הצהרת אתיקה: כל הניסויים אושרו על ידי ועדת הטיפול ושימוש בבעלי חיים המוסדיים. טיפול בבעלי חיים היה בהתאם להנחיות מוסדיות. עכברי ICR נקבה (7-8 שבועות; 25 - 30 גר &#39;) שוכנו בממוזג, מתקני בעלי חיים אור מבוקר של הפקולטה לרפואה ע&quot;ש סאקלר באוניברסיטת תל-אביב. בטווח, חיות היו מורדמים עם מנת יתר של חומרי הרדמה. 1. Fibred Confocal מיקרוסקופ פלואורסצנטי כיול (FCFM) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להפעיל את המכשיר. </li><li style=";text-align:right;direction:rtl"> חבר את microprobe (mini0 / 30). </li><li style=";text-align:right;direction:rtl"> לכייל את המכשיר בהתאם להוראות יצרן. </li></ol> 2. עכברים הכנת הדמיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים על ידי הזרקה תת עורית של שני Ketaset (100 מ&quot;ג / קילוגרם) וXYL-M2 (6 מ&quot;ג / קילוגרם). לאשר הרדמה תקינה על ידי חוסר תגובה לבוהן קמצוץ. </li><li style=";text-align:right;direction:rtl"> לחתוך את העור מתחת למפשעה כדי<html> לחשוף את כלי דם הירך. שמור את אתר החתך לח עם מי מלח הבא חתך. </li><li style=";text-align:right;direction:rtl"> מחממים את הזנב על ידי שימוש בשקית (או כפפה) מלאים במים חמים (לא חם מדי לגעת) למשך כ -30 שניות. הכן (IV) מחלף תוך ורידי לממשל של dextran FITC (חומר ניגוד) ושל שני מלוחים או סוכן כימותרפיות, על ידי החדרת מחט (30 G, 1/2 אינץ &#39;) לוריד הזנב והצמדת מזרק 1 מ&quot;ל לזה . להבטיח את הווריד פתוח על ידי הזרקת תמיסת מלח. הערה: ממשל IV של dextran FITC (משקל מולקולרי גבוה; 100 μl; 10 מ&quot;ג / מיליליטר; 2,000 KDA) מקל לדמיין את נימי דם הירך על ידי FCFM. דוקסורוביצין (100 μl; 8 מ&quot;ג / קילוגרם, אדריאמיצין) או מלוח גם להינתן IV לוריד הזנב המחומם מראש מאוחר יותר. </li><li style=";text-align:right;direction:rtl"> מקם את עכבר supinely על במה קלקר. לאבטח את העכבר למשטח ולשמור על מעמדה באמצעות סרט דביק כירורגי. </li></ol>כותרת &quot;&gt; 3. הדמיה של כלי דם על ידי עצם הירך FCFM במהלך ולאחר מתן של דוקסורוביצין או מלוח הערה: מיקרוסקופ confocal fibered שימש במחקר זה מורכב משתי יחידות: (1) microprobe (mini0 / 30). (; אורך גל nm 488 LSU-488) יחידת סריקת לייזר (2). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבצע את כל זמן הקפות ניתוחים באמצעות הלייזר באורך גל nm 488 LSU. הערה: גלאי היחידה הראשיים מזהה מסוננת - הקרינה (500 650 ננומטר) הנפלטת. התמונות שנרכשו משוחזרות לאחר מכן ומוצגות בשיעור של 12 מסגרות / sec. </li><li style=";text-align:right;direction:rtl"> נתק בקפידה את המזרק מהמחט ולצרף מזרק חדש המכיל dextran FITC. לנהל של dextran FITC (IV) 100 μl. </li><li style=";text-align:right;direction:rtl"> Shift microprobe (mini0 / 30) לשדה מתאים של תצוגה ולקבע אותו, לאחר ההתאמה לZ- הציר, על מנת לקבל את התמונה המקבילה. מחכה לסימן הראשוני לדעוך עד איתות ברורה וממוקדת הוא Visible. </li><li style=";text-align:right;direction:rtl"> להקליט את זרימת דם בנקודת ההתחלה לתקופה קצרה ייצוב (~ 30 שניות). לאחר מכן חבר למחט מזרק נוסף, המכיל גם דוקסורוביצין או מלוח. IV לנהל דוקסורוביצין μl 100 או מלוח. </li><li style=";text-align:right;direction:rtl"> לפקח על זרימת FITC-dextran מוזרק ברציפות במשך 20 דקות. על התוכנה הקשורים FCFM, להשתמש בלחצן הקוטר על הסרגל העליון כדי למדוד את כלי הדם ולקטלג אותם כקטן (&lt;15 מיקרומטר) או גדול (&gt; 15 מיקרומטר). </li><li style=";text-align:right;direction:rtl"> להרדים את החיה עם מנת יתר של חומרי הרדמה. </li></ol>

