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

Todos los procedimientos experimentales fueron aprobados por el IACUC de la Universidad de Auburn, Auburn, Alabama. 1. Preparación de medios de cultivo, platos y Cultura bacteriana <ol><li> Inocular 10 ml de caldo nutritivo en un matraz con una sola colonia de la cultura BSB3 Bacillus subtilis, que se cultiva durante la noche en una placa de agar nutriente. </li><li> Hacer 500 ml de medio de esporulación 19 (por litro: Bacto Nutrient Broth, 8 g; 10% (w / v) de KCl, 10 ml; 1,2% (w / v) MgSO4 x 7H 2 O, 10 ml; agar, 15 g) y autoclave a 121 ° C durante 20 min. Dejar enfriar a 50 ° C y añadir las siguientes soluciones estériles: 1 M de Ca (NO3) 2, 1 ml; 0,01 M MnCl 2, 1 ml; 1 mM de FeSO 4, 1 ml. </li><li> Medio preparado Enfriar a 40 ° C durante 20 min a temperatura ambiente y verter 25 ml en cada una de 20 placas de Petri estériles en el gabinete estéril. Deje que el medio solidifique durante la noche. </li><li> Difundir 0,5 ml de cultivo de una noche de Bacillus subtilis BSB3 sobre la superficie del medio en las placas de Petri. Incubar cultivo a 37 ° C durante 5 días hasta el 90% de esporulación. Revelar esporas de fase brillante de alta resolución de la microscopía. </li><li> bacterias de la cosecha mediante la adición de 1 ml de solución salina tamponada con fosfato estéril (PBS) a la placa y el raspado bacterias de una superficie usando un esparcidor de células estériles. Guarde la suspensión en el refrigerador hasta que sea necesario. </li><li> Confirmar viabilidad de las bacterias en placas 0,1 ml de la dilución apropiada (10 -5-10 -6) de una suspensión bacteriana en placas de medio de esporulación seguido de incubación durante 18-24 horas a 37 ° C </li><li> Recuento de las colonias bacterianas de las placas usando un contador de colonias y calcular el título de las bacterias en la suspensión de la prueba. Preparar suspensión bacteriana con la concentración final 1 x 10 8 UFC / ml en PBS. </li></ol> 2. Animales <ol><li> Utilice 32 ratas Sprague-Dawley macho (250 - 300 g,). Mantener los animales en condiciones libres de patógenos específicos con acceso libre a pienso estándar de pellets y agua. Establecer una temperatura media (21 ± 1 ° C), humedad (50 ± 5%) y un ciclo de luz-oscuridad de 12 horas. </li></ol> 3. Diseño Experimental <ol><li> Iniciar el tratamiento de los animales por sonda oral 11, después de 2 días de aclimatación. Usar la jeringa con la aguja de gavage oral. El tratamiento de las ratas con 16 B. suspensión subtilis BSB3 (1 ml por rata) dos veces al día durante dos días (B. subtilis grupo). Tratar a 16 ratas con PBS (1 ml por rata) dos veces al día durante dos días (grupo PBS) (Figura 1). </li><li> Después del tratamiento, una subdivisión de cada grupo (8 ratas por grupo): Grupo 1- control (PBS / sin estrés), Grupo 2- B. subtilis (B. subtilis / sin estrés), Grupo 3- estrés (PBS / estrés por calor) y el Grupo B. 4- subtilis + estrés (B. subtilis estrés / calor). </li><li> Medir la temperatura rectal de cada rata usando un electronic termómetro digital. Mantener las ratas de los grupos 1 y 2 a temperatura ambiente durante 25 min. Coloque los animales de los grupos 3 y 4 en una cámara climática a 45 ° C y una humedad relativa del 55% durante 25 min. </li><li> Después, medir la temperatura rectal de cada rata en todos los grupos utilizando un termómetro digital electrónico. Coloque todos los animales a temperatura ambiente durante 4 h. </li><li> Anestesiar las ratas con isoflurano (2-4%) en un frasco de anestesia sellable y observar hasta que las ratas se anestesiaron profundamente (ausencia de respuesta a pizca dedo del pie). La eutanasia a las ratas por decapitación rápida con una guillotina. </li><li> Recoger la sangre del tronco de cada rata 20 (15 - 16 ml) con las pipetas apirógenas en tubos apirógenas para obtener suero e inmediatamente tomar muestras de sangre (7 l de cada rata) con una pipeta para el examen microscópico. </li><li> Poner los tubos con la sangre en un refrigerador durante al menos 30 minutos para permitir la coagulación. Tubos de centrifugación a 20 ° C, 7.000 xg durante 10 minutos y rápidamenteeliminar el suero con una pipeta. Suero alícuota obtenida de cada rata en 50 volúmenes l y se almacena