עבודה זו נתמכה על ידי תרומות של המועצה מקסיקני למדע וטכנולוגיה (Conacyt, 207,268 ו 233,395 למיכאל שנור). KFCO היא כלת מלגה Conacyt (396,260) כדי להשיג תואר MSc.

<ol>
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החוקרים אין לי מה לחשוף.

על מנת להשתמש תוספי תזונה במחקרים קליניים, את היתרונות והבטיחות של תוספים כאלה יש להעריך בזהירות in vivo במחקרים בבעלי חיים. במקרה של קוליטיס, מספר דגמים בעלי חיים מתאימים דומות הסימנים הקליניים של מחלת מעי דלקתי הוקמו, כוללים מודלים כימיים באמצעות DSS, TNBS, או חומצה א...

קוליטיס היא מצב דלקתי של המעי הגס שיכול לגרום שלשולים וכאבי בטן. קוליטיס יכולה להיות חריפה, בתגובה לזיהום או ללחץ, או שזה יכול להתפתח מחלה כרונית, כגון קוליטיס כיבית (UC), השייכת לקבוצת מחלות מעי דלקתיות (IBD). למרות הסימנים הקליניים של UC תוארו היטב, בפתוגנזה עדיין לא ידוע ולא מובן 1. מקובל בין מומחי UC היא מחלה מולטיפקטוריאלית, עם מוטציות גנטיות ותגובות חיסון סוטות משחקת תפקיד מרכזי 2. עם זאת, גורמים סביבתיים, כגון סגנון החיים והתזונה, גם לתרום להתפתחות המחלה והתקדמות 3. למרבה הצער, IBD אינה ניתנת לריפוי, אך ישנן אפשרויות טיפול רבות שמטרתן להקל על סימפטומים קליניים. טיפולים שוטפים כוללים תרופות אנטי דלקתיות, כגון sulfasalazine ו סטרואידים; כשמה כן היא, כמו azathioפריין; נוגדנים חד שבטיים כי ציטוקינים פרו-דלקתיים ללכוד, כגון-α tumor necrosis factor (TNF-α), או מולקולות הדבקה לחסום, כגון integrins, להפחית גיוס לויקוציטים מוגזם; ומעכבים כי למקד קינאזות המפעילות מסלולים פרו-דלקתיים, כגון קינאז יאנוס (JAK) 4. לא כל המטופלים מגיבים לכל הטיפולים, כך אסטרטגיות טיפוליות חייבות להיות אינדיבידואלי 5. יתר על כן, רוב התרופות טיפוליות אלו להפריע במטבוליזם תגובות חיסוניות, לעתים קרובות גורמים לתופעות לוואי חמורות. מסיבה זו, נחקרו אפשרויות טיפול אלטרנטיביות. טיפולים אלטרנטיביים לכלול פרוביוטיקה ותוספי תזונה, אשר הוחלו במודלים של בעלי חיים וניסויים קליניים עם רמות שונות של הצלחה 6,7. מצאנו לאחרונה כי היישום של תוספי תזונה יחידים שונים, כגון ויטמינים אנטי-חמצונים או ω3-רב בלתי רוויות וחומצות שומן (PUFAs), הוא נחות היישום של שילוב של תוספים כאלה בהקלת קוליטיס הסימפטומים למחלה קרדיווסקולרית 8,9. מחקרים אלו בוצעו בעכברים, כך מחקרים קליניים חייבות להתבצע בבני אדם כדי לקבוע האם ממצאים אלו יחולו גם על בני אדם. לפני מחקרים קליניים מבוצעים, את היעילות והבטיחות של אפשרויות טיפול חדשות יש לבחון אותה במודלים של בעלי חיים. עבור IBD, נתרן גופרתי dextran (DSS) מודל כבר בשימוש נרחב כדי לחקור את המנגנונים של התפתחות המחלה ואת ההשפעות החיוביות של תרופות ותוספי תזונה 6,10. במרבית המחקרים, רק מחלה חריפה על פני פרק זמן של שבעה ימים מושרים; בכל זאת, הסימנים הקליניים בניסויים בבעלי חיים אלה דומים לאלה שנצפו בחולים במחלות מעי דלקתיות (כלומר, שלשול דמי, ירידה במשקל, חוסר תפקוד אפיתל, וחדירה תא החיסון) 10. DSS גורם ארוזיות הרירית,וכתוצאה מכך תפקוד מחסום בחדירות אפיתל במעי מוגבר 11. המנגנון המדויק אינו ידוע. עם זאת, מחקר חדש כי DSS אינטראקציה עם שרשרת חומצות שומן באורך בינוני כדי ליצור ננו-lipocomplexes כי הם מסוגלים להיכנס לתאי האפיתל וכדי לגרום אותות דלקתיים מסלולי 12. במאמר זה נתאר בפירוט כיצד קוליטיס מושרה ונותח בעכברים, איך תזונתי תוספים מוחל על ידי gavage כדי להבטיח מינון קבוע כל חיה, ואיך את ההשפעות של תוספים כאלה על סימפטומים קוליטיס שונים נבחנות.

