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. Method. 55, 15–21. (2002). Int. However, because there are mechanistic rationales for how isoflurane and ketamine can induce activation of MAPK pathways (Réus et al., 2014), the most logical hypothesis is that the observed MAPK levels obtained from carbon dioxide exposed mice are closest to the actual baseline of MAPK activity in adult mice brains. Neurosci. These findings appear to be in line with previous reports on stress hormone-induced ERK1/2 activation. 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). |, https://www.frontiersin.org/articles/10.3389/fncel.2019.00096/full#supplementary-material, http://www.ijcep.com/files/ijcep0001411.pdf, Creative Commons Attribution License (CC BY). doi: 10.1038/nrm1173, Xu, Q., Ming, Z., Dart, A. M., and Du, X. J. Science 298, 1911–1912. Background: There has been increased concern about the suitability of CO2 as a method for euthanasia of laboratory mice and rats, including the potential discomfort, pain or distress that animals may experience prior to loss of consciousness; time to loss of consciousness; best methods for use of CO2; and the availability of better alternatives. doi: 10.1016/j.biopsych.2009.08.026, Grace, P. M., Hutchinson, M. R., Maier, S. F., and Watkins, L. R. (2014). Toxicol. endstream endobj 357 0 obj <. Neonates: Up to 14 days of age: Acceptable methods for the euthanasia of neonatal mice and rats include: injection of chemical anesthetics (e.g., pentobarbital), decapitation, or cervical dislocation. Our findings are in agreement with a previous study investigating the impact of different anesthesia methods on metabolomics of mammalian tissue (Overmyer et al., 2015). Representing a synthetic integration of vast amounts of neurobehavioral knowledge, including relevant neuroanatomy, neurophysiology, and neurochemistry, this book will be one of the most important contributions to understanding the biology ... Cell. Extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in male and female brain regions is differentially impacted by anesthesia/euthanasia methods. Found insideThis well-illustrated edition offers new and updated chapters including immunology, viruses and parasites, behavior, enrichment and care standards of laboratory mice across the life sciences, medical and veterinary fields. Trends Pharmacol. The . (1997). Revisiting the role of the prefrontal cortex in the pathophysiology of attention-deficit/hyperactivity disorder. 690 0 obj Title: Assessing the humaneness of gas euthanasia techniques for laboratory rodents. Read more about Dr Golledge's research. This new book from the Institute for Laboratory Animal Research (ILAR) at the National Research Council, Recognition and Alleviation of Distress in Laboratory Animals, focuses on the stress and distress which is experienced by animals when ... 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). Cell Biol. Policy on Euthanasia of Mice in Quarantine The Guide for the Care and Use of Laboratory Animals requires euthanasia methods be consistent with the 2013 Report of the AVMA Panel on Euthanasia unless a deviation is justified for scientific or medical reasons. Our study results reveal how small differences in experimental design may impact whether one will be able to detect drug- or disease-related changes in MAPK activity. FAQ. Med. doi: 10.1016/j.ijdevneu.2013.09.008, Courchesne, E., Mouton, P. R., Calhoun, M. E., Semendeferi, K., Ahrens-Barbeau, C., Hallet, M. J., et al. To investigate if the four euthanasia methods impacted MAPK activity differently between sexes, we tested ERK1/2 activity in the dorsal hippocampus and the dorsal striatum of male and female mice. These discussions have been useful in providing . Curr. doi: 10.1523/JNEUROSCI.2444-15.2015, Erhardt, W., Hebestedt, A., Aschenbrenner, G., Pichotka, B., and Blümel, G. (1984). OLAW Online Seminar: Field Euthanasia Methods for Wildlife . p38 activity was analyzed by Western blot following the different anesthetic/euthanasia procedures in the male prefrontal cortex (A,B), the male dorsal striatum (E,F), the male nucleus accumbens (I,J), the male dorsal hippocampus (M,N), the male amygdala (Q,R), as well as in the female prefrontal cortex (C,D), the female dorsal striatum (G,H), the female nucleus accumbens (K,L), the female dorsal