עבודה זו מומנה על ידי מענק מטעם המרכז לחקר חדשנות מניעה וטיפול במחלות דרכי הנשימה, NINDS מענק (NS052574), ומתוך התוכנית סנדלר לחקר אסטמה.

<ol>
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אין ניגוד עניינים הצהיר.

הפרוטוקול המובא כאן מספק הכנה פיזיולוגית להערכת תפקוד דרכי הנשימה השריר. אנו פועלים בדרך כלל 3-4 אמבטיה ההכנות איברים בו זמנית, לעומת זאת, מערכות ארוזים מראש זמינים ממספר ספקים המאפשרים מדידה בו זמנית של עד 8 ההכנות (ADInstruments, דיוק מכשירים בעולם, ומכשירים הרווארד). ניצלנו מספר מתמרים בכוח איברים ורקמות אמבטיות עם תוצאות מקבילים. עם זאת, אנו מוצאים כי גירוי השדה החשמלי מספק כמה שונות בהתבסס על הבדלים קלים בין אלקטרודות גירוי גודל, מרחק בין לוחות אלקטרודה, ואת המיקום של ההכנה בתוך שדה חשמלי. לכן, טיפול נוסף צריך להתבצע כדי להפוך אלקטרודות שדה דומים ככל האפשר. אחד הפרמטרים החשובים ביותר במדידות כוח שווי מידה הוא הנושא של נרמול הצירים כדי לפצות על שינויים מסת שריר, אוהבריאות של רקמת השריר בין הכנות שונות. בין השאר, ההבדלים ניתן למזער על ידי השוואת בעלי החיים בגיל דומה מאותו המין (נקבות עכברים נוטים ליצור מתח מופחת קנה הנשימה). יתר על כן, מצאנו כי נורמליזציה למשקל קנה הנשימה רטוב או יבש חסר דיוק מספיק, אולי בשל גודלו הקטן של העכבר קנה הנשימה. במקום זאת, השימוש מרובות, התכווצויות, אשלגן גבוהה יש יתרון די. התכווצויות אשלגן גבוהה לשרת שתי מטרות. התכווצות אשלגן גבוהה נראה &quot;לעורר&quot; את השריר קנה הנשימה ומבטיח כי ההתכווצויות הן לשחזור לפני שתמשיך עם אתגרים ניסיוניים. התכווצות אשלגן גבוהה גם נראה נורמליזציה מדויק על מסת השריר הפעיל שנמצא בהכנה. לכן, מדידות מתח הניסוי באים לידי ביטוי לעתים קרובות ככוח מנורמל לצמצום אשלגן גבוהה. בנוסף, איכות הכנה ניתן להעריך באמצעות חוזה אשלגן גבוהה הנגרמתיון. לדוגמה, אנו מוצאים כי 8 - 10 גברים ישנים בשבוע עכברים C57BL/6J יש התכווצות אשלגן גבוהה הנגרמת של 20 ± 3.8 MN (ממוצע ± סטיית תקן, n = 17). אם כמה חוזים הכנה קנה הנשימה גם מתחת לטווח זה (להלן 12 mn או שתי סטיות תקן מתחת לממוצע), אז זה נחשב בדרך כלל &quot;פגום&quot; ולא מנוצלים לניסויים. לחלופין, נורמליזציה של מתח למתח המקסימלי על אגוניסט להרוות כולינרגית ניתן להשתמש. זה שימושי לצפייה שינויים רגישות אגוניסט, אבל עלול להחמיץ שינויים שההשפעה התכווצות מקסימלית. שיטות הוצגו להפעיל התכווצות או באמצעות אגוניסט כולינרגית או עם גירוי חשמלי. יישום אגוניסט כולינרגית לאמבטיה רקמות ישירות מפעיל שריר חלק. לעומת זאת, בתדירות EFS ​​גירוי בינוני (עד 25 הרץ) ברוב של התכווצות מתווכת באמצעות הפעלת עצב שחרור neurotransmitter 22. כך החוקר יש את ההזדמנות לחקור סוכני המשפיעים presynaptic / עצב בתיווך התכווצות באמצעות גירוי EFS. לבסוף, מחקרים מראים סוגי תאים אחרים, כגון תא הפיטום 23, ותאי אפיתל 24 גם להשפיע contractility בהכנת קנה הנשימה מבודד. לכן, כהכנה חוץ גופית קנה הנשימה העכבר מספק assay תפקודית חזקים במשך מספר סוגי תאים המשפיעים contractility וחלק דרכי הנשימה השריר. לסיכום, העכבר כהכנה קנה הנשימה חוץ גופית הייתה שימושית במיוחד בניתוח של שינויים גנטיים המשפיעים על תפקוד דרכי הנשימה. כמה דוגמאות כוללות ניתוח knockouts גנים של תעלות יונים 17,20,25,26, קולטנים metabotropic 27,28,29,30, במורד איתות מפלי 31. בנוסף, העכבר אנטיגן תיגר משמש לעתים קרובות ללימודי אסתמה 32 ו במבחנהקנה הנשימה הכנה מספקת assay שימושי שינויים contractility כי מתפתח הפיתוח הבא של אסתמה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> אנלוגי, ממיר דיגיטלי </td><td> ADInstruments </td><td> PowerLab 4/35 </td><td></td></tr><tr><td> Carbachol (כלוריד Carbamoylcholine) </td><td> Sigma-Aldrich </td><td> C4832 </td><td> 10 -2 M במים (aliquots ניתן לאחסן ב -20 ° C) </td></tr><tr><td> Charting תוכנה </td><td> ADInstrtuments </td><td> LabChart </td><td></td></tr><tr><td> חימום Circulator </td><td> Haake </td><td> מיקסר מיל MM400 </td><td></td></tr><tr><td> כוח איזומטרי מתמר </td><td> קנט מדעי </td><td> TRN001 </td><td></td></tr><tr><td> ממריץ </td><td> דשא טכנולוגיות </td><td> S88 פלט כפול Squהם דופק ממריץ </td><td></td></tr><tr><td> רקמות אמבטיה </td><td> WPI </td><td> 47264 </td><td></td></tr></tbody></table>

