עבודה זו נתמכה בחלקה על ידי NIH מענקי מחקר R01AR062207, R01AR061484, R56AI100901, K01AR053210, ומענק מחקר ממוקד מחלה מקרן ראומטולוגיה מחקר (ליו יי).

כל הניתוחים הקשורים לבעלי החיים צריכים להיות מאושר על ידי הטיפול בבעלי חיים ובועדת האתיקה של מוסד המקומי, עם מאמץ שנעשה כדי למזער את כאב ואי נוחות הנגרמת על ידי הניתוח. 1. הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר C57BL / 6 עכברי זכרים 8-12 שבועות ישנים עם משקל גוף של כ 25 גרם לניתוח. </li><li style=";text-align:right;direction:rtl"> להרדים את בעלי החיים באמצעות זריקת intraperitoneal של קוקטייל המכיל גם xylazine (5 מ&quot;ג / קילוגרם) וקטמין (40 מ&quot;ג / קילוגרם). </li><li style=";text-align:right;direction:rtl"> לגלח את הברך עם סכיני גילוח, ולאחר מכן בניתוח לעטוף את בעלי החיים ולעקר את האתר כירורגית עם בטאדין ואלכוהול (3x) ולכסות את עיני עכבר עם משחה. </li></ol> 2. תהליך כירורגי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעשות חתך para-patellar 1 סנטימטר אורך המדיאלי לחשוף את מפרק הברך. </li><li style=";text-align:right;direction:rtl"> פתח את מפרק הברך בעדינות דרך העקירה רוחב של הפיקה ורצועות פיקה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את קופסית מפרק הברך אורכיםדרך חתך para-הפיקה המדיאלי בשלב 2.1. </li><li style=";text-align:right;direction:rtl"> לנתח קופסית מפרק הברך עם מלקחיים. </li><li style=";text-align:right;direction:rtl"> תפוס את חלקו האחורי של כף רגל אחורית עם יד שמאל, ולבצע עקירה לרוחב של הפיקה ורצועות פיקה עם מלקחיים. החזק את כף הרגל האחורית בעדינות ולוודא כדי למנוע טראומה בכפה. טוב יותר קופסית המפרק מנותחת, יידרש פחות הכוח כדי להפוך את העקירה. </li></ol></li><li style=";text-align:right;direction:rtl"> לטפטף מי מלח סטריליים על פני השטח של סחוס במפרק במהלך פעולה, כדי למנוע התייבשות של משטח סחוס. </li><li style=";text-align:right;direction:rtl"> לחתוך את רצועות meniscotibial המדיאלי המעגנת את המניסקוס המדיאלי לרמת השוקה. הימנע מפגיעה בסחוס מתחת המניסקוס המדיאלי. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהה את המניסקוס המדיאלי אשר מאתר בין condyle המדיאלי של עצם ירך ומישור המדיאלי של עצם השוק. </li><li style=";text-align:right;direction:rtl"> זהה את רצועת meniscotibial שמחברת את הצד הלטרלי של המניסקוס המדיאלי עם רוממות intercondylar של שוקה. </li><li style=";text-align:right;direction:rtl"> החזק את ההייnd כפה בעדינות עם יד, ולחתוך את רצועות meniscotibial המדיאלי בקפידה על ידי שימוש בלהב כירורגי מס 10. ודא שלא לגרום לפוצעים במפרק סחוס ורצועות אחרות. במקרים רבים, המכסה את כרית השומן צריכה להיות משכה לצד, אבל לא שיש להסירו על מנת לזהות את הרצועה. </li></ol></li><li style=";text-align:right;direction:rtl"> סגור את קופסית מפרק הברך עם תפר נספג 6-0. </li><li style=";text-align:right;direction:rtl"> סגור את העור עם 6-0 תפר משי. </li></ol> 3. טיפול שלאחר ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תחול ירידה של bupivacaine 0.25% אחד באתר כירורגית של כל עכבר כדי למזער את הכאב שלאחר ניתוח. </li><li style=";text-align:right;direction:rtl"> השאר את העכברים פעלו בחינם כדי לקבל מים ומזון. </li></ol> 4. ניקוד היסטולוגית של דגם DMM כירורגי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להקריב את העכברים בנקודות שצוינו זמן (למשל 4 שבועות, 8 שבועות, ו12 שבועות. כאן אנו הראו תוצאות של 8 שבועות כנציג) לאחר הניתוח. </li><li style=";text-align:right;direction:rtl"> מפרקי הברך כולו הם קבועים, decalcified, לפdded ידי פרפין ולאחר מכן נותח באופן סדרתי ב5 מרווח מיקרומטר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את הגפיים אחוריות שלמים עם מס &#39;10 להבים. </li><li style=";text-align:right;direction:rtl"> לנתח את העור ושרירים של הגפיים האחורי בזהירות, ולתקן את הדגימות עם PFA 4% במשך 3 ימים בRT. </li><li style=";text-align:right;direction:rtl"> הסר את PFA, ולנקות את הדגימות עם מים. לאחר מכן, decalcify הדגימות בEDTA על 4 מעלות צלזיוס למשך 2 שבועות. </li><li style=";text-align:right;direction:rtl"> מייבשים את הדגימות בשיפוע אתנול. בפירוט, לשמור את הדגימות באתנול 70% עבור שעה 1, ולאחר מכן לשנות את אתנול ולשמור את הדגימות בסט חדש של 70% אתנול לO / נ לאחר מכן, הכניס את הדגימות ב80% ו90% אתנול כתוצאה מכך, ולשמור אותם לשעה 1, בהתאמה, ואחריו אתנול 100% לO / נ </li><li style=";text-align:right;direction:rtl"> הסר את אתנול. שמור את הדגימות בoxylene עבור שעה 1, ולעבור לקבוצה חדשה של oxylene. חזור 3x שלב זה. </li><li style=";text-align:right;direction:rtl"> להטביע את הדגימות לתוך תבנית פרפין באמצעות מרכז הטבעה לייקה. לאחר מכן, הדגימות הם מחולק ב5 מיקרומטר באמצעות microtome סיבובי (לייקה RM2255, גרמניה) ולאחר מכן נאסף על גבי שקופיות זכוכית. </li><li style=";text-align:right;direction:rtl"> סעיפי sagittal סידוריים נחתכים עבור כל דגימה פורש אזור ממרכז condyle לרוחב למרכז של condyle המדיאלי. </li></ol></li><li style=";text-align:right;direction:rtl"> מכתים Safranin O מבוצע, ואחריו ניקוד וניתוח סטטיסטי באמצעות שיטת ניקוד OARSI כפי שתואר לעיל 25. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ראשית, deparaffinize השקופיות בoxylene, ומימה בשיפוע אתנול למים מזוקקים. לאחר מכן, הם מוכתמים השקופיות דרך ברזל Hematoxylin הפתרון של וייגרט, פתרון 0.05% ירוקים מהיר (FCF), 1% חומצה אצטית פתרון, ו0.1% O פתרון Safranin. הר השקופיות באמצעות מדיום שרף. </li><li style=";text-align:right;direction:rtl"> ציון השקופיות המוכתמות Safranin O מבוסס על אובדן proteoglycan (צבע אדום) בסחוס ואחוז ההרס במבנה סחוס, לעשות ניתוח סטטיסטי לציון היסטולוגית. </li></ol></li></ol>

