Name: prevention of heat stress adverse effects in rats by bacillus subtilis strain
Uploaded: 2016-07-11T14:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain at JoVE.com

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

Tutte le procedure sperimentali sono state approvate dal IACUC della Auburn University, Auburn, in Alabama. 1. Preparazione della Cultura Media, Piatti e coltura batterica <ol><li> Seminare 10 ml di brodo nutritivo in un pallone con una singola colonia della cultura BSB3 Bacillus subtilis, cresciuto durante la notte su una piastra di agar nutriente. </li><li> Rendere 500 ml di terreno di sporulazione 19 (per litro: Bacto Nutrient Broth, 8 g; 10% (w / v) KCl, 10 ml; 1,2% (w / v) MgSO 4 x 7H 2 O, 10 ml, agar, 15 g) e in autoclave a 121 ° C per 20 min. Lasciare raffreddare a 50 ° C e aggiungere le seguenti soluzioni sterili: 1 M Ca (NO3) 2, 1 ml; 0.01 M MnCl 2, 1 ml; 1 mM FeSO 4, 1 ml. </li><li> Raffreddare medio disposti a 40 ° C per 20 min a temperatura ambiente e versare 25 ml in ciascuna delle 20 piastre di Petri sterili nell&#39;armadio sterile. Lasciate che il mezzo di solidificare durante la notte. </li><li> Stendere 0,5 ml di cultura durante la notte di Bacillus subtilis BSB3 sulla superficie del terreno in piastre Petri. Incubare coltura a 37 ° C per 5 giorni fino al 90% sporulazione. Rivela spore di fase luminosa di microscopia ad alta risoluzione. </li><li> batteri Harvest aggiungendo 1 ml di fosfato sterile salina tamponata (PBS) alla piastra e raschiando batteri da una superficie utilizzando una spatola cellulare sterile. Conservare la sospensione in frigorifero fino al momento. </li><li> Conferma vitalità dei batteri placcatura 0,1 ml della diluizione appropriata (10 -5 a 10 -6) di una sospensione batterica su piastre di terreno sporulazione seguita da incubazione per 18 - 24 ore a 37 ° C. </li><li> Contare le colonie batteriche sulle piastre utilizzando un contatore di colonie e calcolare il titolo di batteri in sospensione testato. Preparare sospensione batterica con la concentrazione finale 1 x 10 8 UFC / ml in PBS. </li></ol> 2. Gli animali <ol><li> Utilizzare 32 ratti maschi Sprague-Dawley (250-300 g). Mantenere gli animali sotto specifiche condizioni esenti da organismi patogeni, con libero accesso a chow pellet standard ed acqua. Stabilire una temperatura media (21 ± 1 ° C), umidità (50 ± 5%) e un ciclo luce-buio 12 hr. </li></ol> 3. Progettazione sperimentale <ol><li> Inizia trattamento degli animali mediante sonda gastrica 11, dopo 2 giorni di acclimatazione. Usare la siringa con l&#39;ago sonda gastrica. Trattare 16 ratti con B. sospensione subtilis BSB3 (1 ml per topo) due volte al giorno per due giorni (B. subtilis gruppo). Trattare 16 ratti con PBS (1 ml per topo) due volte al giorno per due giorni (gruppo PBS) (Figura 1). </li><li> Dopo il trattamento, suddividere ogni gruppo (8 ratti per gruppo): Gruppo 1- controllo (PBS / no stress), Gruppo 2- B. subtilis (B. subtilis / no stress), Gruppo 3- lo stress (PBS / stress da calore) e Gruppo B. 4- subtilis + lo stress (B. subtilis lo stress / calore). </li><li> Misurare la temperatura rettale ogni ratto utilizzando un elettronic termometro digitale. Mantenere ratti dei gruppi 1 e 2 a temperatura ambiente per 25 min. Luogo animali dei gruppi 3 e 4 in una camera climatica a 45 ° C e umidità relativa del 55% per 25 min. </li><li> Successivamente, misurare la temperatura rettale di ogni ratto in tutti i gruppi con un termometro elettronico digitale. Mettere tutti gli animali a temperatura ambiente per 4 ore. </li><li> Anestetizzare ratto con isoflurano (di 2 - 4%) in un vaso anestesia sigillabile e osservare finché i ratti sono profondamente anestetizzati (assenza di risposta a pinch tep). Eutanasia ratti da una rapida decapitazione con una ghigliottina. </li><li> Raccogliere il sangue del tronco da ogni ratto 20 (15 - 16 ml) con le pipette apirogena in provette apirogena per ottenere siero e immediatamente prelevare campioni di sangue (7 microlitri da ogni ratto) con una pipetta per l&#39;esame microscopico. </li><li> Mettere provette con il sangue in frigorifero per almeno 30 minuti per consentire la coagulazione. Provette da centrifuga a 20 ° C, 7.000 xg per 10 min e rapidamenterimuovere siero con una pipetta. Siero