in vitro measurements of tracheal constriction using mice

מהונדס לבין עכברים בנוקאאוט היו כלים רבי עוצמה לחקר הפיזיולוגיה ו פתופיזיולוגיה של דרכי הנשימה 1,2. ב tensometry חוץ גופית ההכנות לקנה הנשימה מבודדים הוכיחה להיות שימושי assay של השריר החלק בדרכי הנשימה (ASM) בתגובה כויץ בעכברים מהונדסים גנטית. אלה בהכנות חוץ גופית לקנה הנשימה הם פשוטים יחסית, לתת מענה חזקים, ולשמור את שני קצות העצבים הכולינרגית תפקודית ותגובות שרירים, גם לאחר incubations ארוכים. Tensometry קנה הנשימה מספקת גם assay תפקודית ללמוד מגוון רחב של השני איתות המסלולים שליח המשפיעות על התכווצות של שריר חלק. התכווצות של קנה הנשימה מתווכת בעיקר על ידי, העצבים הפאראסימפתטית כולינרגית המשחררים אצטילכולין על ASM (איור 1). קולטני אצטילכולין ASM הגדולים הם M2 ו M3 muscarinic שהם G I / O ו GQ קולטנים מצמידים, בהתאמה <sup&gt; 3,4,5. רצפטורים M3 לעורר התכווצות ידי צימוד כדי GQ להפעיל phospholipase C, IP3 גידול הייצור IP3 בתיווך לשחרור סידן reticulum sarcoplasmic 3,6,7. M2 / G I / O איתות הוא האמין כדי לשפר את הצירים על ידי עיכוב של cyclase adenylate מובילה לירידה ברמות הסכמי קמפ 5,8,9,10. מסלולים אלה מהווים את מה שנקרא &quot;pharmaco-התכווצות צימוד&quot; של השריר החלק בדרכי הנשימה 11. בנוסף, איתות כולינרגית באמצעות קולטנים מסוג M2 (ו מווסתת על ידי איתות M3) כוללת מסלולים depolarize ASM אשר בתורו להפעיל מסוג L, מתח תלויי תעלות סידן (איור 1) ואת זרימת סידן (מה שנקרא &quot;עירור-התכווצות צימוד&quot; ) 4,7. סקירות מפורטות יותר על איתות המסלולים השליטה היצרות דרכי הנשימה ניתן למצוא 4,12. המסלולים הנ&quot;ל נראה כי שימור בין עכברים ומינים אחרים. עם זאת, וקנה הנשימה עכבר שונים מינים אחרים אניn חלק איתות המסלולים. הבולט ביותר הוא חוסר התגובה כויץ ל 13,14 ו היסטמין אדנוזין, שניהם ידועים מאפננים ASM בבני אדם ומינים אחרים 5,15. כאן אנו מציגים פרוטוקולים הבידוד של טבעות לקנה הנשימה של Murine ואת במדידה חוץ גופית התפוקה כויץ שלהם. כללו תיאורים של תצורת ציוד, בידוד קנה הנשימה טבעת ומדידות ההתכווצות. דוגמאות ניתנות לעורר התכווצויות בעקיפין באמצעות גירוי אשלגן גבוהה של עצבים ישירות על ידי שלילת קוטביות של שריר ASM להפעיל מתח תלוי זרם סידן (1. גבוהה K +, איור 1). בנוסף, שיטות מוצגים גירויים של העצבים לבד באמצעות גירוי שדה חשמלי (2. EFS, איור 1), או גירוי ישיר של השריר ASM באמצעות מוליך עצבי אקסוגני להחיל אמבטיה (3. אקסוגני אח, איור 1). זה flexibility וקלות ההכנה הופך את המודל טבעת מבודדת קנה הנשימה assay חזק ומתפקד במשך מספר איתות מפלי המעורבים התכווצות שרירים דרכי הנשימה חלקה.

