Summary

מניעת חום מתח תופעות לוואי בחולדות על ידי<em> Subtilis Bacillus</em> זנים

Published: July 11, 2016
doi:

Summary

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Abstract

מחקר זה נועד להעריך את ההשפעה המגנה של זן subtilis Bacillus נגד סיבוכים הקשורים לחום מתח. שלושים ושתיים חולדות Sprague-Dawley שימשו במחקר זה. בעלי החיים טופלו דרך פה פעם ביום למשך ימים עם B. subtilis BSB3 זן או PBS. למחרת, לאחר הטיפול האחרון, כל קבוצה חולקה ושתי קבוצות ניסוי (אחד שטופל PBS ואחד שטופל ב subtilis) הוצבו 45 o צלזיוס למשך 25 דקות. שתי קבוצות בקרה נשאר במשך 25 דקות בטמפרטורת החדר. כל החולדות היו מורדמות ופרמטרים שונים נותחו בכל הקבוצות. השפעות שליליות של עומס חום רשומות על ידי הירידה של גובה villi ועובי רירי הכולל באפיתל המעי; טרנסלוקציה של חיידקים מן לומן; גדל vesiculation של אריתרוציטים והעלאה של lipopolysaccharides (LPS) רמה בדם. יעילות המגן של טיפול מוערכת על ידי מראשvention של תופעות לוואי אלה. הפרוטוקול הוקם לטיפול האוראלי של חולדות עם חיידקים למניעת סיבוכי עומס חום, אבל פרוטוקול זה יכול להיות משונה המשמש מסלולים אחרים של ממשל ועל ניתוח של תרכובות שונות.

Introduction

Different stressors affect human and animal health. Temperature is one of the most stressful factors, causing chronic, acute and even lethal illnesses1. Changes in intestinal morphology and a loss of gut barrier integrity after heat stress were documented in many cases2,3. This protective barrier is responsible for defense against translocation of gut bacteria and their toxins, in particular lipopolysaccharides (LPS), from the lumen to the internal circulation4,5. Stability of the gut microbiota significantly influences intestinal barrier function, the immune response of the host, and tolerance to stress conditions6. Thus, modulation of the intestinal microbiota can provide a novel approach for prevention of stress-related adverse effects.

Beneficial bacteria have been used as a promising strategy to modify the gut microbiota for successful management of various clinical conditions, such as diarrhea, inflammatory bowel disease, atopic dermatitis, metabolic disorders7-10. Probiotic effects of beneficial bacteria include production of essential metabolites to support intestinal health, stimulation of the immune system, promotion of lactose tolerance, restoration of epithelial dysfunction. Probiotics were effective in prevention of the stress-related complications in vitro and in animal models11,12.

Researchers have given more attention to Bacillus bacteria as probiotics because these bacteria support intestinal homeostasis13 and have beneficial effects on the host14. Our previous data demonstrated high efficacy of Bacillus probiotics against pathogens in vitro15,16 and in clinical trials17,18. Here, we aim to evaluate the preventive effect of B. subtilis BSB3 strain against complications after heat stress.

Protocol

כל הפרוצדורות אושרו על ידי IACUC של אוניברסיטת אובורן, אובורן, אלבמה. 1. הכנת תרבות מדיה, צלחות תרבית חיידקים לחסן 10 מ"ל של מרק מזין בבקבוק עם מושבה אחת של תרבות BSB3 subtilis Bacillus, גדל לילה על צל?…

Representative Results

טמפרטורת הגוף הממוצעת של בעלי חיים לפני ומיד אחרי עומס החום היה 36.7 ± 0.07 מעלות צלסיוס ו -40.3 ± 0.17 מעלות צלסיוס, בהתאמה (P <0.05). חשיפה של חולדות לחום (קבוצה 3) הביאה עיכוב משמעותי של גובה villi ועובי רירי כולל (איורים 2, 3). אוראלי של זן BSB3 ל?…

Discussion

חשיפת תוצאות בטמפרטורה גבוהה בתנאים בריאותיים חמורים 29. מניעה וגילוי מוקדם של סיבוכים הקשורים עומס החום הם בעלי חשיבות חיונית 30. הפרוטוקול המובא יכול לשמש כדי להעריך את היעילות של גישות שונות למניעת תופעות לוואי עומס חום.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Auburn University fund FOP 101002-139294-2050.

Materials


 

Phosphate buffered saline (PBS) Sigma-Aldrich, St. Louis, MO P4417
Ethyl Alcohol  Pharmco products Inc. Brookfield, CT, USA  64-17-5
Agar VWR 97064-334
MacConkey agar plates VWR 470180-742
5% blood agar plates VWR 89405-024
Brucella blood with Hemin, and Vitamin K,
 plates
VWR 89405-032
Bouin’s Fixative Electron Microscopy Sciences, Hatfield, PA, USA 15990
IL-10 Rat ELISA kit Invitrogen, Camarillo, CA, USA KRC0101
IL-1beta Rat ELISA Kit Invitrogen, Camarillo, CA, USA KRC0011
IL-6 Rat ELISA Kit Invitrogen, Camarillo, CA, USA KRC0061
INF-gamma Rat ELISA Kit Invitrogen, Camarillo, CA, USA KRC4021
TNF-alpha Rat ELISA Kit Invitrogen, Camarillo, CA, USA KRC3011
Rat LPS ELISA Kit NeoBioLab, MA, USA RL0275
Environmental Chamber 6020-1 Caron, Marietta, OH, USA 6020-1
Centrifuge  Beckman Coulter, Indianapolis, IN, USA Optima L-90K
Ultra Centrifuge
Light microscope optical system CitoViva Technology Inc., Auburn, AL
Colony counter Fisher Scientific , Pittsburgh, PA, USA RE-3325
Freezer Haier, Brooklyn, NY, USA HCMO50LA
Ultra microplate reader Bio-Tek Instrument, Winooski, VT, USA ELx 808
Auto Strip Washer Bio-Tek Instrument, Winooski, VT, USA ELx50
Pipettes Gilson, Pipetman, France P100, P200, P1000
C24 Incubator Shaker New Brunswick Scientific, Enfield, CT, USA Classic C24
Rocking Shaker  Reliable Scientific, Inc., Nesbit, MS, USA 55
Petri dishes Fisher Scientific, Pittsburgh, PA, USA 875713 100mmX15mm
SterilGard III Advance The Baker Company, Sanford, ME, USA SG403
Culture Growing Flasks Corning Incorporated, Corning, NY, USA 4995 PYREX 250mL Erlenmeyer flasks
Optical Spectrometer Genesys 20 Thermo Scientific, Waltham, MA, USA. 4001
Sony DXC-33 Video camera Sony
Richardson test slide Electron Microscopy Science, Hatfield, PA, USA 80303
Millipore water purification system Millipore Direct-Q
Image-Pro Plus software Media Cybernetics, MD, USA
Triple Beam Balance OHAUS Corporation, Parsippany, NJ, USA
Tissue Homogenizer, Dounce VWR 71000-518

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Cite This Article
Sorokulova, I., Globa, L., Pustovyy, O., Vodyanoy, V. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (113), e54122, doi:10.3791/54122 (2016).

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