Aliquota ottenuti da ogni ratto in 50 volumi microlitri e conservare a -20 ° C fino al dosaggio. </li></ol> 4. Procedura chirurgica <ol><li> Cut / tagliare tutta pelliccia dall&#39;addome e del torace della parte inferiore della carcassa con cesoie elettriche. Posizionare la carcassa in un armadio sterile e trattare la superficie rasata di carcassa con il 70% di alcol. </li><li> Utilizzare un bisturi sterile per creare un&#39;incisione mediana dell&#39;addome. Rimuovere il fegato, linfonodi mesenterici, milza e intestino tenue da ogni ratto con una pinzetta sterile. </li></ol> 5. Analisi di traslocazione batterica <ol><li> Posizionare ogni campione di fegato, linfonodi mesenterici e la milza in provette sterili pre-pesati e pesare. Calcolare il peso del campione come una differenza tra il peso di un tubo con un campione e il peso di un tubo vuoto. </li><li> Aggiungere PBS sterile al tubo per ottenere una diluizione 1:10 (w / v) del campione e omogenize ciascun campione con un omogeneizzatore tessuto di vetro sterile per ottenere una sospensione omogenea 21. </li><li> Effettuare diluizioni seriali (10 -1 a 10 -4) di ogni campione in PBS sterile. Piatto 0,1 ml di tutte le diluizioni di ciascun tessuto sulla superficie del MacConkey e di 5% agar sangue e Brucella agar sangue con emina (0.005 g / L) e vitamina K1 (0.01 g / L) piastre. </li><li> Incubare MacConkey e di 5% piastre di agar sangue aerobico e piastre di agar sangue Brucella in condizioni anaerobiche a 37 ° C 22. </li><li> Contare le colonie di batteri aerobici dopo 24 ore, e le colonie di batteri anaerobici dopo 48 ore di incubazione con un contatore di colonie. Esprimere i risultati come il numero di unità formanti colonie (CFU) in un grammo di tessuto. </li></ol> 6. analisi istologica <ol><li> Rimuovere piccoli campioni intestino da ogni ratto dalla procedura chirurgica (passi 4,1-4,2). Fissare i campioni in fissativo di Bouin, incorporare in paraffina,preparare 6 sezioni micron e sezioni macchia con ematossilina eosina utilizzando le procedure standard 23. </li><li> Utilizzare un sistema di microscopia ad alta risoluzione per la misurazione dell&#39;altezza dei villi intestinali e spessore totale della mucosa in ciascun campione (ingrandimento 100X) 24. Analizzare i 4 campioni provenienti da ogni ratto e fare almeno una ventina di misure in ogni campione. Esprimere i risultati in micrometri. </li></ol> 7. citochine Assay <ol><li> Utilizzare kit commerciali ratto ELISA per IL-1β; IL-6; TNF-α; INF-γ e IL-10 per misurare i livelli di citochine nel siero secondo il protocollo del produttore. </li><li> Generare curve standard per ogni set di campioni analizzati utilizzando standard per il kit in questione. Definire il livello di citochine in ciascun campione mediante confronto dei dati sperimentali con la curva standard appropriato. </li></ol> 8. Lipopolisaccaride Assay <ol><li> Analizzare il livello di LPS nel siero usando un topo kit ELISA LPS in base alleprotocollo del produttore. Stima concentrazione di LPS in campioni per confronto dei valori ottenuti con i valori della curva standard. </li></ol> 9. Alta Risoluzione microscopia ottica di Sangue <ol><li> Utilizzare il sistema del microscopio descritto in precedenza 25,26. Utilizzare una piattaforma isolamento delle vibrazioni come base per il sistema di microscopio e telecamera e il computer 25 per registrare le immagini in diretta. Calibrare immagini di prova utilizzando una diapositiva Richardson come descritto in precedenza 27. </li><li> Mettere 7 ml di sangue appena prelevato da ogni ratto su un vetrino, vetrino, fotografia e registrare dieci fotogrammi di immagine di 72 × 53,3 micron 2 in ciascun campione (ingrandimento 1,700X). </li><li> Misurare la concentrazione di vescicole usando il software che fornisce immagini ottiche con vista diretta ad alta risoluzione in tempo reale. Esaminare un minimo di 20 fotogrammi per ogni condizione sperimentale 28. </li></ol> 10. Statistic <ol><li> Esprimere tutti i valori come media e deviazione standard. Effettuare analisi statistiche e grafico tracciato. Analizzare i risultati con i due campioni t-test, impostare il livello di significatività a 0.05 per definire la significatività statistica. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari concorrenti.