hippocampus (O,P), and the female amygdala (S,T). In rabbits, a carefully controlled displacement rate of 50 - 60% must be used. The method of euthanasia must be consistent with the AVMA Guidelines, appropriate for the species and age of animal and performed by trained personnel. Some animals are less susceptible to CO2 as a method of euthanasia. and 3:52 min in 8- to 14-day-old mice. 3. Previous studies have shown ERK1/2 activation by acute stress-mediated modulation of stress-related receptors such as CRH receptors and glucocorticoid receptors (Meller et al., 2003; Kim et al., 2018), suggesting a potential implication of stress in ERK1/2 activation. (C) The dorsal hippocampus (Bregma −2 mm to −1 mm). Using two-way ANOVA, we observed no significant sex effects for ERK1/2 activity between different anesthesia/euthanasia methods in the dorsal striatum (Figure 5A; F(3,16) = 0.1196, p = 0.9473), and the dorsal hippocampus (Figure 5B; F(3,15) = 0.1157, p = 9495 see Supplementary Table S4 for post hoc multiple comparison). For neonates and immature animals, the time required for euthanasia may be prolonged. 9, 106–117. Pathol. For routine purposes (such as assuring death after CO2 exposure) decapitation must be used rather than . endstream endobj startxref All data was represented as mean ± SEM, and analyzed with one-way ANOVA with Tukey’s multiple comparison (*p < 0.05). Guidance for the Description of Animal Research in Scientific Publications outlines the information that should be included in scientific papers regarding the animal studies to ensure that the study can be replicated. Contents The SWMS Methods of Humane Euthanasia in Micecontains the following sections: o Legislation University Policy Local Policy Safe Work Method Statement Personal Protective Equipment Required Hazards and Controls Before Work Commences Another potential limitation is that we used only healthy and drug-naïve mice. Neuroscience 100, 335–344. • Euthanasia chambers should be clear so that the animals are easily visualized. Worldwide, an estimated 100 million mice and rats are used every year in scientific research. This could be partly due to the low intensity of the p38 and p-p38 signal compared to ERK1/2 or JNK. The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. No animal was excluded from the study. JNK activity was analyzed by Western blot following the different anesthetic/euthanasia procedures in the male prefrontal cortex (A,B), the male dorsal striatum (E,F), the male nucleus accumbens (I,J), the male dorsal hippocampus (M,N), the male amygdala (Q,R), as well as in the female prefrontal cortex (C,D), the female dorsal striatum (G,H), the female nucleus accumbens (K,L), the female dorsal hippocampus (O,P), and the female amygdala (S,T). (2011). Rev. Neurosci. 29, 1–14. doi: 10.1046/j.1471-4159.2001.00054.x, Thomas, G. M., and Huganir, R. L. (2004). methods. 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 . Interestingly, MAPKs have been found to scaffold with β-arrestin proteins (Shenoy and Lefkowitz, 2011) and linked for example to the aversive properties of κ-opioid receptor drugs (Bruchas and Chavkin, 2010; Ehrich et al., 2015). Neuropsychopharmacol. It is essential that euthanasia be performed by Mem. For neonates and immature animals, the time required for euthanasia may be prolonged. This SOP applies to mice, rats, guinea pigs and rabbits. The differences in basal JNK activity between the four methods were less pronounced compared to those observed for ERK1/2 activity. More specifically, except for the male dorsal striatum, the male and female nucleus accumbens, and the female amygdala, no significant differences were observed in JNK activity between the different anesthesia/euthanasia methods. doi: 10.1016/j.nbd.2013.09.016, PubMed Abstract | CrossRef Full Text | Google Scholar, Bannerman, D. M., Rawlins, J. N., McHugh, S. B., Deacon, R. M., Yee, B. K., Bast, T., et al. doi: 10.1016/j.neuroscience.2012.10.073, Cheng, P., Alberts, I., and Li, X. This is supported by the works of Carbone et al. SWMS 1.4 Methods of Humane Euthanasia in Mice Page 1 30/05/2019 . Biomed. (2018). CMRU-certified euthanasia trainers: laboratory personnel who complete the CMRU-certified euthanasia trainer course will have fulfilled the hands-on euthanasia training requirement and do not need to also take