a -20 ° C hasta el ensayo. </li></ol> 4. Procedimiento Quirúrgico <ol><li> Cortar / recorte toda la piel del abdomen y el tórax de la parte inferior de la carcasa con unas tijeras eléctricas. Coloque la carcasa en un gabinete estéril y tratar la superficie afeitada de la carcasa con alcohol 70%. </li><li> Utilice un bisturí estéril para crear una incisión en la línea media del abdomen. Eliminar hígado, ganglios linfáticos mesentéricos, el bazo y el intestino delgado de cada rata utilizando pinzas estériles. </li></ol> 5. Análisis de la translocación bacteriana <ol><li> Colocar cada muestra de hígado, ganglios linfáticos mesentéricos y el bazo en tubos estériles tarados y pesar. Calcular el peso de la muestra como una diferencia entre el peso de un tubo con una muestra y el peso de un tubo vacío. </li><li> Añadir PBS estéril al tubo para obtener una dilución 1:10 (w / v) de la muestra y homogeNiza cada muestra con un homogeneizador de tejidos de vidrio estéril para obtener una suspensión homogénea 21. </li><li> Hacer diluciones seriadas (10 -1 a 10 -4) de cada muestra en PBS estéril. Plate 0,1 ml de todas las diluciones de cada tejido sobre la superficie de MacConkey y agar de sangre 5% y agar sangre Brucella con hemina (0,005 g / L) y vitamina K1 (0,01 g / L) placas. </li><li> Incubar MacConkey y de placas de agar sangre 5% aeróbicamente y placas de agar sangre Brucella en condiciones anaeróbicas a 37 ° C 22. </li><li> Recuento de las colonias de bacterias aerobias después de 24 hr, y las colonias de bacterias anaerobias después de 48 horas de incubación usando un contador de colonias. Expresar los resultados como el número de unidades formadoras de colonias (UFC) en un gramo de tejido. </li></ol> 6. Análisis histológico <ol><li> Retire pequeñas muestras de intestino de cada rata por el procedimiento quirúrgico (pasos 4.1 a 4.2). Fijar las muestras en fijador de Bouin, incrustar en parafina,preparar 6 micras secciones y secciones de tinción con hematoxilina y eosina usando procedimientos estándar 23. </li><li> Utilice un sistema de microscopio de alta resolución para medir la altura de las vellosidades intestinales y espesor total de la mucosa en cada muestra (aumento de 100X) 24. Analizar 4 muestras de cada rata y hacer por lo menos veinte mediciones en cada muestra. Expresar los resultados en micrómetros. </li></ol> 7. Ensayo de citoquinas <ol><li> Utilice kits comerciales de ELISA para rata IL-1β; IL-6; TNF-α; INF-γ, y IL-10 para medir los niveles de citoquinas en el suero de acuerdo con el protocolo del fabricante. </li><li> Generar curvas estándar para cada conjunto de muestras ensayadas usando estándares para el kit relevante. Definir el nivel de citocinas en cada muestra por comparación de los datos experimentales con la curva estándar apropiado. </li></ol> 8. Ensayo de lipopolisacárido <ol><li> Analizar el nivel de LPS en el suero usando una rata kit de ELISA de acuerdo con LPSprotocolo del fabricante. Estimar la concentración de LPS en muestras por comparación de los valores obtenidos con los valores de la curva estándar. </li></ol> 9. Alta Resolución Microscopía de Sangre <ol><li> Utilizar el sistema de microscopio descrito previamente 25,26. Utilice una plataforma de aislamiento de vibración como una base para el sistema de microscopio y cámara de vídeo y el ordenador 25 para grabar las imágenes en vivo. Calibrar las imágenes de prueba utilizando una diapositiva Richardson como se ha descrito previamente 27. </li><li> Coloque 7 l de sangre recién extraída de cada rata en un portaobjetos de vidrio, cubreobjetos, fotografiar y grabar diez cuadros de imagen de 72 x 53,3 m 2 en cada muestra (ampliación 1,700X). </li><li> Medir la concentración de vesículas utilizando software que proporciona una alta resolución de visión directa imágenes ópticas en tiempo real. Examinar un mínimo de 20 cuadros de imagen para cada condición experimental 28. </li></ol> 10. Statistics <ol><li> Expresar todos los valores como media y desviación estándar. Realizar el análisis estadístico y el trazado gráfico. Analizar los resultados con la prueba t de dos muestras, ajustar el nivel de significación de 0,05 para definir la significación estadística. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