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אנחנו בזאת מצהירים כי אין לנו שום ניגוד עניינים.

הוא דיווח כי הזן של עכברים הוא חשוב מאוד לזירוז מודל DMM, זנים שונים כמו של עכברים דרגות חומרה שונים של OA אחרי DMM, עם הרמה הגבוהה ביותר במתח 129/SvEv, ואחריו C57BL6, 129/SvInJ ולאחר מכן FVB / n 26. חלק גדול מtransgenes הוקם בעכברי C57BL6, כגון PGRN-/ - עכברים ששמשנו במחקר הנוכחי, שהם יחסית רגישי...

דלקת מפרקים ניוונית (OA), המכונה גם דלקת פרקים ניוונית, משפיע 15% מאוכלוסיית העולם של ומעל 46 מיליון אנשים בתוך ארצות הברית, ומאופיין בדלקת, ניוון סחוס, והיווצרות osteophyte 1. זה יכול להיות תוצאה של יחסי גומלין מורכבים של גורמים גנטיים, מטבולים, ביומכנית וביוכימיים. המנגנון הבסיסי של OA ימשיך להתחמק מהקהילה המדעית. קיימים כיום מודלים של בעלי חיים רבים שיכולים לחקות את הפתוגנזה של OA 2,3. זה חשוב להקים מודלים של בעלי חיים בעכברים בגלל שתי הזמינות של עכברים מהונדסים גנטי שונים ועלות התועלת של ניסויים. בין סוגים שונים של מודלים OA ניסויים השונים, היציבות הנגרמת בניתוח של המניסקוס המדיאלי מודל (DMM) היא מודל OA היטב קיבל בגלל שחזור הטוב שלה והתקדמות איטית יחסית בפיתוח OA. שתי התכונות האלה יש ליהיה מפתח להערכת התקדמות OA בטיפולים שונים או transgenes 3-8. עם זאת, העקביות של מודל OA כירורגית מושפעת מגורמים שונים במהלך הניתוח וכתוצאה מכך, היישום של מודל עכבר כירורגית מוגבל. Progranulin (PGRN) הוא גורם גדילה רב תפקודי המתבטא בתאים שונים. זה ידוע כי PGRN משחק תפקיד קריטי בתהליכים פיסיולוגיים ומחלות שונים, כגון ריפוי פצעים 9, tumorigenesis 10, ודלקת 11-15. מחקרים גם הראו כי אי ספיקה של PGRN יכולה לגרום למחל ניווניות של מערכת עצבים בבני האדם ועכברים 16-18. זה ידוע כי PGRN בא לידי ביטוי בסחוס במפרק אנושי, ורמתו גבוהה באופן משמעותי בסחוס של חולים עם OA ודלקת מפרקים שגרונית 19. בנוסף, PGRN גם ממלא תפקיד מכריע בהתפשטות הכונדרוציטים 20, בידול וendochondralהתאבנות של צלחת צמיחה במהלך פיתוח 21,22. לאחרונה, דיווח כי PGRN קומם TNF-α באמצעות קשירה לקולטנים TNF והציג פונקציה אנטי דלקתית במודלים של דלקת מפרקים דלקתיים 13,14,23,24. עם זאת, תפקידיו של PGRN בOA, במיוחד in vivo, נותר חידה. בזאת, אנו מציגים את ההליך כדי לגרום למודל DMM כירורגית, ולחקור את התפקיד של PGRN בפיתוח OA באמצעות הקמת מודל DMM ב WT וPGRN-/ - עכברים.