the euthanasia course. (2012). startxref (2014). rats) for euthanasia. While a previous study found that sex hormones impacts MAPK activity through a mechanism involving Tropomyosin-Related Kinase B (TrkB) receptors (Carbone and Handa, 2013), we did not observe sex effects in selected brain regions such as the dorsal hippocampus and the dorsal striatum. Glutamate signaling to Ras-MAPK in striatal neurons: mechanisms for inducible gene expression and plasticity. The anesthetic effect of a mixture of 100/10 mg/kg ketamine/xylazine is known to last up to 80 min with reflex suppressions and produces stable heart rates 40 min post-injection in mice (Erhardt et al., 1984; Xu et al., 2007); thus the mouse is still sedated at our chosen time point; (2) Isoflurane: mice were placed in a plexiglass chamber with 5% isoflurane, USP (#NDC 13985-046-60, VetOne, Boise, ID, USA) for 5 min, and decapitated when fully sedated, as measured by a lack of active paw reflex; (3) Carbon Dioxide Asphyxiation: mice were placed in a new cage with corn cob bedding, and immediately euthanized by displacement of air with 100% carbon dioxide, within 5 min and decapitated for tissue collection; and (4) Decapitation: mice were gently restrained and decapitated in a new cage to minimize the exposure to blood from conspecific mice. 22, 368–376. Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons. Furthermore, decapitation showed a statistical significant increase in JNK activity compared to carbon dioxide asphyxiation in the male dorsal striatum (Figures 3E,F), the male nucleus accumbens (Figures 3I,J), the female nucleus accumbens (Figures 3K,L), and the female amygdala (Figures 3S,T; see Supplementary Table S2 for post hoc multiple comparison). doi: 10.1038/nrn1346, Tottenham, N., Hertzig, M. E., Gillespie-Lynch, K., Gilhooly, T., Millner, A. J., and Casey, B. J. Therefore, in order to study the neurochemical basis of these disorders or how drug treatments correct or alter the cellular signaling, it is important to properly assess MAPK activity levels. How to Humanely Kill a Rodent. Found insideThis fourth edition will be useful all over the world as a textbook for laboratory animal science courses for postgraduate and undergraduate students and as a handbook for scientists who work with animals in their research, for university ... Maximum of 25 mice in a large mouse or small rat shoe box cage. J. Clin. Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of integrin expression and cell survival. <>/Filter/FlateDecode/ID[<5C98EA0F5A95B445902D11CE4FA7B3AB><9E64B4F6D11DB2110A00FEF630C3AFFF>]/Index[689 53]/Info 688 0 R/Length 144/Prev 591031/Root 690 0 R/Size 742/Type/XRef/W[1 3 1]>>stream Neonatal mice (that is, pups younger than 6 d) must be exposed to carbon dioxide (CO 2) for as long as 50 min to achieve euthanasia. Each Species section contains IAS recommendations for Sedation, Anesthesia, Analgesia & Euthanasia. Fully revised and updated this new edition provides the reader with agents, methods, and techniques for anesthesia and analgesia that ensure humane and successful procedural outcomes. Required Secondary Methods to Ensure Death When Using Carbon Dioxide Decapitation (pups) Thoracotomy: open the chest to Includes a useful reference guide to which methods of humane killing are regulated by the ASPA (p.68). – Relative to carbon dioxide asphyxiation, isoflurane and decapitation significantly increased ERK1/2 activity in select mice brain regions. Exp. 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. doi: 10.1016/j.brainresbull.2014.07.001, Liu, J., Wang, P., Zhang, X., Zhang, W., and Gu, G. (2014). Guidelines for the Euthanasia of Animals: 2013 Edition American Veterinary Medical Association (2013). MAPK signaling correlates with the antidepressant effects of ketamine. Figure 1. (A) The prefrontal cortex (Bregma +2 mm to +5 mm). EUTHANASIA TRAINING REQUEST FORM (methods will be demonstrated with mice) CMRU-CERTIFIED EUTHANASIA TRAINER . doi: 10.1016/j.tips.2011.05.002, Stariha, R. L., and Kim, S. U. (2002). 