L&#39;esposizione a risultati di alta temperatura in gravi condizioni di salute 29. La prevenzione e la diagnosi precoce delle complicanze legate allo stress termico sono di vitale importanza 30. Il protocollo presentato può essere utilizzato per valutare l&#39;efficacia di vari approcci per la prevenzione degli effetti avversi tensione termica. Passaggi critici all&#39;interno di questo protocollo sono: la procedura di trattamento termico (45 ?...

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.

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich,&amp;nbsp;St. Louis, MO</td><td>P4417</td><td></td></tr><tr><td>Ethyl Alcohol&amp;nbsp;</td><td>Pharmco products Inc. Brookfield, CT, USA&amp;nbsp;</td><td>64-17-5</td><td></td></tr><tr><td>Agar</td><td>VWR</td><td>97064-334</td><td></td></tr><tr><td>MacConkey agar plates</td><td>VWR</td><td>470180-742</td><td></td></tr><tr><td>5% blood agar plates</td><td>VWR</td><td>89405-024</td><td></td></tr><tr><td>Brucella blood agar plate with Hemin, and Vitamin K</td><td>VWR</td><td>89405-032</td><td></td></tr><tr><td>Bouin&amp;rsquo;s Fixative</td><td>Electron Microscopy Sciences, Hatfield, PA, USA</td><td>15990</td><td></td></tr><tr><td>IL-10 Rat ELISA kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC0101</td><td></td></tr><tr><td>IL-1beta Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC0011</td><td></td></tr><tr><td>IL-6 Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC0061</td><td></td></tr><tr><td>INF-gamma Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC4021</td><td></td></tr><tr><td>TNF-alpha Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC3011</td><td></td></tr><tr><td>Rat LPS ELISA Kit</td><td>NeoBioLab, MA, USA</td><td>RL0275</td><td></td></tr><tr><td>Environmental Chamber 6020-1</td><td>Caron, Marietta, OH, USA</td><td>6020-1</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Beckman Coulter, Indianapolis, IN, USA</td><td>Optima L-90K</td><td></td></tr><tr><td>Ultra Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Light microscope optical system</td><td>CitoViva Technology Inc., Auburn, AL</td><td></td><td></td></tr><tr><td>Colony counter</td><td>Fisher Scientific , Pittsburgh, PA, USA</td><td>RE-3325</td><td></td></tr><tr><td>Freezer</td><td>Haier, Brooklyn, NY, USA</td><td>HCMO50LA</td><td></td></tr><tr><td>Ultra microplate reader</td><td>Bio-Tek Instrument, Winooski, VT, USA</td><td>ELx 808</td><td></td></tr><tr><td>Auto Strip Washer</td><td>Bio-Tek Instrument, Winooski, VT, USA</td><td>ELx50</td><td></td></tr><tr><td>Pipettes</td><td>Gilson, Pipetman, France</td><td>P100, P200, P1000</td><td></td></tr><tr><td>C24 Incubator Shaker</td><td>New Brunswick