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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

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, m....

Protocol

All experimental procedures were approved by IACUC of Auburn University, Auburn, Alabama.

1. Preparation of Culture Media, Dishes and Bacterial Culture

  1. Inoculate 10 ml of nutrient broth in a flask with a single colony of Bacillus subtilis BSB3 culture, grown overnight on a nutrient agar plate.
  2. Make 500 ml of sporulation medium19 (per liter: Bacto Nutrient Broth, 8 g; 10% (w/v) KCl, 10 ml; 1.2% (w/v) MgSO4 x 7H2O, 10 ml; agar, 15 g) and autoclave at 12.......

Representative Results

The mean body temperature of animals before and immediately after heat stress was 36.7 ± 0.07 oC and 40.3 ± 0.17 oC, respectively (P < 0.05). Exposure of rats to heat (group 3) resulted in significant inhibition of villi height and total mucosal thickness (Figures 2, 3). Oral administration of BSB3 strain before stress protected intestine from the harmful effect of heat.

Trans.......

Discussion

Exposure to high temperature results in serious health conditions29. Prevention and early detection of complications related to heat stress are of vital importance30. The presented protocol can be used to evaluate the efficacy of various approaches for the prevention of heat stress adverse effects.

Critical steps within this protocol include: the heat treatment procedure (45 oC, 25 min); the usage of apyrogenic materials during collection and p.......

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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

....

Materials


 

NameCompanyCatalog NumberComments
Phosphate buffered saline (PBS)Sigma-Aldrich, St. Louis, MOP4417
Ethyl Alcohol Pharmco products Inc. Brookfield, CT, USA 64-17-5
AgarVWR97064-334
MacConkey agar platesVWR470180-742
5% blood agar platesVWR89405-024
Brucella blood with Hemin, and Vitamin K,
 plates
VWR89405-032
Bouin’s FixativeElectron Microscopy Sciences, Hatfield, PA, USA15990
IL-10 Rat ELISA kitInvitrogen, Camarillo, CA, USAKRC0101
IL-1beta Rat ELISA KitInvitrogen, Camarillo, CA, USAKRC0011
IL-6 Rat ELISA KitInvitrogen, Camarillo, CA, USAKRC0061
INF-gamma Rat ELISA KitInvitrogen, Camarillo, CA, USAKRC4021
TNF-alpha Rat ELISA KitInvitrogen, Camarillo, CA, USAKRC3011
Rat LPS ELISA KitNeoBioLab, MA, USARL0275
Environmental Chamber 6020-1Caron, Marietta, OH, USA6020-1
Centrifuge Beckman Coulter, Indianapolis, IN, USAOptima L-90K
Ultra Centrifuge
Light microscope optical systemCitoViva Technology Inc., Auburn, AL
Colony counterFisher Scientific , Pittsburgh, PA, USARE-3325
FreezerHaier, Brooklyn, NY, USAHCMO50LA
Ultra microplate readerBio-Tek Instrument, Winooski, VT, USAELx 808
Auto Strip WasherBio-Tek Instrument, Winooski, VT, USAELx50
PipettesGilson, Pipetman, FranceP100, P200, P1000
C24 Incubator ShakerNew Brunswick Scientific, Enfield, CT, USAClassic C24
Rocking Shaker Reliable Scientific, Inc., Nesbit, MS, USA55
Petri dishesFisher Scientific, Pittsburgh, PA, USA875713100mmX15mm
SterilGard III AdvanceThe Baker Company, Sanford, ME, USASG403
Culture Growing FlasksCorning Incorporated, Corning, NY, USA4995PYREX 250mL Erlenmeyer flasks
Optical Spectrometer Genesys 20Thermo Scientific, Waltham, MA, USA.4001
Sony DXC-33 Video cameraSony
Richardson test slideElectron Microscopy Science, Hatfield, PA, USA80303
Millipore water purification systemMilliporeDirect-Q
Image-Pro Plus softwareMedia Cybernetics, MD, USA
Triple Beam BalanceOHAUS Corporation, Parsippany, NJ, USA
Tissue Homogenizer, DounceVWR71000-518

References

  1. Crandall, C. G., Gonzalez-Alonso, J. Cardiovascular function in the heat-stressed human. Acta Physiol. 199, 407-423 (2010).
  2. Lambert, G. P. Role of gastrointestinal permeability in exertional heatstroke. E....

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