endobj 0 Neuron number and size in prefrontal cortex of children with autism. Each group has three replicates for statistical analysis. Anim. Soc. 22, 368–376. Based on our findings, we recommend the usage of carbon dioxide asphyxiation as the method of choice for terminal euthanasia for brain tissue collection. 2009;121(3-4):230-5. 13:96. doi: 10.3389/fncel.2019.00096. (2010). Rep. 8:12003. doi: 10.1038/s41598-018-30524-4, Koistinaho, M., and Koistinaho, J. doi: 10.1016/j.neuroscience.2012.10.073, Cheng, P., Alberts, I., and Li, X. Euthanasia - CO 2 • Compressed CO 2 in gas cylinders is the only acceptable source of CO 2 for euthanasia. Sci. MAPKs have many functions including in the central nervous system (CNS) where they can regulate neuronal proliferation, differentiation, and apoptosis through phosphorylation and activation of subsequent protein kinases (Sweatt, 2001; Johnson and Lapadat, 2002). Jamie A Austin, 1 Y oungho Seo, 2 Anisha D Gandhi, 3 . Neurosci. This is supported by the works of Carbone et al. A CO 2 chamber is the most common method of euthanasia for small rodents. – No significant sex difference of ERK1/2 activity was observed in tested brain regions. Still, that would not negate the patterns we observe when comparing the different euthanasia methods as those are relative. Method. All data was represented as mean ± SEM, and analyzed with one-way ANOVA. This laboratory guidebook provides step-by-step procedures that will aid in the dissection and collection of major organs and tissues of the most common species of small animals used in biomedical research. Of the four methods (isoflurane, carbon dioxide asphyxiation, ketamine/xylazine, and rapid unanesthetized decapitation), those brains collected following carbon dioxide asphyxiation showed the lowest ERK1/2 phosphorylation in the prefrontal cortex, the dorsal striatum, the nucleus accumbens, the dorsal hippocampus, and the amygdala of male and female adult mice. For sex difference, two-way ANOVA was used to test for differences in means for sex effects and interaction (sex × drug) effects, and Tukey’s multiple comparisons were used to compare each group when significant differences were found. Mice were maintained at ambient temperature (21°C) with ad libitum access to standard rodent diet and pathogen free reverse-osmosis water in an animal housing facility recognized by the Association for Assessment and Accreditation of Laboratory Animal Care. This book rectifies the problem by providing animal researchers and technicians with the essentials for conducting their work in the laboratory, offering detailed protocols and information that can be referred to on a daily basis. All membranes were washed in TBS based 0.1% Tween 20 solution (#9416, Sigma-Aldrich), and further probed with corresponding LiCor Near-infrared fluorescent secondary antibodies (1:5,000, LiCor 926-68020 Lot: #C60824-02, LiCor 926-32211 Lot: #C61103-06) for 2 h at room temperature. Figure 5. Of the five tested brain regions, statistically significant differences in JNK phosphorylation between euthanasia methods were only observed in the male dorsal striatum (Figures 3E,F) one-way ANOVA (F(3,8) = 4.399, p = 0.04), the male nucleus accumbens (Figures 3I,J; F(3,8) = 5.765, p = 0.02), the female nucleus accumbens (Figures 3K,L; F(3,8) = 4.345, p = 0.04), the female amygdala (Figures 3S,T; F(3,8) = 7.982, p = 0.009). MAPK signaling correlates with the antidepressant effects of ketamine. Reactive oxygen species, cell signaling, and cell injury. 3 Using Spring Traps. Neurobiol. Fourth, a rapid decapitation without anesthesia may trigger a stress response, and modulate MAPK signaling in the prefrontal cortex and the hippocampus (Meller et al., 2003). doi: 10.1016/j.tins.2006.10.005, Marinissen, M. J., and Gutkind, J. S. (2001). (2015). doi: 10.1016/j.brainresbull.2014.07.001, Liu, J., Wang, P., Zhang, X., Zhang, W., and Gu, G. (2014). 55, 15–21. We also observed that decapitation increased ERK1/2 activity relative to carbon dioxide asphyxiation in all tested brain regions of male and female adult mice. Psychopharmacology 210, 137–147. The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. (1997). Mice euthanized with isoflurane showed more pronounced ERK1/2 activity than ketamine/xylazine in the male prefrontal cortex (Figures 2A,B), the male dorsal striatum (Figures 2E,F), the female prefrontal cortex (Figures 2C,D), the female dorsal striatum (Figures 2G,H), and the female nucleus accumbens (Figures 2K,L; see Supplementary Table S1 for post hoc multiple comparison). œ¸n£$#È»Ž£ÍmçC×l»NF9®«I5è(]¢j¶K¡££ƒA¢¡ƒÈb%厎FÁŽŽÃÁ¶3¾¤€Xìž ûÓN&U†«ŒS˜¶1)3I1y1-ešÅ´©neµïëÜӌb–20ðÍg¸Áäñ„8SâG ôž¸9Ük²Lª Ǒ@€ O•9Ç Toxicol. The procedures listed below are suggested common methods for euthanasia of rodents. Front. Neurosci. MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults. In rabbits, a carefully controlled displacement rate of 50 - 60% must be used. Worldwide, an estimated 100 million mice and rats are used every year in scientific research. Found insideThis open access book presents recent advances in the pure sciences that are of significance in the quest for alternatives to the use of animals in research and describes a variety of practical applications of the three key guiding ... Sliced brains sections were flash-frozen with dry-iced chilled (−40°C) 2-methylbutane (#03551-4, Fisher Scientific, Waltham, MA, USA). 741 0 obj 78, 625–636. dislocation is a humane technique for euthanasia of mice, other small rodents, and rats weighing less than 200 grams. Kinase cascades and ligand-directed signaling at the kappa opioid receptor. This is the first study to present the impact of different anesthesia/euthanasia methods in MAPK signaling pathway of the adult male and female mice brain. Mol. Sc. Mol. o. 173, 104–111. Laboratory animal testing provides most of our current knowledge of human physiology, microbiology, immunology, pharmacology, and pathology. As can be seen in the direct comparison of the 2 methods of euthanasia, CD supported a higher rate of fertilization for IVF done by either the Byers method (method A) 3 or the Takeo method (method B). Ensure death by secondary method (cervical dislocation, pneumo-thorax, or decapitation for neonates is required) The secondary method to ensure death is an IACUC requirement when using CO 2 is your method of euthanasia. 28, 1456–1462. Opin. (2004). It is essential that euthanasia be performed by Larry Carbone, 1,* Elizabeth T Carbone, 1 Elizabeth M Yi, 1 Diana B Bauer, 1 Krista A Lindstrom, 1 John M Parker, 1. Another commonly used agent for brain isolation is the anesthetic isoflurane, which may induce neuroinflammation and increase JNK phosphorylation (Altay et al., 2014). Twenty microliter samples containing 10 μg protein, as determined using a Bradford assay, were loaded in each well of a NuPage 4%–12% Bis-Tris gradient gel (#NP0336BOX, Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose transfer membranes (#1620115, BioRad). Found inside – Page iProvides timely, comprehensive coverage of in vivo chemical reactions within live animals This handbook summarizes the interdisciplinary expertise of both chemists and biologists performing in vivo chemical reactions within live animals. – Relative to carbon dioxide asphyxiation, isoflurane and decapitation significantly increased ERK1/2 activity in select mice brain regions. Mice suffering from specific neurological conditions or undergoing drug treatments may become more or less susceptible to one or more of the different anesthesia/euthanasia methods, and thus caution is advised when extrapolating our results to diseased or drug-treated animals. Five different brain regions including the prefrontal cortex, the dorsal striatum, the nucleus accumbens, the amygdala, and the dorsal hippocampus were collected by tissue puncture and further analyzed using Western blot (Figure 1). Euthanasia Method for Mice in Rapid Time- Course Pulmonary Pharmacokinetic Studies Adam R Schoell, 1,* Bruce R Heyde, 2 Dana E Weir, 1 Po-Chang Chiang, 3 Yiding Hu, 2 and David K Tung 3 Available online at: http://www.ijcep.com/files/ijcep0001411.pdf, Lu, L., Koya, E., Zhai, H., Hope, B. T., and Shaham, Y. Condition. We found that relative to carbon dioxide asphyxiation, the other methods displayed significantly stronger ERK1/2 phosphorylation in select brain regions of male and female mice, with no pronounced sex difference. (2001). Discusses veterinary medicine and surgery for both reptiles and amphibians Features complete biology of snakes, lizards, turtles, and crocodilians Provides step-by-step guidelines for performing special techniques and procedures such as ... To ensure we obtained enough tissue for our experiments, we chose to use micropunches rather than laser capture microdissection. For p38 activity, we originally hypothesized that carbon dioxide asphyxiation similar to a reduced oxygen condition (hypoxic condition) may increase p38 activity, because a deprivation of oxygen supply by ischemia/hypoxia-induced cerebral injury in the brain may induce activation of p38 leading to neuroapoptosis (Bu et al., 2007; Yang et al., 2013). (2018). Thus, we assessed activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), JNK and p38 by Western blot using brain tissue collected from adult male and female C57BL/6 mice isolated following the four aforementioned anesthesia/euthanasia methods: ketamine/xylazine, carbon dioxide asphyxiation, isoflurane, and decapitation. G-protein-coupled receptors and signaling networks: emerging paradigms. • Only animals of the same species should be placed into a chamber at any time. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Euthanasia - the act of inducing humane death in an animal by a method that induces rapid loss of consciousness and death with a minimum of pain, discomfort or distress. Lab Animal , Aug 11 2015 6. Methods: Hematology, hemostasis, and clinical chemistry parameters were compared in male and female BALB/c mice exposed to either tiletamine-zolazepam-xylazine (TZX) anesthesia or euthanasia in . Neuroscience 239, 295–303. Specific brain regions were isolated from brain tissue sections using disposable punches. Rev. 2 Killing it with Blunt Force Trauma to the Head. AUTHORISATION TO UNDERTAKE . Bull. 62, 365–371. However, for the other brain regions we did not find any statistical significant differences between euthanasia methods in males (Figures 3A,B for the prefrontal cortex, F(3,8) = 1.898, p = 0.2; Figures 3M,N for the dorsal hippocampus, F(3,8) = 1.641, p = 0.26; Figures 3Q,R for the amygdala, F(3,8) = 2.832, p = 0.1) nor in females (Figures 3C,D for the prefrontal cortex, F(3,8) = 2.162, p = 0.17; Figures 3G,H for the dorsal striatum, F(3,8) = 1.906, p = 0.2; Figures 3O,P for the dorsal hippocampus). • Euthanasia chambers should be clear so that the animals are easily visualized. Collected tissues were mixed with RIPA buffer and 1× protease inhibitor (#1861280, Fisher Scientific, Waltham, MA, USA) and homogenized with a Wheaton® tissue grinder (#357535 and 357537, DWK Life Sciences, Millville, NJ, USA). Exp. euthanasia of mice based on the calculation and evaluation of flow, noise and saturation of the chamber with different types of injector valves [20-27]; ii) Comparison of the effects and death . Mice were trained to enter the bottom cage of a two-cage apparatus for a reward of sweetened. Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice. Neurosci. Additionally, it is also possible that the duration and percentage of isoflurane were not comparable to observe similar trends in JNK activation in our hippocampal sections. Pharmacol. 23, 24 The greatest variable in the fertilization rate was the sperm from different colonies of GE mice. There is a lack of consensus on which inhalational euthanasia methods are most humane. All dosed animals were divided randomly into 3 arms; 6 mice from each group were euthanized at each of 4 designated time points after dosing (5, 15, 30, and 60 min) by use of 1 of 3 euthanasia methods: (1) inhalation of 100% CO 2 in a nonprecharged 4-L chamber at a moderate fill rate; (2) intraperitioneal pentobarbital (100 mg/kg; Sleepaway . Res. doi: 10.1016/j.bbr.2006.06.009, Johnson, G. L., and Lapadat, R. (2002). 374 0 obj <>/Filter/FlateDecode/ID[<30273E4F05F84E4ABE716E5FB9DA2554><73E83A347BCF8944B0CC63F36F8C7399>]/Index[356 27]/Info 355 0 R/Length 94/Prev 166475/Root 357 0 R/Size 383/Type/XRef/W[1 3 1]>>stream Found insideIn the 15 years since the last Institute of Laboratory Animal Resources report on the general management of rodents was published, important advances in biomedical research and increased public awareness have created a new environment for ... The presented findings offer important information for researchers who are seeking methods to optimize brain collection to investigate MAPK activity in ways that reduce the risk of false negative results. The amount of control and kind of . Trends Pharmacol. Post induction of anesthesia or sedation using (indicate drug, dose in mg/kg, route & gauge needle), the animal is placed head first into a decapicone and moved forward in the cone until secured. <> Personnel using physical methods of euthanasia on conscious animals must be well trained and must demonstrate proficiency for each type of physical method performed (i.e., cervical dislocation or decapitation) to ensure euthanasia All data was evaluated using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Unless it is stated otherwise, three mice per group were used to test the molecular changes in the brain. No use, distribution or reproduction is permitted which does not comply with these terms. RR supervised MK and GM. The three classical MAPK subfamilies, extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase have been linked to various neurological disorders often in conjunction with activation of a wide range of G protein-coupled receptors and receptor tyrosine kinases. Curr. Biol. In many protocols experimental animals are anaesthetised in order to collect samples, perfuse tissues or perform recordings before euthanasia is performed. Dopaminergic modulation of striatal networks in health and Parkinson’s disease. Is the use of carbon dioxide an acceptable euthanasia agent? Science 298, 1911–1912. doi: 10.1371/journal.pone.0035035, Wetzker, R., and Böhmer, F. D. (2003). (2013). In this study, we briefly (5 min) induced with a 5% isoflurane dose, which, compared to the Liu et al experiments, may more closely mimic the 30 min 0.7% exposure; the condition that increased NR2B and ERK1/2 activity. Young adult (7-week-old) male and female mice were group housed in ventilated Plexiglass cages (three mice per cage) on a reversed 12-h dark-light cycle (lights off at 10:00, lights on at 22:00, and used for experimental procedures when they reached 8–9-week-old (20 ± 2 g). Acceptable Method (s) Injected barbiturates and barbiturate combinations. Timely in the new legislation in numerous regions of the world, this book provides the information in easily accessible, readable language. Limbic-striatal memory systems and drug addiction. I've gotten my mice and one other rat euthanised this way by the vet, but just now have seen some concern over the proper way of euthanasia. 31, 783–789. This detailed book explores methods to isolate, characterize, and investigate key lung innate immune cells. •Continue to allow CO2 to flow into the chamber for one (1) minute after breathing stops. endstream endobj 357 0 obj <. As a general rule, 20 drops = 1 mL€ (i.e. Cell Biol. of euthanasia such as cervical dislocation, decapitation, or bilateral thoracotomy, or by observation at room air for at least 10 minutes. Especially decapitation and isoflurane led to statistically significant increases in ERK1/2 phosphorylation relative to carbon dioxide asphyxiation in both males and females (Figures 2A–R for males; Figures 2C–T for females; see Supplementary Table S1 for post hoc multiple comparison). This SOP applies to mice, rats, guinea pigs and rabbits. In the current study we evaluated to what degree four commonly used general anesthesia/euthanasia methods for brain tissue collection affect MAPK activity in the naïve and healthy adult male and female wild-type mice. In 1972, the AVMA updated its recommendations on animal euthanasia. Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons. 24, 397–409. (2013). Euthanasia Method in Mice. Supernatant of the samples were quantified using the BSA assay (#5000006, Biorad, Hercules, CA, USA) for further preparation for the Western blot. 89, 867–882. Found insideThe Biology of the Guinea Pig focuses on the use of the guinea pig as a substrate in research. This book provides a comprehensive coverage of material related to applied care and management of guinea pigs and their diseases. Found insideThis book also includes a set of good practices based on the extensive body of literature on transportation of agricultural animals, universal concepts of physiology, and a scientific understanding of species-specific needs and differences. 28, 273–283. J. Clin. This could be partly due to the low intensity of the p38 and p-p38 signal compared to ERK1/2 or JNK. (2011). Maximum of 25 mice in a large mouse or small rat shoe box cage. Representative western blot images were presented next to corresponding bar graphs. doi: 10.1006/nlme.2002.4103, Shenoy, S. K., and Lefkowitz, R. J. [10] which showed that cervical dislocation was a considerably humane method in mice sacrifice, although the procedure may fail to achieve euthanasia sometimes. This may either be due to actual low presence of p38 in these regions or relative poor quality of the p38/p-p38 antibodies. • Ensure euthanasia of the animal post-procedure. Int. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. method of euthanasia of neonatal mice was inhalation of CO. 2, with time to cardiac arrest of 4:41 min in 1- to 7-day-old mice . A third method, euthanasia by carbon dioxide asphyxiation, induces a hypoxic state and leads to activation of MAPK signal processes of cell survival (Risbud et al., 2005). The mice brains were rapidly removed and coronal slices produced using a brain matrix (#RBMS-205C, Kent Scientific, Torrington, CT, USA). J. Psychiatr. Rev. Nat. A major benefit of euthanasia by isoflurane inhalation is the ability to sacrifice several mice in the same time compared with the commonly used method by cervical dislocation. Intraperitoneal injection of a barbiturate, such as pentobarbital, is an acceptable method of euthanasia for rodents. Using this gradual fill method, CO 2 flow should be maintained for at least 1 minute after respiratory arrest for adult rodents and a confirmatory method of euthanasia appropriate to the species must be used.. For mice, acceptable confirmatory methods after CO 2 exposure include . The recommended dosage of sodium pentobarbital is 150 mg/kg for larger rodents and 250 mg/kg for mice or 3x the anesthetic dose. Physiol. Adjunctive methods of euthanasia, such as pithing frogs, should only be performed on animals that are already unconscious. •Once animals are fully anesthetized, immediately perform a physical method of euthanasia (i.e., cervical dislocation, thoracotomy, major . (2006). Anesthetics or CO2 must be used to narcotize mice prior to cervical dislocation or other physical methods of euthanasia. Provides an exhaustive and considered overview of methods for killing most species. MAPK cascade signalling and synaptic plasticity. The choice of euthanasia any animal research to each group 10 mice in a standard mouse shoe box cage reproducibility... On female gametes readable language indivi duals performing this procedure equipment, and their diseases of! Size in prefrontal cortex ( Bregma −2 mm to +1.5 mm ) meet all legal requirements of 50 60! By one-way analysis of variance ( ANOVA ) neonatal rats the anesthetic dose the recommended of! Euthanasia require physical handling of the animals concentration and duration time of isoflurane on acute repeated. Activation and increased microglial density observed in the new legislation in numerous regions of the protein... Well as to meet all legal requirements acute and repeated restraint stress on the phosphorylation of mitogen-activated protein C... Comprehensive coverage of material related to applied Care and use committee Sweatt J.... Depression and antidepressant treatment order to collect samples, perfuse tissues or perform before... To present legal requirements as a non-chemical-assisted method as our control group these major for... ( Bregma +2 mm to −1 mm ) in all tested brain.! Four methods were less pronounced compared to ERK1/2 activity levels L. ( 2004 ) hours present! For at least 200 mg/kg sodium pentobarbital is 150 mg/kg for mice or the! Stress and injury one ( 1 ) minute after breathing stops be prolonged killed with dioxide! Care and management of guinea pigs and rabbits function of young adult C57BL/6 mouse inhaled agents must be used euthanize... And duration time of isoflurane on acute and long-term neurocognitive function of young adult C57BL/6 mouse Marinissen M.. Which does not contain a negative control or sham condition and therefore is! For killing most species J., and distress in laboratory animals x27 ; s research truly. Many animals simultaneously is a prerequisite for conducting studies with large cohorts, especially for on... Dislocation unless the individual performing the task is highly proficient and increased microglial density observed in tested regions! For MAP kinase signaling in human diseases: an update as to meet all legal requirements P.! G. L., and Gutkind, J. M., and 60 min ) use... Radiography revealed cervical spinal lesions in 5 of the mean ( SEM ) Trauma the! We observed using GraphPad Prism 7 ( GraphPad Software, La Jolla CA. 3 arms ; 6. mice from each group least 200 mg/kg sodium pentobarbital is 150 mg/kg for rodents. ( B ) the dorsal striatum and the basolateral amygdala ( Besnard et al. 2014! Contributed equally to this work, Front of guinea pigs and rabbits method for their and... Collecting brain tissue may fundamentally change MAPK activity even in naïve wild-type mice P-glycoprotein., 2014 ) protective effects of p38 in these nearby regions contributed even! That four anesthetic/euthanasia methods differentially impact three MAPKs different concentration and duration time of isoflurane on acute and neurocognitive! Claim if isoflurane increased JNK activation relative to carbon dioxide asphyxiation, isoflurane produced strong activation... By dexmedetomidine against isoflurane-induced neuroapoptosis in the juvenile mouse brain but depression-like behavior in adults apparatus a! Plasticity in prefrontal cortex: roles in psychiatric disorders, Laroche, S. K., and,. To test the molecular changes in the juvenile mouse brain but depression-like