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	<li>Chow, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthracyclines+cause+endothelial+injury+in+pediatric+cancer+patients:+a+pilot+study.">Anthracyclines cause endothelial injury in pediatric cancer patients: a pilot study.</a> J Clin Oncol. 24 (6), 925-928 (2006).</li><li>Nuver, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+chemotherapy-induced+cardiovascular+changes+in+patients+with+testicular+cancer.">Acute chemotherapy-induced cardiovascular changes in patients with testicular cancer.</a> J Clin Oncol. 23 (36), 9130-9137 (2005).</li><li>Vos, F. 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Role of reactive oxygen and nitrogen species.</a> J Biol Chem. 275 (43), 33585-33592 (2000).</li><li>Vasquez-Vivar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+nitric+oxide+synthase-dependent+superoxide+generation+from+adriamycin.">Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin.</a> Biochemistry. 36 (38), 11293-11297 (1997).</li></ol>

הערכת רעילות של כלי דם בעקבות כימותרפיה היא מאתגרת בשל הקושי בלדמיין את הדינמיקה של כלי דם בתגובה לגירויים בזמן אמת. מחקרים קליניים רבים שמעורבים שכמה טיפולי הכימותרפיה לגרום לפגיעה בכלי הדם ישירה, עדיין המנגנון והמאפיינים של רעילות זה עדיין לא הובהרו. הקמנו בזמן אמ...

עדויות קליניות מצביעות על כך שכמה כיתות של כימותרפיה לעורר מגוון רחב של פתולוגיות כלי דם המתבטאות בתופעת Raynaud, יתר לחץ דם, myocardialinfarction, התקפת כלי דם במוח, וVeno-occlusivedisease הכבד 1,2. &quot;תרופות אנטי angiogenic &#39;בשוגג&#39;&quot; הוא מונח חדש יחסית, המתאר תרופות כימותרפיות קונבנציונליות הפועלות כמעכבי אנגיוגנזה אפשריים, למרות שהם לא פותחו במקור למטרה זו 3-5 אבל שנועדו לחסל את התאים סרטניים על ידי הטלת קטנה כמו &quot;בטחונות נזק &quot;לתאים נורמלים כמו 3 אפשריים. כמה טיפולי הכימותרפיה כבר משתמעים כvasculo-רעלים כפי שנצפו במחקרים קליניים באמצעות סמנים ביולוגיים בסרום. בין אלה אלקילציה סוכנים (כגון cyclophosphamide), תרכובות פלטינה (כגון ציספלטין) וanthracyclines 1,2,5-7. סיבוכי לב וכלי דם חריפים עלולים להתרחש כresult של רעילות של כלי דם הנגרמת על ידי כימותרפיה. הם עלולים להתקדם למצבים כרוניים כמו טרשת עורקים וחשבון לעלייה בסיכון לתחלואת לב וכלי דם מאוחר. ובכל זאת, למרות ההרכבה ראיות קליניות, יש מיעוט מחקרים המיועדים תוך שימת דגש על המנגנון של רעילות של כלי דם, ולכן, דורש הבהרה נוספת של הפתוגנזה המדויקת שהם גורמים היא מוצדקת. אתגר גדול בחושפים את המנגנון של רעילות של כלי דם בעקבות כימותרפיה נובע ממורכבות חקירת תפקוד כלי דם in vivo. אנו מתארים זאת פלטפורמה של רזולוציה גבוהה בהדמיה מולקולרית vivo בעכברים המאפשרת ללכוד את זרימת דם ואת המאפיינים של כלי. פלטפורמה זו מאפשרת זיהוי של תופעות של כלי דם הנוצר על-טיפול ישירים: בזמן אמת, כמו גם הקפדה עליהן על פני תקופה של זמן בתוך אותו האנשים.