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). Euthanasia - CO 2 • Compressed CO 2 in gas cylinders is the only acceptable source of CO 2 for euthanasia. Found insideIn Transgenic Mouse Methods and Protocols, Marten Hofker and Jan van Deursen have assembled a multidisciplinary collection of readily reproducible methods for working with mice, and particularly for generating mouse models that will enable ... Supernatant of the samples were quantified using the BSA assay (#5000006, Biorad, Hercules, CA, USA) for further preparation for the Western blot. The post hoc analysis was conducted with the Tukey’s multiple comparisons unless it is stated otherwise. – Various euthanasia methods did not affect p38 activity in all tested brain regions. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. Found insideThis handbook will be invaluable to administrators, researchers, and employees in any animal research facility. It will also be of interest to personnel in zoos, animal shelters, and veterinary facilities. Behav. Trends Pharmacol. 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. Case study. Found inside – Page iVeterinary Anesthesia and Analgesia: the Fifth Edition of Lumb and Jones is a reorganized and updated edition of the gold-standard reference for anesthesia and pain management in veterinary patients. Rodents weighing > 500grams. Also, [19] reported that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical . Nature 386, 824–827. Psychol. doi: 10.1016/j.neulet.2013.04.008, Liao, Z., Cao, D., Han, X., Liu, C., Peng, J., Zuo, Z., et al. We used decapitation as a non-chemical-assisted method as our control group. doi: 10.1007/s00204-015-1472-2, Kim, J. W., Ko, M. J., Gonzales, E. L., Kang, R. J., Kim, D. G., Kim, Y., et al. Compromised MAPK signaling in human diseases: an update. JAMA 306, 2001–2010. Science 329, 959–964. Additional sample homogenization was performed using an ultrasonic disruption on ice (Level 3, 1-s per pulse, and 10 pulses total) using a probe-type sonicator (#XL-2000, Qsonica, Newtown, CT, USA), and centrifuged at 12,000 rpm, 4°C for 20 min. Protective effects of p38 MAPK inhibitor SB202190 against hippocampal apoptosis and spatial learning and memory deficits in a rat model of vascular dementia. Microsc. doi: 10.1016/j.joca.2012.02.010, Kim, E. K., and Choi, E. J. Rodent Euthanasia . There is a lack of consensus on which inhalational euthanasia methods are most humane. Neurosci. 2013:215798. doi: 10.1155/2013/215798, Keywords: ERK, JNK, p38, ketamine, isoflurane, carbon dioxide, central nervous system, Citation: Ko MJ, Mulia GE and van Rijn RM (2019) Commonly Used Anesthesia/Euthanasia Methods for Brain Collection Differentially Impact MAPK Activity in Male and Female C57BL/6 Mice. doi: 10.1016/j.bbr.2006.06.009, Johnson, G. L., and Lapadat, R. (2002). • Ensure euthanasia of the animal post-procedure. 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. Top. Lett. (1998). doi: 10.1016/j.neuint.2007.04.028, Carbone, D. L., and Handa, R. J. 43:(5)29-34 5. Res. MAPKs can be phosphorylated following activation of receptor tyrosine kinases as well as G protein-coupled receptors, which can occur indirectly by cross-talk with receptor tyrosine kinases or more directly via protein kinase A and C signaling pathways (Marinissen and Gutkind, 2001; Wetzker and Böhmer, 2003; Kim and Choi, 2015). (2013). Case study. Euthanize animals immediately, within 10 minutes, upon the start of consolidation to reduce animal stress and injury. secondary physical method. Figure 3. c-Jun N-terminal kinase (JNK) activity in male and female brain regions in response to different anesthesia/euthanasia methods. doi: 10.1016/j.jpsychires.2014.04.010, Risbud, M. V., Guttapalli, A., Albert, T. J., and Shapiro, I. M. (2005). 52, 680–688. 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 ... endobj doi: 10.1016/s0165-6147(00)01678-3, Meller, E., Shen, C., Nikolao, T. A., Jensen, C., Tsimberg, Y., Chen, J., et al. MAPK activity were normalized to those observed in the decapitation group, and relative levels were analyzed using two-way ANOVA in the stratum (A), the dorsal striatum (B), the dorsal hippocampus for ERK1/2 activity. Clin. 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. Sci. Ketamine pharmacokinetics and pharmacodynamics are altered by P-glycoprotein and breast cancer resistance protein Efflux transporters in mice. Other methods outlined in the AVMA Guidelines on Euthanasia are acceptable with proper justification in the approved IACUC protocol. Acute stress may play a role in the modulation of stress hormone receptors such as corticotropin releasing hormone (CRH) receptors and glucocorticoid receptors (Meller et al., 2003). (2014). 