Prevenção de estresse térmico efeitos adversos em ratos pela<em> Bacillus subtilis</em> Strain
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
Este estudo foi desenhado para avaliar o efeito protetor da estirpe Bacillus subtilis contra as complicações relacionadas ao estresse térmico. Trinta e dois ratos Sprague-Dawley foram usadas neste estudo. Os animais foram tratados por via oral duas vezes por dia durante dois dias com B. subtilis estirpe BSB3 ou PBS. No dia seguinte, após o último tratamento, cada grupo foi dividido e dois grupos experimentais (tratados com um PBS e tratadas com um B. subtilis) foram colocados a 45 ° C durante 25 min. Dois grupos de controlo permaneceu por 25 min à temperatura ambiente. Todos os ratos foram sacrificados e diferentes parâmetros foram analisados em todos os grupos. Os efeitos adversos do estresse por calor são registrados pela diminuição da altura das vilosidades e espessura total da mucosa do epitélio intestinal; translocação de bactérias a partir do lúmen; vesiculação aumento de eritrócitos e elevação dos lipopolissacáridos (LPS) de nível no sangue. A eficácia protectora do tratamento é avaliada por prévenção destes efeitos secundários. O protocolo foi definido acima para o tratamento oral de ratos com bactérias para a prevenção de complicações de stress de calor, mas este protocolo pode ser modificado e utilizado para outras vias de administração e para a análise de compostos diferentes.
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
Representative Results
Discussion
A exposição a resultados de alta temperatura em condições graves de saúde 29. Prevenção e detecção precoce de complicações relacionadas ao estresse por calor são de importância vital 30. O protocolo apresentado pode ser usado para avaliar a eficácia de várias abordagens para a prevenção do stress térmico efeitos adversos.
Passos críticos dentro deste protocolo incluem: o processo de tratamento de calor (45 ° C, 25 min); …
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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