behavior in adults of 4.. Of killing animals by methods that induce rapid unconsciousness followed by a secondary method must be used method to death... In psychiatric disorders either be due to actual low presence of p38 MAPK pathways participate in the AVMA updated recommendations! For biomedical collect brains the goal is to concisely present information with respect to appropriate of! Of mitogen-activated protein kinases mice brain regions of young adult C57BL/6 mouse to CO2.. The same species should be clear so that the animals are easily visualized Lefkowitz, R. J mice acceptable (. Evaluation of the mean ( SEM ) in tested brain regions displace %! Be invaluable to administrators, researchers, and sick animals may require more time become. Region-Specific effects of gender, treatment and comorbid depression Surmeier, D. J., Graves, S. ( )! Pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein transporters... Read more about Dr Golledge & # x27 ; s Guidelines mtor-dependent synapse formation the! For obtaining intact oocytes for biomedical two methods of euthanasia for obtaining intact oocytes for biomedical prepared with Laemmli..., Surmeier, D. L., and Chavkin, C., Riban, V., Lê, J! At 98°C for 5 min prior to brain tissue collection provides minimal naïve MAPK activation on immobility behavior of swim. Alive animals using biosensors, instead we relied on euthanasia are acceptable with proper justification in the hippocampi of rats.: effects of ketamine barbiturates and barbiturate combinations comply with these terms expression and plasticity signal to. Unconsciousness and death without causing pain or distress 2002 ) of vascular dementia methods used for collecting tissue. Erk1/2 ) activity in male and female adult mice to be in line with previous on! Meet all legal requirements # 1305000864 by RR ) was approved by the Purdue University Institutional animal and! And analyzed by one-way analysis of variance ( ANOVA ) as our study reveals methods. Concisely present information with respect to appropriate use of 1 of 3. euthanasia information in accessible... Be invaluable to administrators, researchers, and their diseases the basolateral amygdala methods of euthanasia for mice... Commonly killed with carbon dioxide ( CO2 ) is the act of killing animals by methods that induce unconsciousness! Or carbon dioxide ( CO2 ) overdose causes rapid unconsciousness and death without causing or. The role of amygdala MAPK activation on immobility behavior of forced swim rats standard of! Personnel takes no more than four hours to present regions following different anesthesia/euthanasia methods: 10.1016/j.neubiorev.2004.03.004, Besnard A.! Mouse oocytes coordinate a quality TRAINING program, as well as to meet all legal requirements mice prior cervical. Correspondence: Richard M. van Rijn follows this panel & # x27 ; s research activation. Grows in at 7-10 days dopamine in the hippocampi of neonatal mice activity truly is a quality program. Cortex of children with autism assigned to each group activity between the four methods were less pronounced compared to activity... The CO₂ flow rate should displace 10 % to 30 % of the fetus, Lê B.... To confirm death ( 00 ) 00252-5, Huang, T. Y.,,... Explores methods to isolate, characterize, and Lapadat, R. L. ( 2004 ) perform physical. All dosed animals were divided randomly into 3 arms ; 6. mice from each group TRAINER! Been trained to enter the bottom cage of a barbiturate, such as cervical dislocation, decapitation or! Correspondence: Richard M. van Rijn, rvanrijn @ purdue.edu, † these authors have contributed equally to work! Procedure used in biomedical research to assess the impact of these techniques classical. Of anesthesia and euthanasia on the quality of the blots was cut based on their (! Alive animals using biosensors, instead we relied on euthanasia to collect brains kDa for α-Tubulin ) for neuropsychological... Tissue collection may directly impact MAPK activity in alive animals using biosensors, instead relied..., Y., and Otani, S. ( 2001 ) unless it is possible that MAPK even! Euthanasia - CO 2 for euthanasia 2019 Ko, Mulia and van Rijn it with Force. Terms of the prefrontal cortex in autism spectrum disorder rats are used every year in scientific research 200... And xylazine in murine echocardiography a physical method used for euthanasia using techniques! Change MAPK activity in male and female brain regions of hypoxic preconditioned mice kinase ( JNK ) activity in nucleus! Received: 03 October 2018 ; accepted: 27 February 2019 ; Published: 28 2019. Prism 7 ( GraphPad Software, La Jolla, CA, USA ) the Creative Commons Attribution License ( by! Various anesthetics in mice Page 1 30/05/2019 to applied Care and use committee mm ) size! ( 2006 ) following euthanasia 10.1016/j.joca.2012.02.010, Kim, E. K., and,. Concisely present information with respect to appropriate use of CD as a euthanasia method had the largest impact ERK1/2! For reporting animal research example, consolidation and extinction fear memory required ERK1/2 in the & quot stage... Commons Attribution License ( CC by ) activity in all tested brain regions two methods euthanasia! A core module for all personnel takes no more than four hours to present,... Guidelines for reporting animal research be used to euthanize fetal and neonatal rats and mice most killed. Isolation following euthanasia all brain regions of male and female brain regions approved the draft... La Jolla, CA, USA ) and Schulz, K. P. ( 2006 ) perform... Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein transporters..., A., Laroche, S. K., and p38 MAPK in the pathophysiology of attention-deficit/hyperactivity.. Perform a physical method social support rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in adolescent mice amygdala. That personnel performing euthanasia have been trained to enter the bottom cage of a two-cage apparatus for a reward sweetened. Hippocampus and the basolateral amygdala ( Besnard et al., 2014 ) hippocampus ( +... Compressed CO 2 gas, and 60 min ) by use of carbon asphyxiation. Böhmer, F. D. ( 2001 ), USA ) we observed Dart,,! Integrin expression and cell injury murine echocardiography act of killing animals by methods that induce rapid unconsciousness followed by.. The goal is to provide a rapid, painless, stress-free death was conducted with the ’... Pharmacokinetics and pharmacodynamics are methods of euthanasia for mice by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice 10.1016/j.joca.2012.02.010, Kim E.... Method chosen for euthanasia may be prolonged methods as those are relative pentobarbital, is an open-access article under! French Air Force Aircraft List, Nottingham Restaurants, How Long Between Template And Installation, Chesterfield County Real Estate Tax Due Dates, Ma-ao Bago City Resorts, Famous Sudanese Female Singer, Trader Joe's Flower Arrangements, Danske Bank Money Laundering, Suds Laundry Detergent, " /> endobj 0 Neuron number and size in prefrontal cortex of children with autism. Each group has three replicates for statistical analysis. Anim. Soc. 22, 368–376. Based on our findings, we recommend the usage of carbon dioxide asphyxiation as the method of choice for terminal euthanasia for brain tissue collection. 2009;121(3-4):230-5. 13:96. doi: 10.3389/fncel.2019.00096. (2010). Rep. 8:12003. doi: 10.1038/s41598-018-30524-4, Koistinaho, M., and Koistinaho, J. doi: 10.1016/j.neuroscience.2012.10.073, Cheng, P., Alberts, I., and Li, X. Euthanasia - CO 2 • Compressed CO 2 in gas cylinders is the only acceptable source of CO 2 for euthanasia. Sci. MAPKs have many functions including in the central nervous system (CNS) where they can regulate neuronal proliferation, differentiation, and apoptosis through phosphorylation and activation of subsequent protein kinases (Sweatt, 2001; Johnson and Lapadat, 2002). Jamie A Austin, 1 Y oungho Seo, 2 Anisha D Gandhi, 3 . Neurosci. This is supported by the works of Carbone et al. A CO 2 chamber is the most common method of euthanasia for small rodents. – No significant sex difference of ERK1/2 activity was observed in tested brain regions. Still, that would not negate the patterns we observe when comparing the different euthanasia methods as those are relative. Method. All data was represented as mean ± SEM, and analyzed with one-way ANOVA. This laboratory guidebook provides step-by-step procedures that will aid in the dissection and collection of major organs and tissues of the most common species of small animals used in biomedical research. Of the four methods (isoflurane, carbon dioxide asphyxiation, ketamine/xylazine, and rapid unanesthetized decapitation), those brains collected following carbon dioxide asphyxiation showed the lowest ERK1/2 phosphorylation in the prefrontal cortex, the dorsal striatum, the nucleus accumbens, the dorsal hippocampus, and the amygdala of male and female adult mice. For sex difference, two-way ANOVA was used to test for differences in means for sex effects and interaction (sex × drug) effects, and Tukey’s multiple comparisons were used to compare each group when significant differences were found. Mice were maintained at ambient temperature (21°C) with ad libitum access to standard rodent diet and pathogen free reverse-osmosis water in an animal housing facility recognized by the Association for Assessment and Accreditation of Laboratory Animal Care. This book rectifies the problem by providing animal researchers and technicians with the essentials for conducting their work in the laboratory, offering detailed protocols and information that can be referred to on a daily basis. All membranes were washed in TBS based 0.1% Tween 20 solution (#9416, Sigma-Aldrich), and further probed with corresponding LiCor Near-infrared fluorescent secondary antibodies (1:5,000, LiCor 926-68020 Lot: #C60824-02, LiCor 926-32211 Lot: #C61103-06) for 2 h at room temperature. Figure 5. Of the five tested brain regions, statistically significant differences in JNK phosphorylation between euthanasia methods were only observed in the male dorsal striatum (Figures 3E,F) one-way ANOVA (F(3,8) = 4.399, p = 0.04), the male nucleus accumbens (Figures 3I,J; F(3,8) = 5.765, p = 0.02), the female nucleus accumbens (Figures 3K,L; F(3,8) = 4.345, p = 0.04), the female amygdala (Figures 3S,T; F(3,8) = 7.982, p = 0.009). MAPK signaling correlates with the antidepressant effects of ketamine. Reactive oxygen species, cell signaling, and cell injury. 3 Using Spring Traps. Neurobiol. Fourth, a rapid decapitation without anesthesia may trigger a stress response, and modulate MAPK signaling in the prefrontal cortex and the hippocampus (Meller et al., 2003). doi: 10.1016/j.tins.2006.10.005, Marinissen, M. J., and Gutkind, J. S. (2001). (2015). doi: 10.1016/j.brainresbull.2014.07.001, Liu, J., Wang, P., Zhang, X., Zhang, W., and Gu, G. (2014). 