B involvement 15 minutes (test session 1) and 30 minutes (test session 2) after the end of decompression: this test uses a 60 cm-long wire cord suspended at a height of 40 cm. The mouse is placed in the middle of the wire cord hanging from its front paws. Suspension time more than 30 sec is considered as a successful grip. Mice which escape by climbing up the cord FCCP during both test sessions are also considered asymptomatic and are given the highest score of 30 sec. In order to alleviate the distress experienced by the animals, all the mice were anesthetized after this period of observations.Materials and Methods Animals and Ethics StatementAll procedures involving experimental animals were in line with European Union rules (Directive 86/609) and French law (Decree 87/848). The ethics committee of IRBA approved this study. Our investigator (NV) is associated to the agreement number 83.6 delivered by the Health and Safety Directorate of our department, as stated in the French rules R.214-93, R-214-99 and R.214-102. Only C57Bl6 (Harlan Laboratories, France) males were used in this experiment in order to avoid fluctuations due to female hormone cycles. All the mice were housed in a common cage and kept oth during rest and during the experiments n a regular day (6h00?8h00)/night (12 hours) cycle. Food (AO3, UAR) and water were ad libitum and the temperature was maintained at 2261uC. A total of 91 mice (6? weeks of age) were exposed to compressed air to induce DCS. The mice were randomly divided into two groups and numbered: 46 for the group treated with fluoxetine and 45 for the controls. Weight was similar in both groups (23.862.3 g for fluoxetine vs 24.362.3 g for controls, p = 0.304). The experimental group received a per os 50 mg/kg fluoxetine solution in the form of buy Gracillin ProzacTM (Lilly laboratories, France) 18 hours before hyperbaric exposition while the control group had a similar saccharine solution (7.4 g/kg) without fluoxetine. We opted to use a high dose of fluoxetine based on previous research in a mouse model of ischemia [18,20].Blood Cell TestsBlood tests were carried out in an automatic analyzer (ABCvet, SCIL, France) on samples taken before the dive and then again 35 minutes after surfacing. Red cells, leukocytes and platelets were counted in 20 ml samples taken from the tip of the tail and diluted in an equivalent volume of 2 mM EDTA (Sigma, France).Hyperbaric ProcedureOur hyperbaric procedure 18325633 was based on previous studies using short and relatively deep no-stop dives that favour neurological symptoms of DCS [26,27]. Batches of 18?0 freely-moving mice (9?0 per cage and per group) were subjected to the hyperbaric protocol in a 200-liter tank fitted with three ports for observation. The compression protocol involved a rise of 10 kPa.min21 up to absolute pressure of 200 kPa (equivalent depth of 10 msw), then 100 kPa.min21 up to 1000 kPa (equivalent depth of 90 msw), maximal absolute pressure at which the animals were kept for 45 minutes before decompression. The decompression rate (100 kPa.sec21) was automatically controlled by a computer linked to an Analogical/Digital converter (NIUSB-6211, National Instrument, USA) itself connected to a solenoid valve (Belino LR24A-SR, Switzerland) and a pressure transmitter (Pressure Transmitter 8314, Burket Fluid Control System, Germany). The program used to control decompression rate was designed on DasyLab (DasyLab National Instrument, USA) by our engineer.Circulating IL-6 Detection40 min afte.B involvement 15 minutes (test session 1) and 30 minutes (test session 2) after the end of decompression: this test uses a 60 cm-long wire cord suspended at a height of 40 cm. The mouse is placed in the middle of the wire cord hanging from its front paws. Suspension time more than 30 sec is considered as a successful grip. Mice which escape by climbing up the cord during both test sessions are also considered asymptomatic and are given the highest score of 30 sec. In order to alleviate the distress experienced by the animals, all the mice were anesthetized after this period of observations.Materials and Methods Animals and Ethics StatementAll procedures involving experimental animals were in line with European Union rules (Directive 86/609) and French law (Decree 87/848). The ethics committee of IRBA approved this study. Our investigator (NV) is associated to the agreement number 83.6 delivered by the Health and Safety Directorate of our department, as stated in the French rules R.214-93, R-214-99 and R.214-102. Only C57Bl6 (Harlan Laboratories, France) males were used in this experiment in order to avoid fluctuations due to female hormone cycles. All the mice were housed in a common cage and kept oth during rest and during the experiments n a regular day (6h00?8h00)/night (12 hours) cycle. Food (AO3, UAR) and water were ad libitum and the temperature was maintained at 2261uC. A total of 91 mice (6? weeks of age) were exposed to compressed air to induce DCS. The mice were randomly divided into two groups and numbered: 46 for the group treated with fluoxetine and 45 for the controls. Weight was similar in both groups (23.862.3 g for fluoxetine vs 24.362.3 g for controls, p = 0.304). The experimental group received a per os 50 mg/kg fluoxetine solution in the form of ProzacTM (Lilly laboratories, France) 18 hours before hyperbaric exposition while the control group had a similar saccharine solution (7.4 g/kg) without fluoxetine. We opted to use a high dose of fluoxetine based on previous research in a mouse model of ischemia [18,20].Blood Cell TestsBlood tests were carried out in an automatic analyzer (ABCvet, SCIL, France) on samples taken before the dive and then again 35 minutes after surfacing. Red cells, leukocytes and platelets were counted in 20 ml samples taken from the tip of the tail and diluted in an equivalent volume of 2 mM EDTA (Sigma, France).Hyperbaric ProcedureOur hyperbaric procedure 18325633 was based on previous studies using short and relatively deep no-stop dives that favour neurological symptoms of DCS [26,27]. Batches of 18?0 freely-moving mice (9?0 per cage and per group) were subjected to the hyperbaric protocol in a 200-liter tank fitted with three ports for observation. The compression protocol involved a rise of 10 kPa.min21 up to absolute pressure of 200 kPa (equivalent depth of 10 msw), then 100 kPa.min21 up to 1000 kPa (equivalent depth of 90 msw), maximal absolute pressure at which the animals were kept for 45 minutes before decompression. The decompression rate (100 kPa.sec21) was automatically controlled by a computer linked to an Analogical/Digital converter (NIUSB-6211, National Instrument, USA) itself connected to a solenoid valve (Belino LR24A-SR, Switzerland) and a pressure transmitter (Pressure Transmitter 8314, Burket Fluid Control System, Germany). The program used to control decompression rate was designed on DasyLab (DasyLab National Instrument, USA) by our engineer.Circulating IL-6 Detection40 min afte.
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