Watch this Scientific Journal Video about In vitro Measurements of Tracheal Constriction Using Mice at JoVE.com

In vitro Measurements of Tracheal Constriction Using Mice

העכברים הטרנסגניים להיות שימושי מאוד ייחוס תפקיד פיזיולוגי ל הגנים. כמו מחקר, כגון בכלל, מחקרים תפקודיות של דרכי הנשימה, במיוחד, עברו שינוי מדהים לכיוון מודלים Murine. כאן אנו מספקים פרוטוקולים<em&gt; במבחנה</em&gt; מחקרים קנה הנשימה התכווצות כדי להעריך את תפקוד השריר החלק בדרכי הנשימה murine.

במדידות חוץ גופית של היצרות קנה הנשימה באמצעות עכברים

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

In vitro Measurements of Tracheal Constriction Using Mice

20.7K Views.  UT Health Science Center, San Antonio. העכברים הטרנסגניים להיות שימושי מאוד ייחוס תפקיד פיזיולוגי ל הגנים. כמו מחקר, כגון בכלל, מחקרים תפקודיות של דרכי הנשימה, במיוחד, עברו שינוי מדהים לכיוון מודלים Murine. כאן אנו מספקים פרוטוקולים במבחנה מחקרים קנה הנשימה התכווצות כדי להעריך את תפקוד השריר הח?...

Video: In vitro Measurements of Tracheal Constriction Using Mice

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

היצרות כרונית של העצב Sciatic ואת רגישות יתר לכאב בדיקה אצל חולדות

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

עולה אבי העורקים תעוקה בחולדות ליצירת עומס יתר לחץ לב היפרטרופיה הדגם

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

חליטות עורק הראשי לpharmacokinetic וpharmacodynamic ניתוח של taxanes בעכברים

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

מדידה עולה אבי העורקים נוקשים<em&gt; In vivo</em&gt; בעכברים באמצעות אולטראסאונד

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

מודל של שיפוץ לב באמצעות התכווצות של אבי העורקים בבטן בחולדות

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

פולשנית רוחבי אבי העורקים התכווצות עכברים

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

הטכניקה של פולשנית התרחבות אבי העורקים רוחבי בעכברים עבור אינדוקציה של גדילת

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

במבחנה and In Vivo זיהוי של Mitophagy ב תאים אנושיים, C. Elegansועכברים

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

דגם סגור-החזה לזירוז רוחבי. התרחבות אבי העורקים בעכברים

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...