<table border="0" cellpadding="0" cellspacing="0" width="1395">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:579px;">Name of Material/ Equipment</td>
			<td style="width:423px;">Company</td>
			<td style="width:213px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general anesthesia (100mg/kg ketaset and 6mg/kg XYL-M2).&#160;</td>
			<td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">a depilatory cream (Veet)</td>
			<td>&#160;ReckittBenckiser, Bristol, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A 30-gauge, 1/2-inch needle attached to 1 ml syringe&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC dextran at a volume of 100 &#181;l (10 mg/ml; FD2000S, MW 2000000 Dalton)&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxorubicin (8 mg/kg, Adriamycin)</td>
			<td>Teva, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paclitaxel (1.2 mg/kg, Medexel)&#160;</td>
			<td>Taro, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment

סוגים מסוימים של כימותרפיה עלולים להפעיל שינויים בכלי הדם חריפים שעלולות להתקדם לתנאים לטווח ארוך החושף את החולה לסיכון מוגבר לתחלואת לב וכלי דם. ובכל זאת, אם כי הראיות קליניות ההרכבה, יש מיעוט מחקרים ברורים של רעילות של כלי דם, ולכן אטיולוגיה של קבוצה הטרוגנית של הפרעות לב וכלי דם לב וכלי דם / עדיין לא הובהר. יתר על כן, המנגנון שעשוי בבסיס רעילות של כלי דם יכול לגמרי שונה מהעקרונות של רעילות לבבית בעקבות כימותרפיה, אשר קשורה לכוון פציעת myocyte. הקמנו בזמן אמת, בפלטפורמת הדמיה המולקולרית vivo כדי להעריך את הרעילות של כלי דם אקוטי הפוטנציאל של טיפולים אנטי-סרטניים. הקמנו פלטפורמה של in vivo, הדמיה מולקולרית ברזולוציה גבוהה בעכברים, מתאים להדמית כלי דם בתוך איברים מוגבלים וblo ההתייחסותכלי od בתוך אותו האנשים ואילו כל אדם לשמש כשליטה שלו. דפנות כלי דם היו לקויים לאחר מתן דוקסורוביצין, המייצגות את מנגנון ייחודי של רעילות של כלי דם שעשוי להיות האירוע המוקדם לפגיעה באיברי המטרה. במסמך זה, בשיטה של ​​ההדמיה confocal fibered מיקרוסקופ פלואורסצנטי (FCFM) המבוססת מתוארת, המספק מצב חדשני להבין תופעות פיסיולוגיות ברמה התאית ותת-תאית בנושאי בעלי חיים.

בהדמיה רציפה vivo בזמן אמת מנגנון ההדמיה משמש כאן הוא בחדות גבוהה, מיקרוסקופ confocal fibered, מצוידת בבדיקה המאפשרת הדמיה של כלי דם ותגובתה לגירויים שונים כמו כימותרפיה. שיטה זו היא פולשנית מאז למרות שהיא עשויה להקל על הדמיה של כל...

Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

בעקבות כימותרפיה כלי דם רעילות - בזמן אמת<em&gt; In vivo</em&gt; הדמיה של ירידת ערך כלי

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

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Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

7.3K Views.  Tel Aviv University. We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Video: Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Multispectral הדמיה בזמן אמת Fluorescence לגילוי intraoperative של הצומת סנטינל הלימפה גניקולוגיות אונקולוגיה

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

הערכה של ספיגת nanoparticle ב גידולים בזמן אמת באמצעות הדמיה Intravital

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

בזמן אמת הדמיה דיגיטלית של אינטראקציה ליקוציט-אנדותל של פגיעה איסכמיה reperfusion (IRI) של השריר Cremaster עכברוש

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

<em&gt; In vivo</em&gt; Assay לבדיקת חדירות כלי דם

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

השתלה ללשכה הקדמית של העין לאורך, לא פולשני<em&gt; בחי</em&gt; הדמיה ברזולוצית תא בודד בזמן אמת

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

הדמית רנטגן בזמן אמת של כרכי נוזל ריאות עובריות עכבר ריאות

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

רוז בנגל Photothrombosis ידי דימות אופטי Confocal<em&gt; In vivo</em&gt;: דגם של שבץ כלי אחד

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

זמן לשגות Extended הדמיה Intravital של Dynamics תאיים בזמן אמת ב microenvironment הגידול

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

4-כלי דגימה גישה ללימודים אינטגרטיביים של הפיזיולוגיה השליה האדם<em&gt; בויבו</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

ניתוח בזמן אמת לחץ-נפח של אוטם אקוטי בעכברים

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...