוליכות במקביל אינן משתנות במהלך מחזור הלב. שיטת הקבלה מסתמכת על שינויים בשלב בתחום החשמל כדי להעריך את התרומה של קיר החדר לאות ההיקף הכולל. שיטה זו מסתמכת על מגוון של קבועים מראש עבור המוליכות של הדם לשריר הלב כדי לקבוע את עוצמת הקול הסופי, אבל עושה צעדים רציפים של מוליכות במקביל במהלך מחזור הלב. שתי מערכות אלה לספק הערכה טובה של נפח חדר השמאלי ואת ההבדלים ביניהם הם לא עשויים להיות משמעותיים מבחינה פיזיולוגית. המודל הגלילי של החדר והנחות אחרות העושה גישות אלה מבוססות קטטר לא מדויקות ככל שיטות אחרות, אך נתונים אלה הניתנים על בסיס פעימה אחרת פעימה כי הוא חיוני להערכת צעדים עצמאיים עומס של תפקוד לב. ההליך המתואר כאן משמש במעבדה שלי ספק נתונים עבור מספר רב של מחקרים הבוחנים את מנגנוני pathophysiological הבסיסיים של קרדיומיופתיה דיסטרופי 12-18. ההליך המתואר להלן הוא אחד משני שניתן להשתמש בהם כדי לקבל נתונים לולאה PV. בעוד רבים מן העקרונות חלים גם גישה, פרוטוקול זה יתמקד בגישת פסגה פתוח חזה; פרוטוקול החזה סגור כבר מפורט במקומות אחרים 19,20. בעוד הליך יתוארו בפירוט, עקרונות העל חשובים כדי לחשוף את הלב עם נזק מינימלי או ללב או לריאות. לאורך כל הפרוטוקול זה חשוב לזכור כי מדובר בהליך בלתי-הישרדות כי לאחר חשיפה טובה של הלב יש חשיבות קריטית עבור המיקום הנכון של הקטטר.

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measuring pressure volume loops in the mouse

הבנת הגורמים וההתקדמות של מחלת לב מהווה אתגר משמעותי לקהילה ביו. הגמישות הגנטית של העכבר מספקת פוטנציאל גדול כדי לחקור תפקוד לב ברמה המולקולרית. גודלו הקטן של העכבר עושה להציג כמה אתגרים בכל הקשור בביצוע phenotyping הלב מפורט. מזעור פיתוחים אחרים טכנולוגיה הפכו שיטות רבות של הערכה לבבית אפשרית העכבר. מבין אלה, אוסף סימולטני של נתונים בלחץ ונפח מספק תמונה מפורטת של תפקוד לב שאינו זמינה דרך כל אפנות אחרות. הנה נוהל מפורט לאיסוף נתוני לולאה לחץ-נפח מתואר. כולל דיון של העקרונות שבבסיס המדידים ואת המקורות הפוטנציאליים של שגיאה. ניהול הרדמה וגישות כירורגית נדונים בפירוט רב כפי שהם שניהם קריטי מדידה המודינמי באיכות גבוהה בהשגהים. עקרונות של פיתוח פרוטוקול המודינמי ואת ההיבטים הרלוונטיים של ניתוח נתונים גם מופנים.

לפי אמנה, נפח הוא זמם על ציר ה- X ואת הלחץ על ציר Y כמו באיור 1. הלולאות לחצו-הנפח כתוצאה מלחץ התוויית נגד נפח צריך להיות דומה מלבן, את הקצוות האנכיים המייצגים שינויי isovolumic בלחץ (כלומר, כאשר הן המיטרלי שסתומי עורקים סגורים). האופקי התחתון מי...

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

Measuring Pressure Volume Loops in the Mouse

כתב יד זה מתאר פרוטוקול מפורט לאיסוף נתונים לחץ-נפח מהעכבר.

מדידת לולאות נפח לחץ העכבר

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

Research

JoVE Journal

Biology

16.2K Views.  University of Minnesota.  כתב יד זה מתאר פרוטוקול מפורט לאיסוף נתונים לחץ-נפח מהעכבר. 

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.

מדידת לחץ דם בעכברים באמצעות לחץ נפח הקלטה, שיטת זנב השרוול

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.

צנתור של לולאות מעיים של מעלי גירה

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.

בשנת vivo ההדמיה לטיפולים במודל עכבר orthotopic של סרטן השחלות

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.

מדידת Caenorhabditis elegans חיים טווח של 96 צלחות microtiter טוב

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.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

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.

מדידת אוסמוטי המים החדירות מקדם (P<sub&gt; F</sub&gt;) של תאים כדוריים: Protoplasts צמח מבודד כדוגמה

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.

מדידת תגובת האכלה מושרה גלוטתיון בהידרה

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.

לחץ-נפח לב ניתוח לולאה באמצעות מוליכות קטטרים בעכברים

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.

מדידת הקשיחות של<em&gt; Ex Vivo</em&gt; עכבר Aortas באמצעות מיקרוסקופ כוח אטומי

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.

טכניקת כלית עכבר המבודדת perfused

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 ...