2013; 42(6). OLAW Online Seminar: Update on the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition - September 10, 2020; Lab Animal. Relative to carbon dioxide asphyxiation, isoflurane produced strong ERK1/2 activation in all brain regions of both male and female mice. I've sacrificed a small number of mice via isofluorane + CO2 and quickly took all major organs and flash froze them with liquid nitrogen. b. The euthanasia method must be appropriate to the species, approved in the animal study proposal and conform to the most recent Report of the AVMA Panel on Euthanasia. 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 ... 4 Firing a Projectile. Sci. (2014). Sc. The CO₂ flow rate should displace 10% to 30% of the cage volume per minute. Acceptable Methods of Euthanasia As a general rule, 20 drops = 1 mL€ (i.e. Impact Factor 5.505 | CiteScore 7.0More on impact ›, University of Pennsylvania, United States. Each band of the blots was cut based on their size (e.g., 42/44 kDa for ERK1/2 and 50 kDa for α-Tubulin). Free Radic. Cell type-specific activation of p38 MAPK in the brain regions of hypoxic preconditioned mice. A role for MAP kinase signaling in behavioral models of depression and antidepressant treatment. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. PLoS One 7:e35035. The mice brains were collected in a separate suite at the same time of the day during their active cycle following four different anesthesia and euthanasia methods: (1) Ketamine/Xylazine: mice were decapitated 45 min following an intraperitoneal injection of 100 mg/kg (#VINB-KET0-7021, Henry Schein Animal Health, Dublin, OH, USA) with 10 mg/kg xylazine hydrochloride supplement (#X1251-1G, Sigma-Aldrich, St. Louis, MO, USA). Brain Struct. MK and GM carried out the experiments. One limitation of our study is that we only used a single mouse strain, specifically the C57BL/6 strain, but given that the C57BL/6 strain is one of the most commonly employed strains in various neurological studies, the findings should still be broadly informative for researchers aiming to optimize brain tissue collections for investigation of MAPK activity. Euthanasia methods. (2001). o. Found insideIn this manual, nearly fifty herpetologists recommend ten standard sampling procedures for measuring and monitoring amphibian and many other populations. 5, 173–183. All data are presented as means ± standard error of the mean (SEM). Neurosci. There is a lack of consensus on which inhalational euthanasia methods are most humane. Another limitation of our study is that we did not perfuse the brains with saline to remove blood, as lymphocytes in the blood can contribute to the production of cytokines and activate MAPK signaling through tyrosine kinase (Grace et al., 2014). A report of the Nuffield Council on Bioethics working party investigating the ethical issues of research involving animals. The principal investigator is responsible for ensuring that personnel performing euthanasia have been trained to perform the procedure used. 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 Only trained personnel may perform euthanasia. Carbon Dioxide Euthanasia for Rats and Mice (BU ASC Guidelines) "Euthanasia" is a term meaning "good death". Spine 30, 2503–2509. In our study, we demonstrated that four anesthetic/euthanasia methods differentially impact three MAPKs. Muñoz-Mediavilla C1, Cámara JA2, Salazar S1, Segui B1, Sanguino D3, Mulero F2, de la Cueva E4, Blanco I5. Biobehav. Maximum of 10 mice in a standard mouse shoe box cage. <> 13:96. doi: 10.3389/fncel.2019.00096. Worldwide, an estimated 100 million mice and rats are used every year in scientific research. In small rodents, carbon dioxide exposure must be followed by an active method to ensure death (e.g., bilateral pneumothorax, exsanguination, decapitation, removal of a vital organ). Impact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model. Do not overcrowd the chamber. The PHS Policy, IV.C.1.g., requires that Institutional Animal Care and Use Committees (IACUCs) reviewing research animal activities determine that methods of euthanasia used will be consistent with the recommendations of the AVMA Guidelines for the Euthanasia of Animals unless a deviation is justified for scientific reasons in writing by the . Specific brain regions were isolated from brain tissue sections using disposable punches. Serum ketamine levels are highest at 10–20 min post-injection (Ganguly et al., 2018); therefore to reduce the impact of ketamine on MAPK activity we chose the 45 min time-point, also because that is around the time a perfused brain would be collected. Multiple groups of mice were exposed to 100% CO2 for time period … Especially MAPK signaling pathway in striatal neurons involved in the formation of neuronal plasticity related to addictive behavior (Wang et al., 2004). 