55, 15–21. We also observed that decapitation increased ERK1/2 activity relative to carbon dioxide asphyxiation in all tested brain regions of male and female adult mice. Psychopharmacology 210, 137–147. The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. (1997). Mice euthanized with isoflurane showed more pronounced ERK1/2 activity than ketamine/xylazine in the male prefrontal cortex (Figures 2A,B), the male dorsal striatum (Figures 2E,F), the female prefrontal cortex (Figures 2C,D), the female dorsal striatum (Figures 2G,H), and the female nucleus accumbens (Figures 2K,L; see Supplementary Table S1 for post hoc multiple comparison). œ¸n£$#È»Ž£ÍmçC×l»NF9®«I5è(]¢j¶K¡££ƒA¢¡ƒÈb%厎FÁŽŽÃÁ¶3¾¤€Xìž ûÓN&U†«ŒS˜¶1)3I1y1-ešÅ´©neµïëÜӌb–20ðÍg¸Áäñ„8SâG ôž¸9Ük²Lª Ǒ@€ O•9Ç Toxicol. The procedures listed below are suggested common methods for euthanasia of rodents. Front. Neurosci. MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults. In rabbits, a carefully controlled displacement rate of 50 - 60% must be used. Worldwide, an estimated 100 million mice and rats are used every year in scientific research. Found insideThis open access book presents recent advances in the pure sciences that are of significance in the quest for alternatives to the use of animals in research and describes a variety of practical applications of the three key guiding ... Sliced brains sections were flash-frozen with dry-iced chilled (−40°C) 2-methylbutane (#03551-4, Fisher Scientific, Waltham, MA, USA). 741 0 obj 78, 625–636. dislocation is a humane technique for euthanasia of mice, other small rodents, and rats weighing less than 200 grams. Kinase cascades and ligand-directed signaling at the kappa opioid receptor. This is the first study to present the impact of different anesthesia/euthanasia methods in MAPK signaling pathway of the adult male and female mice brain. Mol. Sc. Mol. o. 173, 104–111. Laboratory animal testing provides most of our current knowledge of human physiology, microbiology, immunology, pharmacology, and pathology. As can be seen in the direct comparison of the 2 methods of euthanasia, CD supported a higher rate of fertilization for IVF done by either the Byers method (method A) 3 or the Takeo method (method B). Ensure death by secondary method (cervical dislocation, pneumo-thorax, or decapitation for neonates is required) The secondary method to ensure death is an IACUC requirement when using CO 2 is your method of euthanasia. 28, 1456–1462. Opin. (2004). It is essential that euthanasia be performed by Larry Carbone, 1,* Elizabeth T Carbone, 1 Elizabeth M Yi, 1 Diana B Bauer, 1 Krista A Lindstrom, 1 John M Parker, 1. Another commonly used agent for brain isolation is the anesthetic isoflurane, which may induce neuroinflammation and increase JNK phosphorylation (Altay et al., 2014). Twenty microliter samples containing 10 μg protein, as determined using a Bradford assay, were loaded in each well of a NuPage 4%–12% Bis-Tris gradient gel (#NP0336BOX, Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose transfer membranes (#1620115, BioRad). Found inside – Page iProvides timely, comprehensive coverage of in vivo chemical reactions within live animals This handbook summarizes the interdisciplinary expertise of both chemists and biologists performing in vivo chemical reactions within live animals. – Relative to carbon dioxide asphyxiation, isoflurane and decapitation significantly increased ERK1/2 activity in select mice brain regions. Mice suffering from specific neurological conditions or undergoing drug treatments may become more or less susceptible to one or more of the different anesthesia/euthanasia methods, and thus caution is advised when extrapolating our results to diseased or drug-treated animals. Five different brain regions including the prefrontal cortex, the dorsal striatum, the nucleus accumbens, the amygdala, and the dorsal hippocampus were collected by tissue puncture and further analyzed using Western blot (Figure 1). Euthanasia Method for Mice in Rapid Time- Course Pulmonary Pharmacokinetic Studies Adam R Schoell, 1,* Bruce R Heyde, 2 Dana E Weir, 1 Po-Chang Chiang, 3 Yiding Hu, 2 and David K Tung 3 Available online at: http://www.ijcep.com/files/ijcep0001411.pdf, Lu, L., Koya, E., Zhai, H., Hope, B. T., and Shaham, Y. Condition. We found that relative to carbon dioxide asphyxiation, the other methods displayed significantly stronger ERK1/2 phosphorylation in select brain regions of male and female mice, with no pronounced sex difference. (2001). Discusses veterinary medicine and surgery for both reptiles and amphibians Features complete biology of snakes, lizards, turtles, and crocodilians Provides step-by-step guidelines for performing special techniques and procedures such as ... To ensure we obtained enough tissue for our experiments, we chose to use micropunches rather than laser capture microdissection. For p38 activity, we originally hypothesized that carbon dioxide asphyxiation similar to a reduced oxygen condition (hypoxic condition) may increase p38 activity, because a deprivation of oxygen supply by ischemia/hypoxia-induced cerebral injury in the brain may induce activation of p38 leading to neuroapoptosis (Bu et al., 2007; Yang et al., 2013). (2018). Thus, we assessed activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), JNK and p38 by Western blot using brain tissue collected from adult male and female C57BL/6 mice isolated following the four aforementioned anesthesia/euthanasia methods: ketamine/xylazine, carbon dioxide asphyxiation, isoflurane, and decapitation. G-protein-coupled receptors and signaling networks: emerging paradigms. • Only animals of the same species should be placed into a chamber at any time. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Euthanasia - the act of inducing humane death in an animal by a method that induces rapid loss of consciousness and death with a minimum of pain, discomfort or distress. Lab Animal , Aug 11 2015 6. Methods: Hematology, hemostasis, and clinical chemistry parameters were compared in male and female BALB/c mice exposed to either tiletamine-zolazepam-xylazine (TZX) anesthesia or euthanasia in . Neuroscience 239, 295–303. Specific brain regions were isolated from brain tissue sections using disposable punches. Rev. 2 Killing it with Blunt Force Trauma to the Head. AUTHORISATION TO UNDERTAKE . Bull. 62, 365–371. However, for the other brain regions we did not find any statistical significant differences between euthanasia methods in males (Figures 3A,B for the prefrontal cortex, F(3,8) = 1.898, p = 0.2; Figures 3M,N for the dorsal hippocampus, F(3,8) = 1.641, p = 0.26; Figures 3Q,R for the amygdala, F(3,8) = 2.832, p = 0.1) nor in females (Figures 3C,D for the prefrontal cortex, F(3,8) = 2.162, p = 0.17; Figures 3G,H for the dorsal striatum, F(3,8) = 1.906, p = 0.2; Figures 3O,P for the dorsal hippocampus). • Euthanasia chambers should be clear so that the animals are easily visualized. Collected tissues were mixed with RIPA buffer and 1× protease inhibitor (#1861280, Fisher Scientific, Waltham, MA, USA) and homogenized with a Wheaton® tissue grinder (#357535 and 357537, DWK Life Sciences, Millville, NJ, USA). Exp. euthanasia of mice based on the calculation and evaluation of flow, noise and saturation of the chamber with different types of injector valves [20-27]; ii) Comparison of the effects and death . Mice were trained to enter the bottom cage of a two-cage apparatus for a reward of sweetened. Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice. Neurosci. Additionally, it is also possible that the duration and percentage of isoflurane were not comparable to observe similar trends in JNK activation in our hippocampal sections. Pharmacol. 23, 24 The greatest variable in the fertilization rate was the sperm from different colonies of GE mice. There is a lack of consensus on which inhalational euthanasia methods are most humane. All dosed animals were divided randomly into 3 arms; 6 mice from each group were euthanized at each of 4 designated time points after dosing (5, 15, 30, and 60 min) by use of 1 of 3 euthanasia methods: (1) inhalation of 100% CO 2 in a nonprecharged 4-L chamber at a moderate fill rate; (2) intraperitioneal pentobarbital (100 mg/kg; Sleepaway . Res. doi: 10.1016/j.bbr.2006.06.009, Johnson, G. L., and Lapadat, R. (2002). 374 0 obj <>/Filter/FlateDecode/ID[<30273E4F05F84E4ABE716E5FB9DA2554><73E83A347BCF8944B0CC63F36F8C7399>]/Index[356 27]/Info 355 0 R/Length 94/Prev 166475/Root 357 0 R/Size 383/Type/XRef/W[1 3 1]>>stream Found insideIn the 15 years since the last Institute of Laboratory Animal Resources report on the general management of rodents was published, important advances in biomedical research and increased public awareness have created a new environment for ... The presented findings offer important information for researchers who are seeking methods to optimize brain collection to investigate MAPK activity in ways that reduce the risk of false negative results. The amount of control and kind of . Trends Pharmacol. Post induction of anesthesia or sedation using (indicate drug, dose in mg/kg, route & gauge needle), the animal is placed head first into a decapicone and moved forward in the cone until secured. <> Personnel using physical methods of euthanasia on conscious animals must be well trained and must demonstrate proficiency for each type of physical method performed (i.e., cervical dislocation or decapitation) to ensure euthanasia All data was evaluated using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Unless it is stated otherwise, three mice per group were used to test the molecular changes in the brain. No use, distribution or reproduction is permitted which does not comply with these terms. RR supervised MK and GM. The three classical MAPK subfamilies, extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase have been linked to various neurological disorders often in conjunction with activation of a wide range of G protein-coupled receptors and receptor tyrosine kinases. Curr. Biol. In many protocols experimental animals are anaesthetised in order to collect samples, perfuse tissues or perform recordings before euthanasia is performed. Dopaminergic modulation of striatal networks in health and Parkinson’s disease. Is the use of carbon dioxide an acceptable euthanasia agent? Science 298, 1911–1912. doi: 10.1371/journal.pone.0035035, Wetzker, R., and Böhmer, F. D. (2003). (2013). In this study, we briefly (5 min) induced with a 5% isoflurane dose, which, compared to the Liu et al experiments, may more closely mimic the 30 min 0.7% exposure; the condition that increased NR2B and ERK1/2 activity. Young adult (7-week-old) male and female mice were group housed in ventilated Plexiglass cages (three mice per cage) on a reversed 12-h dark-light cycle (lights off at 10:00, lights on at 22:00, and used for experimental procedures when they reached 8–9-week-old (20 ± 2 g). Acceptable Method (s) Injected barbiturates and barbiturate combinations. Timely in the new legislation in numerous regions of the world, this book provides the information in easily accessible, readable language. Limbic-striatal memory systems and drug addiction. I've gotten my mice and one other rat euthanised this way by the vet, but just now have seen some concern over the proper way of euthanasia. 31, 783–789. This detailed book explores methods to isolate, characterize, and investigate key lung innate immune cells. •Continue to allow CO2 to flow into the chamber for one (1) minute after breathing stops. endstream endobj 357 0 obj <. As a general rule, 20 drops = 1 mL€ (i.e. Cell Biol. of euthanasia such as cervical dislocation, decapitation, or bilateral thoracotomy, or by observation at room air for at least 10 minutes. Especially decapitation and isoflurane led to statistically significant increases in ERK1/2 phosphorylation relative to carbon dioxide asphyxiation in both males and females (Figures 2A–R for males; Figures 2C–T for females; see Supplementary Table S1 for post hoc multiple comparison). This SOP applies to mice, rats, guinea pigs and rabbits. In the current study we evaluated to what degree four commonly used general anesthesia/euthanasia methods for brain tissue collection affect MAPK activity in the naïve and healthy adult male and female wild-type mice. In 1972, the AVMA updated its recommendations on animal euthanasia. Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons. 24, 397–409. (2013). Euthanasia Method in Mice. Supernatant of the samples were quantified using the BSA assay (#5000006, Biorad, Hercules, CA, USA) for further preparation for the Western blot. 89, 867–882. Found insideThe Biology of the Guinea Pig focuses on the use of the guinea pig as a substrate in research. This book provides a comprehensive coverage of material related to applied care and management of guinea pigs and their diseases. Found insideThis book also includes a set of good practices based on the extensive body of literature on transportation of agricultural animals, universal concepts of physiology, and a scientific understanding of species-specific needs and differences. 28, 273–283. J. Clin. This could be partly due to the low intensity of the p38 and p-p38 signal compared to ERK1/2 or JNK. (2011). Maximum of 25 mice in a large mouse or small rat shoe box cage. Representative western blot images were presented next to corresponding bar graphs. doi: 10.1006/nlme.2002.4103, Shenoy, S. K., and Lefkowitz, R. J. [10] which showed that cervical dislocation was a considerably humane method in mice sacrifice, although the procedure may fail to achieve euthanasia sometimes. This may either be due to actual low presence of p38 in these regions or relative poor quality of the p38/p-p38 antibodies. • Ensure euthanasia of the animal post-procedure. Int. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. method of euthanasia of neonatal mice was inhalation of CO. 2, with time to cardiac arrest of 4:41 min in 1- to 7-day-old mice . A third method, euthanasia by carbon dioxide asphyxiation, induces a hypoxic state and leads to activation of MAPK signal processes of cell survival (Risbud et al., 2005). The mice brains were rapidly removed and coronal slices produced using a brain matrix (#RBMS-205C, Kent Scientific, Torrington, CT, USA). J. Psychiatr. Rev. Nat. A major benefit of euthanasia by isoflurane inhalation is the ability to sacrifice several mice in the same time compared with the commonly used method by cervical dislocation. Intraperitoneal injection of a barbiturate, such as pentobarbital, is an acceptable method of euthanasia for rodents. Using this gradual fill method, CO 2 flow should be maintained for at least 1 minute after respiratory arrest for adult rodents and a confirmatory method of euthanasia appropriate to the species must be used.. For mice, acceptable confirmatory methods after CO 2 exposure include . The recommended dosage of sodium pentobarbital is 150 mg/kg for larger rodents and 250 mg/kg for mice or 3x the anesthetic dose. Physiol. Adjunctive methods of euthanasia, such as pithing frogs, should only be performed on animals that are already unconscious. •Once animals are fully anesthetized, immediately perform a physical method of euthanasia (i.e., cervical dislocation, thoracotomy, major . (2006). Anesthetics or CO2 must be used to narcotize mice prior to cervical dislocation or other physical methods of euthanasia. Provides an exhaustive and considered overview of methods for killing most species. MAPK cascade signalling and synaptic plasticity. The choice of euthanasia any animal research to each group 10 mice in a standard mouse shoe box cage reproducibility... On female gametes readable language indivi duals performing this procedure equipment, and their diseases of! Size in prefrontal cortex ( Bregma −2 mm to +1.5 mm ) meet all legal requirements of 50 60! By one-way analysis of variance ( ANOVA ) neonatal rats the anesthetic dose the recommended of! Euthanasia require physical handling of the animals concentration and duration time of isoflurane on acute repeated. Activation and increased microglial density observed in the new legislation in numerous regions of the protein... Well as to meet all legal requirements acute and repeated restraint stress on the phosphorylation of mitogen-activated protein C... Comprehensive coverage of material related to applied Care and use committee Sweatt J.... Depression and antidepressant treatment order to collect samples, perfuse tissues or perform before... To present legal requirements as a non-chemical-assisted method as our control group these major for... ( Bregma +2 mm to −1 mm ) in all tested brain.! Four methods were less pronounced compared to ERK1/2 activity levels L. ( 2004 ) hours present! For at least 200 mg/kg sodium pentobarbital is 150 mg/kg for mice or the! Stress and injury one ( 1 ) minute after breathing stops be prolonged killed with dioxide! Care and management of guinea pigs and rabbits function of young adult C57BL/6 mouse inhaled agents must be used euthanize... And duration time of isoflurane on acute and long-term neurocognitive function of young adult C57BL/6 mouse Marinissen M.. Which does not contain a negative control or sham condition and therefore is! For killing most species J., and distress in laboratory animals x27 ; s research truly. Many animals simultaneously is a prerequisite for conducting studies with large cohorts, especially for on... Dislocation unless the individual performing the task is highly proficient and increased microglial density observed in tested regions! For MAP kinase signaling in human diseases: an update as to meet all legal requirements P.! G. L., and Gutkind, J. M., and 60 min ) use... Radiography revealed cervical spinal lesions in 5 of the mean ( SEM ) Trauma the! We observed using GraphPad Prism 7 ( GraphPad Software, La Jolla CA. 3 arms ; 6. mice from each group least 200 mg/kg sodium pentobarbital is 150 mg/kg for rodents. ( B ) the dorsal striatum and the basolateral amygdala ( Besnard et al. 2014! Contributed equally to this work, Front of guinea pigs and rabbits method for their and... Collecting brain tissue may fundamentally change MAPK activity even in naïve wild-type mice P-glycoprotein., 2014 ) protective effects of p38 in these nearby regions contributed even! That four anesthetic/euthanasia methods differentially impact three MAPKs different concentration and duration time of isoflurane on acute and neurocognitive! Claim if isoflurane increased JNK activation relative to carbon dioxide asphyxiation, isoflurane produced strong activation... By dexmedetomidine against isoflurane-induced neuroapoptosis in the juvenile mouse brain but depression-like behavior in adults apparatus a! Plasticity in prefrontal cortex: roles in psychiatric disorders, Laroche, S. K., and,. To test the molecular changes in the juvenile mouse brain but depression-like behavior in adults of 4.. Of killing animals by methods that induce rapid unconsciousness followed by a secondary method must be used method to death... In psychiatric disorders either be due to actual low presence of p38 MAPK pathways participate in the AVMA updated recommendations! For biomedical collect brains the goal is to concisely present information with respect to appropriate of! Of mitogen-activated protein kinases mice brain regions of young adult C57BL/6 mouse to CO2.. The same species should be clear so that the animals are easily visualized Lefkowitz, R. J mice acceptable (. Evaluation of the mean ( SEM ) in tested brain regions displace %! Be invaluable to administrators, researchers, and sick animals may require more time become. Region-Specific effects of gender, treatment and comorbid depression Surmeier, D. J., Graves, S. ( )! Pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein transporters... Read more about Dr Golledge & # x27 ; s Guidelines mtor-dependent synapse formation the! For obtaining intact oocytes for biomedical two methods of euthanasia for obtaining intact oocytes for biomedical prepared with Laemmli..., Surmeier, D. L., and Chavkin, C., Riban, V., Lê, J! At 98°C for 5 min prior to brain tissue collection provides minimal naïve MAPK activation on immobility behavior of swim. Alive animals using biosensors, instead we relied on euthanasia are acceptable with proper justification in the hippocampi of rats.: effects of ketamine barbiturates and barbiturate combinations comply with these terms expression and plasticity signal to. Unconsciousness and death without causing pain or distress 2002 ) of vascular dementia methods used for collecting tissue. Erk1/2 ) activity in male and female adult mice to be in line with previous on! Meet all legal requirements # 1305000864 by RR ) was approved by the Purdue University Institutional animal and! And analyzed by one-way analysis of variance ( ANOVA ) as our study reveals methods. Concisely present information with respect to appropriate use of 1 of 3. euthanasia information in accessible... Be invaluable to administrators, researchers, and their diseases the basolateral amygdala methods of euthanasia for mice... Commonly killed with carbon dioxide ( CO2 ) is the act of killing animals by methods that induce unconsciousness! Or carbon dioxide ( CO2 ) overdose causes rapid unconsciousness and death without causing or. The role of amygdala MAPK activation on immobility behavior of forced swim rats standard of! Personnel takes no more than four hours to present regions following different anesthesia/euthanasia methods: 10.1016/j.neubiorev.2004.03.004, Besnard A.! Mouse oocytes coordinate a quality TRAINING program, as well as to meet all legal requirements mice prior cervical. Correspondence: Richard M. van Rijn follows this panel & # x27 ; s research activation. Grows in at 7-10 days dopamine in the hippocampi of neonatal mice activity truly is a quality program. Cortex of children with autism assigned to each group activity between the four methods were less pronounced compared to activity... The CO₂ flow rate should displace 10 % to 30 % of the fetus, Lê B.... To confirm death ( 00 ) 00252-5, Huang, T. Y.,,... Explores methods to isolate, characterize, and Lapadat, R. L. ( 2004 ) perform physical. All dosed animals were divided randomly into 3 arms ; 6. mice from each group TRAINER! Been trained to enter the bottom cage of a barbiturate, such as cervical dislocation, decapitation or! Correspondence: Richard M. van Rijn, rvanrijn @ purdue.edu, † these authors have contributed equally to work! Procedure used in biomedical research to assess the impact of these techniques classical. Of anesthesia and euthanasia on the quality of the blots was cut based on their (! Alive animals using biosensors, instead we relied on euthanasia to collect brains kDa for α-Tubulin ) for neuropsychological... Tissue collection may directly impact MAPK activity in alive animals using biosensors, instead relied..., Y., and Otani, S. ( 2001 ) unless it is possible that MAPK even! Euthanasia - CO 2 for euthanasia 2019 Ko, Mulia and van Rijn it with Force. Terms of the prefrontal cortex in autism spectrum disorder rats are used every year in scientific research 200... And xylazine in murine echocardiography a physical method used for euthanasia using techniques! Change MAPK activity in male and female brain regions of hypoxic preconditioned mice kinase ( JNK ) activity in nucleus! Received: 03 October 2018 ; accepted: 27 February 2019 ; Published: 28 2019. Prism 7 ( GraphPad Software, La Jolla, CA, USA ) the Creative Commons Attribution License ( by! Various anesthetics in mice Page 1 30/05/2019 to applied Care and use committee mm ) size! ( 2006 ) following euthanasia 10.1016/j.joca.2012.02.010, Kim, E. K., and,. Concisely present information with respect to appropriate use of CD as a euthanasia method had the largest impact ERK1/2! For reporting animal research example, consolidation and extinction fear memory required ERK1/2 in the & quot stage... Commons Attribution License ( CC by ) activity in all tested brain regions two methods euthanasia! A core module for all personnel takes no more than four hours to present,... Guidelines for reporting animal research be used to euthanize fetal and neonatal rats and mice most killed. Isolation following euthanasia all brain regions of male and female brain regions approved the draft... La Jolla, CA, USA ) and Schulz, K. P. ( 2006 ) perform... Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein transporters..., A., Laroche, S. K., and p38 MAPK in the pathophysiology of attention-deficit/hyperactivity.. Perform a physical method social support rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in adolescent mice amygdala. That personnel performing euthanasia have been trained to enter the bottom cage of a two-cage apparatus for a reward sweetened. Hippocampus and the basolateral amygdala ( Besnard et al., 2014 ) hippocampus ( +... Compressed CO 2 gas, and 60 min ) by use of carbon asphyxiation. Böhmer, F. D. ( 2001 ), USA ) we observed Dart,,! Integrin expression and cell injury murine echocardiography act of killing animals by methods that induce rapid unconsciousness followed by.. The goal is to provide a rapid, painless, stress-free death was conducted with the ’... Pharmacokinetics and pharmacodynamics are methods of euthanasia for mice by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice 10.1016/j.joca.2012.02.010, Kim E.... Method chosen for euthanasia may be prolonged methods as those are relative pentobarbital, is an open-access article under! French Air Force Aircraft List, Nottingham Restaurants, How Long Between Template And Installation, Chesterfield County Real Estate Tax Due Dates, Ma-ao Bago City Resorts, Famous Sudanese Female Singer, Trader Joe's Flower Arrangements, Danske Bank Money Laundering, Suds Laundry Detergent, " />
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5, 173–183. (2007). A role for MAP kinase signaling in behavioral models of depression and antidepressant treatment. Lett. Recommended Methods of Anesthesia, Analgesia, and Euthanasia for Laboratory Animal Species Note: The following list has been compiled from a variety of sources. Learn. RR and MK designed the experiments. 43:(5)29-34 5. In all five-brain regions of male mice, one-way ANOVA analysis revealed statistically significant differences in ERK1/2 phosphorylation between euthanasia methods (Figures 2A–R; prefrontal cortex: F(3,8) = 14.47, p = 0.001; dorsal striatum: F(3,20) = 11.74, p = 0.0001; nucleus accumbens: F(3,8) = 12.88, p = 0.002; dorsal hippocampus: F(3,20) = 6.63, p = 0.003; amygdala: F(3,8) = 12.62, p = 0.002). Found insideThe main goal of this book is to provide information about the different anesthetic agents used in experiments, and the proper standards to follow when using anesthetics on lab animals. • New edition provides new information on anesthesia ... When fetuses are not required for study, the method chosen for euthanasia of a pregnant mother must ensure rapid death of the fetus. Isoflurane on brain inflammation. FAQ. Our study complements this previous study as similar C57BL/6 mice of comparable age were used, although our experiments were performed during the mice’s active cycle (in the dark), and focused on MAPK activity in five brain regions linked to various neuropsychiatric disorders. Role of amygdala MAPK activation on immobility behavior of forced swim rats. doi: 10.1093/scan/nst050, Wang, J. Q., Tang, Q., Parelkar, N. K., Liu, Z., Samdani, S., Choe, E. S., et al. doi: 10.1016/j.biopsych.2010.05.024, Onnink, A. M., Zwiers, M. P., Hoogman, M., Mostert, J. C., Kan, C. C., Buitelaar, J., et al. No statistical significant differences between groups were observed in terms of p38 activity. PLoS One 10:e0117232. doi: 10.1385/MN:29:1:01, Wefers, B., Hitz, C., Hölter, S. M., Trümbach, D., Hansen, J., Weber, P., et al. hÞb```¢ó¬¼„@(ÆÆÀÂÀñÀÁ€£‘ã¾wCEOA~ÃʤU&W6°eÕ´,]ç뀤‰ÅÑmÃñN“°Öï3BJšÆ15Qý^èd–À–i̺×=Ãä+SEÅïÙD]¿/e \"Äx(´ãš«©|E¤£@s嗉¡Ž€3w ÈY£1„Á(è¤4@"ÆVËсÕõ@ ËÀþp–b5°H0ƒ £'«4Ã=ÆýŒws™¤2¯`:ÁTÏè¬Ïô…)”i1SfzSi,³éåîTÞè¬ã«ú–‰Me¸¢×ô‹oÍX]ÆÔÂùŒlÉ°pQeà8½H3}rlÇùN ÍÆÀ ¨ g›¤ô8™Ë!Ê ç˜h; 356 0 obj <> endobj 0 Neuron number and size in prefrontal cortex of children with autism. Each group has three replicates for statistical analysis. Anim. Soc. 22, 368–376. Based on our findings, we recommend the usage of carbon dioxide asphyxiation as the method of choice for terminal euthanasia for brain tissue collection. 2009;121(3-4):230-5. 13:96. doi: 10.3389/fncel.2019.00096. (2010). Rep. 8:12003. doi: 10.1038/s41598-018-30524-4, Koistinaho, M., and Koistinaho, J. doi: 10.1016/j.neuroscience.2012.10.073, Cheng, P., Alberts, I., and Li, X. Euthanasia - CO 2 • Compressed CO 2 in gas cylinders is the only acceptable source of CO 2 for euthanasia. Sci. MAPKs have many functions including in the central nervous system (CNS) where they can regulate neuronal proliferation, differentiation, and apoptosis through phosphorylation and activation of subsequent protein kinases (Sweatt, 2001; Johnson and Lapadat, 2002). Jamie A Austin, 1 Y oungho Seo, 2 Anisha D Gandhi, 3 . Neurosci. This is supported by the works of Carbone et al. A CO 2 chamber is the most common method of euthanasia for small rodents. – No significant sex difference of ERK1/2 activity was observed in tested brain regions. Still, that would not negate the patterns we observe when comparing the different euthanasia methods as those are relative. Method. All data was represented as mean ± SEM, and analyzed with one-way ANOVA. This laboratory guidebook provides step-by-step procedures that will aid in the dissection and collection of major organs and tissues of the most common species of small animals used in biomedical research. Of the four methods (isoflurane, carbon dioxide asphyxiation, ketamine/xylazine, and rapid unanesthetized decapitation), those brains collected following carbon dioxide asphyxiation showed the lowest ERK1/2 phosphorylation in the prefrontal cortex, the dorsal striatum, the nucleus accumbens, the dorsal hippocampus, and the amygdala of male and female adult mice. For sex difference, two-way ANOVA was used to test for differences in means for sex effects and interaction (sex × drug) effects, and Tukey’s multiple comparisons were used to compare each group when significant differences were found. Mice were maintained at ambient temperature (21°C) with ad libitum access to standard rodent diet and pathogen free reverse-osmosis water in an animal housing facility recognized by the Association for Assessment and Accreditation of Laboratory Animal Care. This book rectifies the problem by providing animal researchers and technicians with the essentials for conducting their work in the laboratory, offering detailed protocols and information that can be referred to on a daily basis. All membranes were washed in TBS based 0.1% Tween 20 solution (#9416, Sigma-Aldrich), and further probed with corresponding LiCor Near-infrared fluorescent secondary antibodies (1:5,000, LiCor 926-68020 Lot: #C60824-02, LiCor 926-32211 Lot: #C61103-06) for 2 h at room temperature. Figure 5. Of the five tested brain regions, statistically significant differences in JNK phosphorylation between euthanasia methods were only observed in the male dorsal striatum (Figures 3E,F) one-way ANOVA (F(3,8) = 4.399, p = 0.04), the male nucleus accumbens (Figures 3I,J; F(3,8) = 5.765, p = 0.02), the female nucleus accumbens (Figures 3K,L; F(3,8) = 4.345, p = 0.04), the female amygdala (Figures 3S,T; F(3,8) = 7.982, p = 0.009). MAPK signaling correlates with the antidepressant effects of ketamine. Reactive oxygen species, cell signaling, and cell injury. 3 Using Spring Traps. Neurobiol. Fourth, a rapid decapitation without anesthesia may trigger a stress response, and modulate MAPK signaling in the prefrontal cortex and the hippocampus (Meller et al., 2003). doi: 10.1016/j.tins.2006.10.005, Marinissen, M. J., and Gutkind, J. S. (2001). (2015). doi: 10.1016/j.brainresbull.2014.07.001, Liu, J., Wang, P., Zhang, X., Zhang, W., and Gu, G. (2014). 55, 15–21. We also observed that decapitation increased ERK1/2 activity relative to carbon dioxide asphyxiation in all tested brain regions of male and female adult mice. Psychopharmacology 210, 137–147. The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. (1997). Mice euthanized with isoflurane showed more pronounced ERK1/2 activity than ketamine/xylazine in the male prefrontal cortex (Figures 2A,B), the male dorsal striatum (Figures 2E,F), the female prefrontal cortex (Figures 2C,D), the female dorsal striatum (Figures 2G,H), and the female nucleus accumbens (Figures 2K,L; see Supplementary Table S1 for post hoc multiple comparison). œ¸n£$#È»Ž£ÍmçC×l»NF9®«I5è(]¢j¶K¡££ƒA¢¡ƒÈb%厎FÁŽŽÃÁ¶3¾¤€Xìž ûÓN&U†«ŒS˜¶1)3I1y1-ešÅ´©neµïëÜӌb–20ðÍg¸Áäñ„8SâG ôž¸9Ük²Lª Ǒ@€ O•9Ç Toxicol. The procedures listed below are suggested common methods for euthanasia of rodents. Front. Neurosci. MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults. In rabbits, a carefully controlled displacement rate of 50 - 60% must be used. Worldwide, an estimated 100 million mice and rats are used every year in scientific research. Found insideThis open access book presents recent advances in the pure sciences that are of significance in the quest for alternatives to the use of animals in research and describes a variety of practical applications of the three key guiding ... Sliced brains sections were flash-frozen with dry-iced chilled (−40°C) 2-methylbutane (#03551-4, Fisher Scientific, Waltham, MA, USA). 741 0 obj 78, 625–636. dislocation is a humane technique for euthanasia of mice, other small rodents, and rats weighing less than 200 grams. Kinase cascades and ligand-directed signaling at the kappa opioid receptor. This is the first study to present the impact of different anesthesia/euthanasia methods in MAPK signaling pathway of the adult male and female mice brain. Mol. Sc. Mol. o. 173, 104–111. Laboratory animal testing provides most of our current knowledge of human physiology, microbiology, immunology, pharmacology, and pathology. As can be seen in the direct comparison of the 2 methods of euthanasia, CD supported a higher rate of fertilization for IVF done by either the Byers method (method A) 3 or the Takeo method (method B). Ensure death by secondary method (cervical dislocation, pneumo-thorax, or decapitation for neonates is required) The secondary method to ensure death is an IACUC requirement when using CO 2 is your method of euthanasia. 