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			<td height="21" style="height:21px;width:439px;">0.3% of gelatin&#160;</td>
			<td style="width:184px;">Bioxon</td>
			<td style="width:139px;">158</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">10% formaldehyde</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">2106-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">96-well plate with flat bottom</td>
			<td style="width:184px;">Corning</td>
			<td style="width:139px;">3368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Absolute ethanol&#160;</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9000-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Absolute xylene</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9490-03</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">ABTS</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H5882</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Bovine Serum Albumin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">9048-46-8</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">ColoScreen</td>
			<td style="width:184px;">Helena Laboratories</td>
			<td style="width:139px;">5072</td>
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			<td height="21" style="height:21px;width:439px;">Corabion (Kindly provided by Merck, Naucalpan, Mexico)</td>
			<td style="width:184px;">Merck</td>
			<td style="width:139px;"></td>
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			<td height="21" style="height:21px;width:439px;">Dextran Sulfate Sodium Salt</td>
			<td style="width:184px;">Affymetrix</td>
			<td style="width:139px;">9011-18-1</td>
			<td style="width:191px;">(M.W. 40,000-50,000)</td>
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			<td height="21" style="height:21px;width:439px;">Dihydroethidium</td>
			<td style="width:184px;">Life Technology</td>
			<td style="width:139px;">D11347</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Eosin-Y&#160;</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">L087-03</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Evans blue</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">314-13-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Feeding&#160; needle</td>
			<td style="width:184px;">Cadence Science Inc.</td>
			<td style="width:139px;">9921</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Glass slide rack with handle</td>
			<td style="width:184px;">Electron Microscopy</td>
			<td style="width:139px;">70312-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Glass Slides</td>
			<td style="width:184px;">Corning</td>
			<td style="width:139px;">2947</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Harris hematoxylin&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">H3136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hexadecyltrimethyammonium (HTAB)</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H6269</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Histosette (Embedding cassette)</td>
			<td style="width:184px;">Simport</td>
			<td style="width:139px;">M498.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hydrochloric acid 1 M</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">9535-62</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hydrogen peroxide</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:139px;">H3410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Ketamine</td>
			<td style="width:184px;">PiSA Agropecuaria</td>
			<td style="width:139px;">Q-7833-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Liquid paraffin&#160;</td>
			<td style="width:184px;">Paraplast</td>
			<td style="width:139px;">39501-006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Lithium carbonate&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">2362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">N,N-dimethylformamide</td>
			<td style="width:184px;">J.T. Baker</td>
			<td style="width:139px;">68-12-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">N-acetylcysteine</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">A9165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plastic cubes</td>
			<td style="width:184px;">Electron Microscopy</td>
			<td style="width:139px;">70181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Poly-L-Lysine Solution</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">25988-63-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Prism 5 statistical software</td>
			<td style="width:184px;">GraphPad Software</td>
			<td style="width:139px;">Prism 5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Saline Solution 0.9% NaCl</td>
			<td style="width:184px;">CS PiSA</td>
			<td style="width:139px;">Q-7833-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sodium citrate</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">W302600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Synthetic resin&#160;</td>
			<td style="width:184px;">Poly Mont</td>
			<td style="width:139px;">7987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Taq DNA polymerase</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:139px;">11615-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tissue-tek. O.C.T Compound</td>
			<td style="width:184px;">Sakura Finetek</td>
			<td style="width:139px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tuberculine Syringe</td>
			<td style="width:184px;">BD Plastipak</td>
			<td style="width:139px;">305945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tween 20</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:139px;">9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td style="width:184px;">Vector Laboratories</td>
			<td style="width:139px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Xylazine</td>
			<td style="width:184px;">PiSA Agropecuaria</td>
			<td style="width:139px;">Q-7833-099</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis

מחלות מעי דלקתיות (IBD), כולל מחלת קרוהן וקוליטיס כיבית, הן הפרעות relapsing כרונית של המעיים. הם לגרום לבעיות חמורות, כגון התכווצויות בבטן, שלשול דמי, וירידה במשקל, בקרב אנשים מושפעים. למרבה הצער, אין תרופה עדיין, וטיפולים רק שואפים להקל על התסמינים. טיפולים שוטפים כוללים תרופות אנטי דלקתיות אימונוסופרסיבי שעשויות לגרום לתופעות לוואי חמורות. זה מצדיק את החיפוש אחר אפשרויות טיפול אלטרנטיביות, כגון תוספי תזונה, כי לא לגרום לתופעות לוואי. לפני היישום שלהם במחקרים קליניים, תרכובות כאלה חייבים להיבדק בקפדנות עבור יעילות וביטחון במודלים של בעלי חיים. דגם ניסיוני אמין הוא נתרן גופרתי dextran (DSS) מודל קוליטיס בעכברים, אשר מתרבה רבי הסימנים הקליניים של קוליטיס כיבתה בבני אדם. לאחרונה הוחל במודל זה כדי לבחון את ההשפעות המיטיבות של תוסף תזונה המכילויטמינים C ו- E, L-arginine, וחומצות שומן רב בלתי רווי-ω3 (PUFA). ניתחנו פרמטרים מחלה שונים ומצאו כי תוספת זו הצליח לשפר היווצרות בצקת, נזק לרקמות, חדירת לויקוציטים, עקה חמצונית, ואת הייצור של ציטוקינים פרו-דלקתיים, שמוביל שיפור כללי במדד פעילות המחלה. במאמר זה, נסביר בפירוט את היישום הנכון של תוספי תזונה באמצעות מודל קוליטיס DSS C57BL / 6 עכברים, כמו גם כיצד פרמטרים המחלה כגון היסטולוגיה, סטרס חמצוני, ודלקת מוערכים. ניתוח ההשפעות המיטיבות של תוספי תזונה שונים עשוי אז בסופו של דבר לפתוח אפיקים חדשים לפיתוח אסטרטגיות טיפול אלטרנטיביים כי להקל על תסמיני IBD ו / או כי להאריך את השלבים כפרה בלי לגרום לתופעות לוואי חמורות.

תוספי תזונה יכול להגן מפני DSS קוליטיס ואנטי דלקתי תוספי תזונה אנטי חמצונית, כגון ויטמין C, ויטמין E, תחמוצת החנקן (NO) -source L-arginine, וחומצות שומן רב בלתי רווי-ω3 (ω3-PUFAs), שימשו בהצלחה משתנה חיה מודלים כדי ...

Watch this Scientific Journal Video about Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis at JoVE.com

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

טיפולים עכשוויים של מחלות מעי דלקתיות (IBD) מכוונים להקל תסמיני מחלה אך הוא עשוי לגרום לתופעות לוואי חמורות. לכן, אסטרטגיות חלופיות נחקרות במודלי קוליטיס חיה. הנה, נסביר כיצד את ההשפעות המיטיבות של תוספי תזונה על סימנים קליניים IBD מנותחים כגון מודל קוליטיס.

ניתוח מועיל אפקטים של תוספי תזונה על פונקציות מעיים אפיתל מכשול במהלך ניסיוני קוליטיס

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

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12.1K Views.  Center for Research and Advanced Studies of the National Polytechnic Institute.  טיפולים עכשוויים של מחלות מעי דלקתיות (IBD) מכוונים להקל תסמיני מחלה אך הוא עשוי לגרום לתופעות לוואי חמורות. לכן, אסטרטגיות חלופיות נחקרות במודלי קוליטיס חיה. הנה, נסביר כיצד את ההשפעות המיטיבות של תוספי תזונה על סימנים קליניים IBD מנותחים כגו...

Video: Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

מודלים DNBS / TNBS קוליטיס: מתן תובנות מחלת מעי דלקתי והשפעות של התזונה שומן

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

צנתור לטווח ארוך של טרונק לימפה במעי וגביית הלימפה חזירים ילודים

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

צעד אחר פרוטוקול שלב לכירורגיה subretinal בארנבונים

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

ההשפעות של טסיות דם עשיר Allogeneic פלזמה (PRP) על תהליך ריפוי של אכילס משרטוטי הגידים של חולדות: תיאור מתודולוגיים

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

בדיקת אולטרה סאונד בחקירות הרבייה ניסויים על חולדות

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

שיפור שיטה להקמת במבחנה מחסום הדם - מוח מודל המבוסס על תאי אנדותל המוח חזירי

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

מיקרוסקופ המבוסס על שיטות להערכה של נדידת תאים אפיתל במהלך במבחנה ריפוי הפצע

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

מדידת ההשפעות של חיידקים וכימיקלים על חדירות המעי של האלמרנים

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

חקירת פירוק מחסום המעיים באורגנואידים חיים

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.