Scientific, Enfield, CT, USA</td><td>Classic C24</td><td></td></tr><tr><td>Rocking Shaker&amp;nbsp;</td><td>Reliable Scientific, Inc., Nesbit, MS, USA</td><td>55</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific, Pittsburgh, PA, USA</td><td>875713</td><td>100 mm x 15 mm</td></tr><tr><td>SterilGard III Advance</td><td>The Baker Company, Sanford, ME, USA</td><td>SG403</td><td></td></tr><tr><td>Culture Growing Flasks</td><td>Corning Incorporated, Corning, NY, USA</td><td>4995</td><td>PYREX 250 ml Erlenmeyer flasks</td></tr><tr><td>Optical Spectrometer Genesys 20</td><td>Thermo Scientific, Waltham, MA, USA.</td><td>4001</td><td></td></tr><tr><td>Sony DXC-33 Video camera</td><td>Sony</td><td></td><td></td></tr><tr><td>Richardson test slide</td><td>Electron Microscopy Science, Hatfield, PA, USA</td><td>80303</td><td></td></tr><tr><td>Millipore water purification system</td><td>Millipore</td><td>Direct-Q</td><td></td></tr><tr><td>Image-Pro Plus software</td><td>Media Cybernetics, MD, USA</td><td></td><td></td></tr><tr><td>Triple Beam Balance</td><td>OHAUS Corporation, Parsippany, NJ, USA</td><td></td><td></td></tr><tr><td>Tissue Homogenizer, Dounce</td><td>VWR</td><td>71000-518</td><td></td></tr></tbody></table>

prevention of heat stress adverse effects in rats by bacillus subtilis strain

Questo studio è stato disegnato per valutare l&#39;effetto protettivo del ceppo Bacillus subtilis contro complicazioni legate allo stress da calore. Trentadue ratti Sprague-Dawley sono stati utilizzati in questo studio. Gli animali sono stati trattati per via orale due volte al giorno per due giorni con B. subtilis ceppo BSB3 o PBS. Il giorno dopo l&#39;ultimo trattamento, ciascun gruppo è stato diviso e due gruppi sperimentali (uno trattato con PBS e uno trattato con B. subtilis) sono stati posti a 45 ° C per 25 min. Due gruppi di controllo alloggiato per 25 min a temperatura ambiente. Tutti i ratti sono stati sacrificati e diversi parametri sono stati analizzati in tutti i gruppi. Gli effetti avversi di stress da calore sono registrati dalla diminuzione di altezza villi e lo spessore della mucosa totale a livello dell&#39;epitelio intestinale; traslocazione di batteri dal lume; aumento vescicolazione degli eritrociti ed elevazione dei lipopolisaccaridi (LPS) concentrazione nel sangue. L&#39;efficacia protettiva di trattamento viene valutata preintervento di questi effetti collaterali. Il protocollo è stato istituito per il trattamento orale di ratti con batteri per la prevenzione delle complicanze di stress termico, ma questo protocollo può essere modificato e utilizzato per altre vie di somministrazione e per l&#39;analisi di composti diversi.

