Preventie van hittestress schadelijke effecten bij ratten door<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
Deze studie werd ontworpen om de beschermende werking van de Bacillus subtilis stam tegen complicaties bij hittestress evalueren. Tweeëndertig Sprague-Dawley ratten werden gebruikt in deze studie. De dieren werden oraal behandeld tweemaal daags gedurende twee dagen met B. subtilis BSB3 stam of PBS. De volgende dag na de laatste behandeling, elke groep werd gesplitst en twee experimentele groepen (één behandeld met PBS en behandeld met een B. subtilis) werden bij 45 ° C gedurende 25 min geplaatst. Twee controlegroepen bleven 25 minuten bij kamertemperatuur. Alle ratten werden gedood en verschillende parameters werden geanalyseerd in alle groepen. Nadelige effecten van hittestress zijn geregistreerd door de daling van de villi hoogte en totale mucosale dikte in het darmepitheel; translocatie van bacteriën uit het lumen; grotere blaasjes van erytrocyten en verhoging van de lipopolysacchariden (LPS) in het bloed. De beschermende werkzaamheid van de behandeling wordt geëvalueerd door preinterventie van deze bijwerkingen. Het protocol werd opgezet voor de orale behandeling van ratten met bacteriën voorkomen hittestress complicaties, maar dit protocol kan worden gemodificeerd en gebruikt voor andere toedieningswijzen en voor de analyse van verschillende verbindingen.
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
Blootstelling aan hoge temperaturen leidt tot ernstige gezondheidsproblemen 29. Preventie en vroege opsporing van complicaties in verband met hittestress zijn van vitaal belang 30. Het gepresenteerde protocol kan worden gebruikt om de werkzaamheid van verschillende benaderingen voor het voorkomen van hittestress nadelige effecten ervan.
Kritische stappen in dit protocol zijn onder meer: de warmtebehandeling procedure (45 o 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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