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establishment of a surgically-induced model in mice to investigate the protective role of progranulin in osteoarthritis

יציבות של המניסקוס המדיאלי מודל (DMM) הוא כלי חשוב ללימוד תפקידי pathophysiological של מולקולות דלקת פרקים רבות הקשורים בפתוגנזה של דלקת מפרקים ניוונית (OA) in vivo. עם זאת, מפורט, במיוחד הפרוטוקול דמיינו להקמת מודל מסובך זה בעכברים, אינו זמין. במסמך זה אנו ניצלנו wildtype וprogranulin (PGRN) - / - עכברים כדוגמאות להציג את פרוטוקול לגרימת מודל DMM בעכברים, ולעומת תחילתה של OA לאחר הקמת מודל מושרה בניתוח זה. הפעולות שבוצעו על עכברים היו או פעולת דמה, שנפתחה רק כמוסה משותפת, או בהפעלת DMM, אשר חתכה את רצועת menisco-השוקה וגרמה להתערערות של המניסקוס המדיאלי. חומרת דלקת מפרקים ניוונית הוערכה באמצעות assay היסטולוגית (למשל צביעת Safranin O), ביטויים של גנים הקשורים OA, צורת השפלה של מולקולות תאי מטריקס סחוס, וosteophyteation. פעולת DMM מושרה בהצלחה ייזום OA והתקדמות בשני wildtype וPGRN-/ - עכברים, ואובדן של גורם גדילת PGNR הוביל לפנוטיפ OA חמור יותר במודל מושרה בניתוח זה.

מודל DMM הוקם בהצלחה בעכברים, וחסר של PGRN מוגזם פיתוח OA בניתוח מושרה. פעולות דמה וDMM (איור 1) בוצעו ב WT וPGRN-/ - עכברים. 8 שבועות לאחר הניתוח, העכברים הוקרבו, ומכתים Safranin O בוצע על החלקים ממפרקי הברך, ואחרי ניתוח סטטיסטי ש?...

Watch this Scientific Journal Video about Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis at JoVE.com

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

אנו מתארים פרוטוקול ליציבות של המניסקוס המדיאלי מודל (DMM) בעכברים, כלי יעיל למחקר דלקת מפרקים ניוונית (OA). בנוסף, יש לנו הפגנו מחסור זה של פיתוח progranulincan להגזים OA והתקדמות באמצעות מודל זה, המציין כי progranulin משחק תפקיד מגן בפתוגנזה של OA.

הקמת דגם בניתוח מושרה בעכברים כדי לחקור את תפקיד מגן של progranulin בדלקת מפרקים ניוונית

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

establishment-surgically-induced-model-mice-to-investigate-protective

Research

JoVE Journal

Bioengineering

27.3K Views.  NYU Hospital for Joint Diseases.  אנו מתארים פרוטוקול ליציבות של המניסקוס המדיאלי מודל (DMM) בעכברים, כלי יעיל למחקר דלקת מפרקים ניוונית (OA). בנוסף, יש לנו הפגנו מחסור זה של פיתוח progranulincan להגזים OA והתקדמות באמצעות מודל זה, המציין כי progranulin משחק תפקיד מגן בפתוגנזה של OA. 

Video: Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparametric מיפוי אופטי של הלב Langendorff-perfused הארנב

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

יצירת דו מימדי מצעים Patterned עבור החזקת חלבון ו-Cell

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

עיצוב של bioreactor לחץ על מחזורי Ex Vivo מחקר של מסתמי הלב אבי העורקים

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

כימות של פונקציה הגלובלית הדיאסטולי על ידי ניתוח המבוסס על דוגמנות Kinematic של Transmitral זרימה באמצעות הפורמליזם מילוי Parametrized הדיאסטולי

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

מישורי צבע מערכת דיפוזיה לחקירת Chemotaxis בקולגן מטריקס 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

<em&gt; במבחנה</em&gt; דגם תרבות איברים של הדיסק הבין חולייתי Murine

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

יצירת מודל גיאומטרי מבנית מציאותי סופיים של Cardiomyocyte ללמוד את התפקיד של אדריכלות הסלולר Cardiomyocyte במערכות ביולוגיות

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: גישה חזקה, לא פולשנית כדי לחקור את חילוף החומרים האנרגיה התאית

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

Nanoemulsion מיוצב Tripeptide של חומצה אולאית

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

שלושה תלת-ממד של מטריקס-Adipocyte מודל תרבות המודל ללימוד תא מטריקס-מטבולית הסלולר

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...