Summary

Предотвращение теплового стресса побочные эффекты у крыс<em> Сенная палочка</em> Штамм

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

Данное исследование было разработано для оценки защитного эффекта штамма Bacillus Сенная против осложнений , связанных с тепловому стрессу. Тридцать два крыс Sprague-Dawley были использованы в этом исследовании. Животных перорально два раза в день в течение двух дней с Б. Сенная штамм BSB3 или PBS. На следующий день после последней обработки, каждая группа была разделена и две экспериментальные группы (один обрабатывали PBS и один обрабатывали B. Сенная) помещали при 45 ° С в течение 25 мин. Две контрольные группы остались в течение 25 мин при комнатной температуре. Все крысы были умерщвлены, и различные параметры были проанализированы во всех группах. Побочные эффекты теплового стресса регистрируются уменьшением ворсинок высоты и общей толщины слизистой оболочки в кишечном эпителии; транслокации бактерий из просвета; увеличилась везикул эритроцитов и возвышение липополисахаридов (ЛПС), уровень в крови. Защитная эффективность лечения оценивали путем предварительнойщению этих побочных эффектов. Протокол был создан для перорального лечения крыс с бактериями для предотвращения теплового стресса осложнений, но этот протокол может быть модифицирован и использован для других путей введения и для анализа различных соединений.

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

Все экспериментальные процедуры были одобрены IACUC из Auburn University, Auburn, Алабама. 1. Подготовка медиа-культуры, посуда и бактериальная культура Инокулируйте 10 мл питательного бульона в колбе с одной колонии Сенная палочка BSB3 культуры, выращенной в течение ночи на питательным …

Representative Results

Средняя температура тела животных до и сразу после теплового стресса был 36,7 ± 0,07 ° С и 40,3 ± 0,17 ° С, соответственно (р <0,05). Экспонирование крыс тепла (группа 3) привела к значительному ингибированию роста ворсинок и общей толщины слизистой оболочки (фиг.2,…

Discussion

Воздействие высоких температур приводит к серьезным состоянием здоровья 29. Профилактика и раннее выявление осложнений , связанных с тепловым стрессом имеют жизненно важное значение 30. Представленный протокол может быть использован для оценки эффективности раз?…

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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