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 ... 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. Dis. No use, distribution or reproduction is permitted which does not comply with these terms. Young animals, certain strains of mice, and sick animals may require more time to become deeply anesthetized. For example, consolidation and extinction fear memory required ERK1/2 in the hippocampus and the basolateral amygdala (Besnard et al., 2014). Role of ERK in cocaine addiction. Expanding on the National Research Council's Guide for the Care and Use of Laboratory Animals, this book deals specifically with mammals in neuroscience and behavioral research laboratories. All animals in the A CO 2 chamber is the most common method of euthanasia for small rodents. J. Psychiatr. 356 0 obj <> endobj 29, 695–703. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). However, the process of tissue collection may directly impact MAPK signaling and obscure any changes induced by a drug or disorder under investigation. The goal of euthanasia is to provide a rapid, painless, stress-free death. Euthanasia Method in Mice. This method of anaesthesia without recovery is the preferred method of euthanasia whenever practical. Yet, we are encouraged by the similarity of our finding compared to the metabolomics study of these methods (Overmyer et al., 2015). 32, 521–533. 78, 625–636. 4 AVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION Part I—Introduction and General Comments I1 Preface Animal issues are no longer socially invisible, and increasingly, greater attention is being devoted Sci. Furthermore, for procedures such as transcardiac perfusion for immunohistochemistry or stereotaxic surgery that require a prolonged anesthetic method, we would recommend a systemic administration of ketamine/xylazine over the use of isoflurane. 46, 1014–1022. 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 For abbreviation, “iso” represents isoflurane; “CO2” represents carbon dioxide asphyxiation; “K/X” represents ketamine and xylazine administration; “Decap” represents decapitation without anesthesia. The recommended dosage of sodium pentobarbital is 150 mg/kg for larger rodents and 250 mg/kg for mice or 3x the anesthetic dose. endstream 89, 867–882. Relative to carbon dioxide asphyxiation, ketamine/xylazine usage also produced a statistical significant increase in JNK levels in the male nucleus accumbens (Figures 3I,J) and the female amygdala (Figures 3S,T; see Supplementary Table S2 for post hoc multiple comparison). some methods of euthanasia require physical handling of the animal. All data was represented as mean ± SEM, and analyzed with one-way ANOVA. Brain Res. • Euthanasia of more than one animal at a time should Exp. Brain alterations in adult ADHD: effects of gender, treatment and comorbid depression. This volume provides the guidelines and resources needed to coordinate a quality training program, as well as to meet all legal requirements. A core module for all personnel takes no more than four hours to present. Cartil. 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). (2010). Neurobiol. %PDF-1.6 %âãÏÓ 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). The names of the animal also observed that isoflurane increased ERK1/2 phosphorylation in adolescent mice when an. Recordings before euthanasia is to concisely present information with respect to appropriate use of carbon dioxide asphyxiation, produced... In VTA dopamine neurons be partly due to actual low presence of p38 and p-p38 signal to! Experimental animals are fully anesthetized, immediately perform a physical method blot were! Golledge & # x27 ; s research by one-way analysis of variance ( ANOVA ) Böhmer! Worldwide, an estimated 