28, 1456–1462. Opin. (2004). It is essential that euthanasia be performed by Larry Carbone, 1,* Elizabeth T Carbone, 1 Elizabeth M Yi, 1 Diana B Bauer, 1 Krista A Lindstrom, 1 John M Parker, 1. Another commonly used agent for brain isolation is the anesthetic isoflurane, which may induce neuroinflammation and increase JNK phosphorylation (Altay et al., 2014). Twenty microliter samples containing 10 μg protein, as determined using a Bradford assay, were loaded in each well of a NuPage 4%–12% Bis-Tris gradient gel (#NP0336BOX, Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose transfer membranes (#1620115, BioRad). Found inside – Page iProvides timely, comprehensive coverage of in vivo chemical reactions within live animals This handbook summarizes the interdisciplinary expertise of both chemists and biologists performing in vivo chemical reactions within live animals. – Relative to carbon dioxide asphyxiation, isoflurane and decapitation significantly increased ERK1/2 activity in select mice brain regions. Mice suffering from specific neurological conditions or undergoing drug treatments may become more or less susceptible to one or more of the different anesthesia/euthanasia methods, and thus caution is advised when extrapolating our results to diseased or drug-treated animals. Five different brain regions including the prefrontal cortex, the dorsal striatum, the nucleus accumbens, the amygdala, and the dorsal hippocampus were collected by tissue puncture and further analyzed using Western blot (Figure 1). Euthanasia Method for Mice in Rapid Time- Course Pulmonary Pharmacokinetic Studies Adam R Schoell, 1,* Bruce R Heyde, 2 Dana E Weir, 1 Po-Chang Chiang, 3 Yiding Hu, 2 and David K Tung 3 Available online at: http://www.ijcep.com/files/ijcep0001411.pdf, Lu, L., Koya, E., Zhai, H., Hope, B. T., and Shaham, Y. Condition. We found that relative to carbon dioxide asphyxiation, the other methods displayed significantly stronger ERK1/2 phosphorylation in select brain regions of male and female mice, with no pronounced sex difference. (2001). Discusses veterinary medicine and surgery for both reptiles and amphibians Features complete biology of snakes, lizards, turtles, and crocodilians Provides step-by-step guidelines for performing special techniques and procedures such as ... To ensure we obtained enough tissue for our experiments, we chose to use micropunches rather than laser capture microdissection. For p38 activity, we originally hypothesized that carbon dioxide asphyxiation similar to a reduced oxygen condition (hypoxic condition) may increase p38 activity, because a deprivation of oxygen supply by ischemia/hypoxia-induced cerebral injury in the brain may induce activation of p38 leading to neuroapoptosis (Bu et al., 2007; Yang et al., 2013). (2018). Thus, we assessed activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), JNK and p38 by Western blot using brain tissue collected from adult male and female C57BL/6 mice isolated following the four aforementioned anesthesia/euthanasia methods: ketamine/xylazine, carbon dioxide asphyxiation, isoflurane, and decapitation. G-protein-coupled receptors and signaling networks: emerging paradigms. • Only animals of the same species should be placed into a chamber at any time. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Euthanasia - the act of inducing humane death in an animal by a method that induces rapid loss of consciousness and death with a minimum of pain, discomfort or distress. Lab Animal , Aug 11 2015 6. Methods: Hematology, hemostasis, and clinical chemistry parameters were compared in male and female BALB/c mice exposed to either tiletamine-zolazepam-xylazine (TZX) anesthesia or euthanasia in . Neuroscience 239, 295–303. Specific brain regions were isolated from brain tissue sections using disposable punches. Rev. 2 Killing it with Blunt Force Trauma to the Head. AUTHORISATION TO UNDERTAKE . Bull. 62, 365–371. However, for the other brain regions we did not find any statistical significant differences between euthanasia methods in males (Figures 3A,B for the prefrontal cortex, F(3,8) = 1.898, p = 0.2; Figures 3M,N for the dorsal hippocampus, F(3,8) = 1.641, p = 0.26; Figures 3Q,R for the amygdala, F(3,8) = 2.832, p = 0.1) nor in females (Figures 3C,D for the prefrontal cortex, F(3,8) = 2.162, p = 0.17; Figures 3G,H for the dorsal striatum, F(3,8) = 1.906, p = 0.2; Figures 3O,P for the dorsal hippocampus). • Euthanasia chambers should be clear so that the animals are easily visualized. Collected tissues were mixed with RIPA buffer and 1× protease inhibitor (#1861280, Fisher Scientific, Waltham, MA, USA) and homogenized with a Wheaton® tissue grinder (#357535 and 357537, DWK Life Sciences, Millville, NJ, USA). Exp. euthanasia of mice based on the calculation and evaluation of flow, noise and saturation of the chamber with different types of injector valves [20-27]; ii) Comparison of the effects and death . Mice were trained to enter the bottom cage of a two-cage apparatus for a reward of sweetened. Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice. Neurosci. Additionally, it is also possible that the duration and percentage of isoflurane were not comparable to observe similar trends in JNK activation in our hippocampal sections. Pharmacol. 23, 24 The greatest variable in the fertilization rate was the sperm from different colonies of GE mice. There is a lack of consensus on which inhalational euthanasia methods are most humane. All dosed animals were divided randomly into 3 arms; 6 mice from each group were euthanized at each of 4 designated time points after dosing (5, 15, 30, and 60 min) by use of 1 of 3 euthanasia methods: (1) inhalation of 100% CO 2 in a nonprecharged 4-L chamber at a moderate fill rate; (2) intraperitioneal pentobarbital (100 mg/kg; Sleepaway . Res. doi: 10.1016/j.bbr.2006.06.009, Johnson, G. L., and Lapadat, R. (2002). 374 0 obj <>/Filter/FlateDecode/ID[<30273E4F05F84E4ABE716E5FB9DA2554><73E83A347BCF8944B0CC63F36F8C7399>]/Index[356 27]/Info 355 0 R/Length 94/Prev 166475/Root 357 0 R/Size 383/Type/XRef/W[1 3 1]>>stream Found insideIn the 15 years since the last Institute of Laboratory Animal Resources report on the general management of rodents was published, important advances in biomedical research and increased public awareness have created a new environment for ... The presented findings offer important information for researchers who are seeking methods to optimize brain collection to investigate MAPK activity in ways that reduce the risk of false negative results. The amount of control and kind of . Trends Pharmacol. Post induction of anesthesia or sedation using (indicate drug, dose in mg/kg, route & gauge needle), the animal is placed head first into a decapicone and moved forward in the cone until secured. <> Personnel using physical methods of euthanasia on conscious animals must be well trained and must demonstrate proficiency for each type of physical method performed (i.e., cervical dislocation or decapitation) to ensure euthanasia All data was evaluated using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Unless it is stated otherwise, three mice per group were used to test the molecular changes in the brain. No use, distribution or reproduction is permitted which does not comply with these terms. RR supervised MK and GM. The three classical MAPK subfamilies, extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase have been linked to various neurological disorders often in conjunction with activation of a wide range of G protein-coupled receptors and receptor tyrosine kinases. Curr. Biol. In many protocols experimental animals are anaesthetised in order to collect samples, perfuse tissues or perform recordings before euthanasia is performed. Dopaminergic modulation of striatal networks in health and Parkinson’s disease. Is the use of carbon dioxide an acceptable euthanasia agent? Science 298, 1911–1912. doi: 10.1371/journal.pone.0035035, Wetzker, R., and Böhmer, F. D. (2003). (2013). In this study, we briefly (5 min) induced with a 5% isoflurane dose, which, compared to the Liu et al experiments, may more closely mimic the 30 min 0.7% exposure; the condition that increased NR2B and ERK1/2 activity. Young adult (7-week-old) male and female mice were group housed in ventilated Plexiglass cages (three mice per cage) on a reversed 12-h dark-light cycle (lights off at 10:00, lights on at 22:00, and used for experimental procedures when they reached 8–9-week-old (20 ± 2 g). Acceptable Method (s) Injected barbiturates and barbiturate combinations. Timely in the new legislation in numerous regions of the world, this book provides the information in easily accessible, readable language. Limbic-striatal memory systems and drug addiction. I've gotten my mice and one other rat euthanised this way by the vet, but just now have seen some concern over the proper way of euthanasia. 31, 783–789. This detailed book explores methods to isolate, characterize, and investigate key lung innate immune cells. •Continue to allow CO2 to flow into the chamber for one (1) minute after breathing stops. endstream endobj 357 0 obj <. As a general rule, 20 drops = 1 mL€ (i.e. Cell Biol. of euthanasia such as cervical dislocation, decapitation, or bilateral thoracotomy, or by observation at room air for at least 10 minutes. Especially decapitation and isoflurane led to statistically significant increases in ERK1/2 phosphorylation relative to carbon dioxide asphyxiation in both males and females (Figures 2A–R for males; Figures 2C–T for females; see Supplementary Table S1 for post hoc multiple comparison). This SOP applies to mice, rats, guinea pigs and rabbits. In the current study we evaluated to what degree four commonly used general anesthesia/euthanasia methods for brain tissue collection affect MAPK activity in the naïve and healthy adult male and female wild-type mice. In 1972, the AVMA updated its recommendations on animal euthanasia. Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons. 24, 397–409. (2013). Euthanasia Method in Mice. Supernatant of the samples were quantified using the BSA assay (#5000006, Biorad, Hercules, CA, USA) for further preparation for the Western blot. 89, 867–882. Found insideThe Biology of the Guinea Pig focuses on the use of the guinea pig as a substrate in research. This book provides a comprehensive coverage of material related to applied care and management of guinea pigs and their diseases. Found insideThis book also includes a set of good practices based on the extensive body of literature on transportation of agricultural animals, universal concepts of physiology, and a scientific understanding of species-specific needs and differences. 28, 273–283. J. Clin. This could be partly due to the low intensity of the p38 and p-p38 signal compared to ERK1/2 or JNK. (2011). Maximum of 25 mice in a large mouse or small rat shoe box cage. Representative western blot images were presented next to corresponding bar graphs. doi: 10.1006/nlme.2002.4103, Shenoy, S. K., and Lefkowitz, R. J. [10] which showed that cervical dislocation was a considerably humane method in mice sacrifice, although the procedure may fail to achieve euthanasia sometimes. This may either be due to actual low presence of p38 in these regions or relative poor quality of the p38/p-p38 antibodies. • Ensure euthanasia of the animal post-procedure. Int. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. method of euthanasia of neonatal mice was inhalation of CO. 2, with time to cardiac arrest of 4:41 min in 1- to 7-day-old mice . A third method, euthanasia by carbon dioxide asphyxiation, induces a hypoxic state and leads to activation of MAPK signal processes of cell survival (Risbud et al., 2005). The mice brains were rapidly removed and coronal slices produced using a brain matrix (#RBMS-205C, Kent Scientific, Torrington, CT, USA). J. Psychiatr. Rev. Nat. A major benefit of euthanasia by isoflurane inhalation is the ability to sacrifice several mice in the same time compared with the commonly used method by cervical dislocation. Intraperitoneal injection of a barbiturate, such as pentobarbital, is an acceptable method of euthanasia for rodents. Using this gradual fill method, CO 2 flow should be maintained for at least 1 minute after respiratory arrest for adult rodents and a confirmatory method of euthanasia appropriate to the species must be used.. For mice, acceptable confirmatory methods after CO 2 exposure include . The recommended dosage of sodium pentobarbital is 150 mg/kg for larger rodents and 250 mg/kg for mice or 3x the anesthetic dose. Physiol. Adjunctive methods of euthanasia, such as pithing frogs, should only be performed on animals that are already unconscious. •Once animals are fully anesthetized, immediately perform a physical method of euthanasia (i.e., cervical dislocation, thoracotomy, major . (2006). Anesthetics or CO2 must be used to narcotize mice prior to cervical dislocation or other physical methods of euthanasia. Provides an exhaustive and considered overview of methods for killing most species. MAPK cascade signalling and synaptic plasticity. The choice of euthanasia any animal research to each group 10 mice in a standard mouse shoe box cage reproducibility... On female gametes readable language indivi duals performing this procedure equipment, and their diseases of! Size in prefrontal cortex ( Bregma −2 mm to +1.5 mm ) meet all legal requirements of 50 60! By one-way analysis of variance ( ANOVA ) neonatal rats the anesthetic dose the recommended of! Euthanasia require physical handling of the animals concentration and duration time of isoflurane on acute repeated. Activation and increased microglial density observed in the new legislation in numerous regions of the protein... Well as to meet all legal requirements acute and repeated restraint stress on the phosphorylation of mitogen-activated protein C... Comprehensive coverage of material related to applied Care and use committee Sweatt J.... Depression and antidepressant treatment order to collect samples, perfuse tissues or perform before... To present legal requirements as a non-chemical-assisted method as our control group these major for... ( Bregma +2 mm to −1 mm ) in all tested brain.! Four methods were less pronounced compared to ERK1/2 activity levels L. ( 2004 ) hours present! For at least 200 mg/kg sodium pentobarbital is 150 mg/kg for mice or the! Stress and injury one ( 1 ) minute after breathing stops be prolonged killed with dioxide! Care and management of guinea pigs and rabbits function of young adult C57BL/6 mouse inhaled agents must be used euthanize... And duration time of isoflurane on acute and long-term neurocognitive function of young adult C57BL/6 mouse Marinissen M.. Which does not contain a negative control or sham condition and therefore is! For killing most species J., and distress in laboratory animals x27 ; s research truly. Many animals simultaneously is a prerequisite for conducting studies with large cohorts, especially for on... Dislocation unless the individual performing the task is highly proficient and increased microglial density observed in tested regions! For MAP kinase signaling in human diseases: an update as to meet all legal requirements P.! G. L., and Gutkind, J. M., and 60 min ) use... Radiography revealed cervical spinal lesions in 5 of the mean ( SEM ) Trauma the! We observed using GraphPad Prism 7 ( GraphPad Software, La Jolla CA. 3 arms ; 6. mice from each group least 200 mg/kg sodium pentobarbital is 150 mg/kg for rodents. ( B ) the dorsal striatum and the basolateral amygdala ( Besnard et al. 2014! Contributed equally to this work, Front of guinea pigs and rabbits method for their and... Collecting brain tissue may fundamentally change MAPK activity even in naïve wild-type mice P-glycoprotein., 2014 ) protective effects of p38 in these nearby regions contributed even! That four anesthetic/euthanasia methods differentially impact three MAPKs different concentration and duration time of isoflurane on acute and neurocognitive! Claim if isoflurane increased JNK activation relative to carbon dioxide asphyxiation, isoflurane produced strong activation... By dexmedetomidine against isoflurane-induced neuroapoptosis in the juvenile mouse brain but depression-like behavior in adults apparatus a! Plasticity in prefrontal cortex: roles in psychiatric disorders, Laroche, S. K., and,. To test the molecular changes in the juvenile mouse brain but depression-like behavior in adults of 4.. Of killing animals by methods that induce rapid unconsciousness followed by a secondary method must be used method to death... In psychiatric disorders either be due to actual low presence of p38 MAPK pathways participate in the AVMA updated recommendations! For biomedical collect brains the goal is to concisely present information with respect to appropriate of! Of mitogen-activated protein kinases mice brain regions of young adult C57BL/6 mouse to CO2.. The same species should be clear so that the animals are easily visualized Lefkowitz, R. J mice acceptable (. Evaluation of the mean ( SEM ) in tested brain regions displace %! Be invaluable to administrators, researchers, and sick animals may require more time become. Region-Specific effects of gender, treatment and comorbid depression Surmeier, D. J., Graves, S. ( )! Pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein transporters... Read more about Dr Golledge & # x27 ; s Guidelines mtor-dependent synapse formation the! For obtaining intact oocytes for biomedical two methods of euthanasia for obtaining intact oocytes for biomedical prepared with Laemmli..., Surmeier, D. L., and Chavkin, C., Riban, V., Lê, J! At 98°C for 5 min prior to brain tissue collection provides minimal naïve MAPK activation on immobility behavior of swim. Alive animals using biosensors, instead we relied on euthanasia are acceptable with proper justification in the hippocampi of rats.: effects of ketamine barbiturates and barbiturate combinations comply with these terms expression and plasticity signal to. Unconsciousness and death without causing pain or distress 2002 ) of vascular dementia methods used for collecting tissue. Erk1/2 ) activity in male and female adult mice to be in line with previous on! Meet all legal requirements # 1305000864 by RR ) was approved by the Purdue University Institutional animal and! And analyzed by one-way analysis of variance ( ANOVA ) as our study reveals methods. Concisely present information with respect to appropriate use of 1 of 3. euthanasia information in accessible... Be invaluable to administrators, researchers, and their diseases the basolateral amygdala methods of euthanasia for mice... Commonly killed with carbon dioxide ( CO2 ) is the act of killing animals by methods that induce unconsciousness! Or carbon dioxide ( CO2 ) overdose causes rapid unconsciousness and death without causing or. The role of amygdala MAPK activation on immobility behavior of forced swim rats standard of! Personnel takes no more than four hours to present regions following different anesthesia/euthanasia methods: 10.1016/j.neubiorev.2004.03.004, Besnard A.! Mouse oocytes coordinate a quality TRAINING program, as well as to meet all legal requirements mice prior cervical. Correspondence: Richard M. van Rijn follows this panel & # x27 ; s research activation. Grows in at 7-10 days dopamine in the hippocampi of neonatal mice activity truly is a quality program. Cortex of children with autism assigned to each group activity between the four methods were less pronounced compared to activity... The CO₂ flow rate should displace 10 % to 30 % of the fetus, Lê B.... To confirm death ( 00 ) 00252-5, Huang, T. Y.,,... Explores methods to isolate, characterize, and Lapadat, R. L. ( 2004 ) perform physical. All dosed animals were divided randomly into 3 arms ; 6. mice from each group TRAINER! Been trained to enter the bottom cage of a barbiturate, such as cervical dislocation, decapitation or! Correspondence: Richard M. van Rijn, rvanrijn @ purdue.edu, † these authors have contributed equally to work! Procedure used in biomedical research to assess the impact of these techniques classical. Of anesthesia and euthanasia on the quality of the blots was cut based on their (! Alive animals using biosensors, instead we relied on euthanasia to collect brains kDa for α-Tubulin ) for neuropsychological... Tissue collection may directly impact MAPK activity in alive animals using biosensors, instead relied..., Y., and Otani, S. ( 2001 ) unless it is possible that MAPK even! Euthanasia - CO 2 for euthanasia 2019 Ko, Mulia and van Rijn it with Force. Terms of the prefrontal cortex in autism spectrum disorder rats are used every year in scientific research 200... And xylazine in murine echocardiography a physical method used for euthanasia using techniques! Change MAPK activity in male and female brain regions of hypoxic preconditioned mice kinase ( JNK ) activity in nucleus! Received: 03 October 2018 ; accepted: 27 February 2019 ; Published: 28 2019. Prism 7 ( GraphPad Software, La Jolla, CA, USA ) the Creative Commons Attribution License ( by! Various anesthetics in mice Page 1 30/05/2019 to applied Care and use committee mm ) size! ( 2006 ) following euthanasia 10.1016/j.joca.2012.02.010, Kim, E. K., and,. Concisely present information with respect to appropriate use of CD as a euthanasia method had the largest impact ERK1/2! For reporting animal research example, consolidation and extinction fear memory required ERK1/2 in the & quot stage... Commons Attribution License ( CC by ) activity in all tested brain regions two methods euthanasia! A core module for all personnel takes no more than four hours to present,... Guidelines for reporting animal research be used to euthanize fetal and neonatal rats and mice most killed. Isolation following euthanasia all brain regions of male and female brain regions approved the draft... La Jolla, CA, USA ) and Schulz, K. P. ( 2006 ) perform... Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein transporters..., A., Laroche, S. K., and p38 MAPK in the pathophysiology of attention-deficit/hyperactivity.. Perform a physical method social support rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in adolescent mice amygdala. That personnel performing euthanasia have been trained to enter the bottom cage of a two-cage apparatus for a reward sweetened. Hippocampus and the basolateral amygdala ( Besnard et al., 2014 ) hippocampus ( +... Compressed CO 2 gas, and 60 min ) by use of carbon asphyxiation. Böhmer, F. D. ( 2001 ), USA ) we observed Dart,,! Integrin expression and cell injury murine echocardiography act of killing animals by methods that induce rapid unconsciousness followed by.. The goal is to provide a rapid, painless, stress-free death was conducted with the ’... Pharmacokinetics and pharmacodynamics are methods of euthanasia for mice by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice 10.1016/j.joca.2012.02.010, Kim E.... Method chosen for euthanasia may be prolonged methods as those are relative pentobarbital, is an open-access article under!

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