La exposición a altas temperaturas en condiciones graves de salud 29. La prevención y la detección precoz de complicaciones relacionadas con el estrés por calor son de vital importancia 30. El protocolo presentado se puede utilizar para evaluar la eficacia de los diversos enfoques para la prevención del estrés por calor efectos adversos. Los pasos críticos dentro de este protocolo son: el procedimiento de tratamiento térmico (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

Este estudio fue diseñado para evaluar el efecto protector de la cepa de Bacillus subtilis contra las complicaciones relacionadas con el estrés por calor. Treinta y dos ratas Sprague-Dawley se utilizaron en este estudio. Los animales se trataron por vía oral dos veces al día durante dos días con B. subtilis cepa BSB3 o PBS. El día siguiente después del último tratamiento, cada grupo se dividió y se colocaron dos grupos experimentales (uno tratado con PBS y uno tratado con B. subtilis) a 45 ° C durante 25 min. Dos grupos de control permanecieron durante 25 min a temperatura ambiente. Todas las ratas se sacrificaron y los diferentes parámetros se analizaron en todos los grupos. Los efectos adversos del estrés por calor son registrados por la disminución de la altura de las vellosidades y el grosor total de la mucosa en el epitelio intestinal; translocación de bacterias desde el lumen; aumento de la vesiculación de los eritrocitos y la elevación de los lipopolisacáridos (LPS) el nivel en la sangre. La eficacia protectora de tratamiento se evaluó por prevención de estos efectos secundarios. El protocolo se creó para el tratamiento oral de ratas con bacterias para la prevención de complicaciones de estrés térmico, pero este protocolo puede ser modificado y utilizado para otras vías de administración y para el análisis de diferentes compuestos.

La temperatura corporal media de los animales antes e inmediatamente después del estrés por calor fue de 36,7 ± 0,07 ° C y 40,3 ± 0,17 ° C, respectivamente (P &lt;0,05). La exposición de ratas al calor (grupo 3) resultó en una inhibición significativa de la altura de las vellosidades y el grosor de la mucosa total de (Figuras 2, 3). La administración oral de la cepa BSB3 antes protegida estrés intestino de los efectos nocivos de calor. </...

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.

Prevención de Estrés Térmico efectos adversos en ratas<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

7.5K vistas.  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 plataforma de metil-Seq de rata para identificar cambios epigenéticos asociados a la exposición de estrés

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 ...

Investigación

Bioquímica

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.

Métodos estandarizados para medir la inducción de la respuesta de choque térmico en Caenorhabditis elegans

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

Medio 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>Aislamiento, purificación e identificación de Bacillus licheniformis productor de bacitracina a partir de heces frescas de cerdos sanos

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.

Aislamiento, purificación e identificación de Bacillus licheniformis productor de bacitracina a partir de heces frescas de cerdos sanos

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

Bioingeniería

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;

Inducción de hipotermia de corta duración en ratas utilizando modelos para estudios que examinan la relevancia clínica y los mecanismos

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 modelo preclínico de golpe de calor por esfuerzo en ratones

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.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el celular microscopia

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

Biología

Establecimiento y validación de un modelo de rata de estrés crónico e impredecible

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.

Autor destacado: Establecimiento de un modelo de roedores para investigar los factores de depresión en la medicina tradicional 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.

Prevención de Estrés Térmico efectos adversos en ratas

Resumen

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