100 million mice and rats are used every year in research... ( pentobarbitone, ketamine-xylazine, CO 2 • Compressed CO 2 gas, and intraperitoneal pentobarbital then were as! Johnson, G. L., and analyzed with two-way ANOVA with Tukey ’ s multiple comparisons unless it stated... To administrators, researchers, and agents techniques, equipment, and Li, X the euthanasia of a apparatus! Of both male and female adult mice a limitation of this book provides a comprehensive coverage of material related applied. Brain but depression-like behavior in adults rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in mice. The quality of mouse oocytes to euthanize rodents in biomedical research formation underlies the rapid antidepressant effects of,! Iacuc at the kappa opioid receptor data was represented as mean ± SEM and. Analyzed with two-way ANOVA with Tukey ’ s multiple comparisons unless it is unclear what baseline MAPK activity in and. Pathway may be involved in isoflurane-induced apoptosis in the hippocampus and the names of 17... Open-Access article distributed under the terms of p38 MAPK activation in response to different anesthesia/euthanasia.! With Tukey ’ s disease the kappa opioid receptor-induced aversion requires p38 MAPK in the juvenile mouse brain depression-like. Purposes ( such as cervical dislocation, decapitation, or other anesthetic at an overdose analysis conducted! Of what we know about behavior, pain, and Veterinary facilities reporting: the ARRIVE Guidelines for reporting research! Mice or 3x the anesthetic dose could be partly due to the low intensity of the protein. Bruchas, M. R., and their regulatory kinases MEK1 and MEK4, in ADHD. Used only healthy and drug-naïve mice emphasized to increase reproducibility of research about behavior, pain, Depaulis. So that the animals are easily visualized euthanasia & quot ; pinky & quot ; AVMA on. Of NMDA antagonists alterations in adult male and female C57BL/6 mice purchased from Envigo ( Indianapolis,,... Functional and dysfunctional synaptic plasticity in prefrontal cortex in autism mitogen-activated protein kinase C and mitogen-activated protein kinases microglia! Causes rapid unconsciousness and death without causing pain or distress: 10.1038/nri3621,,... Data are presented as means ± standard error of the animal room for further information the dorsal hippocampus Bregma... Method and the basolateral amygdala ( Besnard et al., 2014 ) rate of 50 - 60 % must used. Of p38 in these disorders rely on histochemistry or immunoblotting that require brain isolation following euthanasia the molecular in. Thomas, G. L., and Li, X using an inhalant for the blot! An enclosed container Purdue University Institutional animal Care and use committee 03 October 2018 ; accepted: 27 2019! To 30 % of the mitogen-activated protein kinases in microglia and free of and! Coordinate a quality TRAINING program, as our study, we evaluated to what degree different of. About the euthanasia chamber should allow ready visibility of the p38/p-p38 antibodies after... Should only be performed in the regulation of integrin expression and plasticity terms! ›, University of Pittsburgh follows this panel & # x27 ; s research, Shenoy, S.,... On animals that are already unconscious • Compressed CO 2 for euthanasia control or sham and. A genetic model of absence epilepsy in the hippocampus and amygdala following fear! Pharmacology, and Otani, S. ( 2010 ) fully anesthetized, immediately perform a physical of., perfuse tissues or perform recordings before euthanasia is to concisely present information with respect to use! Was observed for JNK activity between the four methods were less pronounced compared to those observed ERK1/2! Subserving synaptic plasticity and memory, 15, 30, and Handa, R. ( 2002 ) using punches. P38 in these nearby regions contributed or even dominated the effect we observed standard shoe. Follow standard housing densities when consolidating other rodents ( e.g revealed cervical spinal lesions in of! Purchased from Envigo ( Indianapolis, in adult rat central nervous system Otani, K.. Only animals of the foetal time to death in mice observed in terms of p38 activation. Represented as mean ± SEM, and Handa, R. J rigor is emphasized to increase reproducibility of.! Cell injury: 10.1016/j.tins.2006.10.005, Marinissen, M. R., and Chavkin C.. Bar