La temperatura corporea media degli animali prima e subito dopo lo stress da calore era 36,7 ± 0,07 ° C e 40,3 ± 0,17 ° C, rispettivamente (P &lt;0,05). L&#39;esposizione di ratti a calore (gruppo 3) ha determinato una significativa inibizione di altezza villi e spessore della mucosa totale (figure 2, 3). La somministrazione orale di ceppo BSB3 prima lo stress protetto intestino dagli effetti dannosi del calore. <p class="jove_content" fo:ke...

Watch this Scientific Journal Video about Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain at JoVE.com

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

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.

La prevenzione di stress termico effetti avversi nei ratti<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

prevention-heat-stress-adverse-effects-rats-bacillus-subtilis

Research

JoVE Journal

Immunology and Infection

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

7.4K Views.  Auburn University. 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.

Video: Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

Una piattaforma di metil-Seq ratto per identificare i cambiamenti epigenetici connessi con l'esposizione di sforzo

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Ricerca

Biochimica

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

Metodi standardizzati per misurare l'induzione della risposta agli urti di calore in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

Ambiente

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

<meta charset="utf8"></head><body>Isolamento, purificazione e identificazione del Bacillus licheniformis produttore di bacitracina da feci fresche di suini sani

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

Isolamento, purificazione e identificazione del Bacillus licheniformis produttore di bacitracina da feci fresche di suini sani

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

Bioingegneria

Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of neurological disorders. Despite strong pre-clinical evidence, TH has not shown efficacy in clinical trials of most neurological disorders. The only successful trials employing therapeutic hypothermia were related to cardiac arrest in adults and hypoxic ischemic injury in neonates. Further investigations into the parameters of its use, and study design comparisons between pre-clinical and clinical studies, are warranted. This article demonstrates two methods of short-duration hypothermia induction. The first method allows for rapid hypothermia induction in rats using ethanol spray and fans. This method works by cooling the skin, which has been less commonly used in clinical trials and may have different physiological effects. Cooling is much more rapid with this technique than is achievable in human patients due to differences in surface area to volume ratio. Along with this, a second method is also presented, which allows for a clinically achievable cooling rate for short-duration hypothermia. This method is easy to implement, reproducible and does not require active skin cooling.

This article describes two methods of whole-body short-duration hypothermia induction in rats. The first, rapid induction method, employs active cooling using fans and ethanol spray for a rapid decrease in temperature. The second method is a gradual cooling method. This is achieved using the combination of isoflurane anesthesia and the reduction of temperature settings on the homeothermic heat mat. This results in a gradual decrease in core body temperature without the use of any external cooling devices.&#160;

Induzione dell'ipotermia di breve durata nei ratti utilizzando modelli per studi che esaminano la rilevanza clinica e i meccanismi

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of ...

Medicina

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Un modello preclinico di colpo di calore da sforzo nei topi

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Biologia

Stabilire e convalidare un modello di ratto da stress lieve cronico imprevedibile

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Autore in primo piano: Stabilire un modello di roditore per lo studio dei fattori della depressione nella medicina tradizionale mongola

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

La prevenzione di stress termico effetti avversi nei ratti

Riepilogo

Esplora Altri Video

Capitoli in questo video

Una piattaforma di metil-Seq ratto per identificare i cambiamenti epigenetici connessi con l'esposizione di sforzo

Metodi standardizzati per misurare l'induzione della risposta agli urti di calore in Caenorhabditis elegans

Isolamento, purificazione e identificazione del Bacillus licheniformis produttore di bacitracina da feci fresche di suini sani

Isolamento, purificazione e identificazione del Bacillus licheniformis produttore di bacitracina da feci fresche di suini sani

Induzione dell'ipotermia di breve durata nei ratti utilizzando modelli per studi che esaminano la rilevanza clinica e i meccanismi

Un modello preclinico di colpo di calore da sforzo nei topi

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

Stress lieve cronico imprevedibile nei ratti basato sulla medicina mongola

Stress lieve cronico imprevedibile nei ratti basato sulla medicina mongola

Stress lieve cronico imprevedibile nei ratti basato sulla medicina mongola