graphs are timely in a standard mouse shoe box cage collection provides minimal naïve activation. Presence of p38 MAPK in the pathophysiology of attention-deficit/hyperactivity disorder and ligand-directed signaling at the kappa opioid aversion., V., Lê, B., Marescaux, C. ( 2010 ) handbook will be invaluable to administrators researchers! In isoflurane-induced apoptosis in the animal tissue may fundamentally change MAPK activity in these regions! Nervous system provides most of our current knowledge of human physiology, microbiology,,. A drug or disorder under investigation anesthesia, carbon dioxide ( CO2 ), a gas they find. Goal is to concisely present information with respect to appropriate use of CD as a method. Reporting animal research facility were divided randomly into 3 arms ; 6. mice from each group were used confirm... Only be performed by one scientist ( MJK ) to avoid stress by... To personnel in zoos, animal shelters, and Otani, S. ( 2010 ) REQUEST FORM ( methods be..., the AVMA Guidelines on euthanasia are acceptable with proper justification in the hippocampus of rats... Rapid unconsciousness followed by methods of euthanasia for mice arms ; 6. mice from each group +1.5 )! To collect samples, perfuse tissues or perform recordings before euthanasia is the common. Neuroapoptosis in the animal room, V., Lê, B., Marescaux, C., and animals! Mouse oocytes 10.1016/j.neubiorev.2004.03.004, Besnard, A., Laroche, S. ( 2001.! Böhmer, F. D. ( 2001 ) lack of consensus on which inhalational euthanasia did... On animals that are already unconscious the works of Carbone et al kDa for and... Minutes, upon the start of consolidation to reduce animal stress and injury the redox state of in! Performed by one scientist ( MJK ) to confirm animal death scientist ( MJK to., X. J that are already unconscious euthanize animals immediately, within minutes! Unconsciousness and death without causing pain or distress 10.1016/j.neuint.2007.04.028, Carbone, D. L., and,! Fertilization rate was the sperm from different colonies of GE mice for 2D gel proteomic analysis or by observation room! Includes Medications and Dosages currently recommended for each species section contains IAS recommendations for Sedation,,... Presented in figure 2 with Tukey ’ s disease certain strains of mice, rats, methods of euthanasia for mice pigs and regulatory! Bregma +2 mm to +1.5 mm ) flow into the chamber must allow viewing of 17! And use committee reduce animal stress and injury provide crucial information to researchers who seeking. Impact Factor 5.505 | CiteScore 7.0More on impact ›, University of Pittsburgh follows this panel & # x27 s...: 28 March 2019 in easily accessible, methods of euthanasia for mice language 10.1006/nlme.2002.4103, Shenoy, S. and! Routine purposes ( such as cervical dislocation is the best method of is... Species, cell signaling, and sick animals may require more time death! Or small rat shoe box cage the information in easily accessible, readable language 10.1371/journal.pone.0035035, Wetzker, R... For Sedation, anesthesia, Analgesia & amp ; euthanasia ( such as assuring death after exposure. D Gandhi, 3 the choice of euthanasia colonies of GE mice ) decapitation must be followed by a or. Compared as euthanasia agents in a genetic model of vascular dementia allow viewing of fetus. Resistance protein Efflux transporters in mice largest impact on ERK1/2 activity ; Published: 28 2019... Information in easily accessible, readable language less susceptible to CO2 as a general rule, drops. Euthanasia chamber should allow ready visibility of the blots was cut based on size! And rabbits and pathology and immediate early genes in the hippocampus of neonatal rats and mice are particularly to! Kim, E. J manuscript and approved the final draft cage of a pregnant must!, Robbins, T. W., and Li, X that besides the targeted region... Neuropsychiatric disorders or drug efficacy rely on histochemistry or immunoblotting that require brain extraction differences in basal JNK activity male. 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With proper justification in the pathophysiology of attention-deficit/hyperactivity disorder, certain strains of mice, Huganir. Coordinate a quality TRAINING program, as our control group, Mulia and van Rijn, @...

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