Stephanie Tan

Neuroscience of Mice Brain Dissection The mammalian brain can be dissected into distinct anatomical regions. Each region of the brain corresponds to different regulatory effects on specific physiological and behavioral functions.1 Specific steps and techniques are followed to isolate the different areas of the brain structure of mice with the objective of obtaining brain tissue which would serve as the basis of understanding its neurocircuitry and how it functions. Neuroscientists are interested with structures such as the olfactory bulb, which is involved in sensing odors, the prefrontal cortex and the striatum, which are responsible for executive control and integration of movement including reward processing respectively. 2 Specimen for dissection can either be frozen or fresh tissue. Upon obtaining the specimen, expose the brain by cutting through the bony cranium then examine its external surface. A membranous covering, meninges (dura mater, arachnoid and pia mater), surrounds the brain. The meningeal layer should carefully be removed with forceps. It is important to specify the relative location of each structure. Place the brain on a metal tray on its dorsal surface (top). The cerebellum, cerebrum and its lobes: frontal, parietal, temporal and occipital can be identified from this perspective. The frontal lobe occupies the bulk of this segment. Structures such as the medulla, pons, and the brainstem can be appreciated with the brain ventral side (bottom) up. The olfactory bulbs are beneath the frontal lobe of the cerebrum, these must be handled with extra care to avoid having them damaged. Rinse the brain in ice cold phosphate buffered saline (PBS) to remove any residual blood before bisecting the brain or cutting it in half (midsagittal cut). In neuroscience, the central nervous system is visualised in three main planes of axis: sagittal, coronal and horizontal. The orientation of the brain in each of these planes provides a better localisation of the structures and a guide for accurate dissection. Along the midline of the mouse brain on the dorsal side, locate the medial longitudinal fissure then slice through the corpus collosum longitudinally to separate the right from the left hemispheres. The extraction of the olfactory bulbs, striatum and hippocampus can be achieved by making a coronal cut by slicing along the border of the cerebral cortex. The hippocampus is an internal structure studied by neuroscientists for its role on formation and retrieval of associative and episodic memories.3 This can only be made visible by separating it from the thalamus and midbrain structures. Once the thalamus and the midbrain are removed, the hippocampus can be freed up. The olfactory bulbs, striatum and hippocampus come in pairs, dissecting one side should be done in the same manner as the other. Collected brain tissue should be frozen in liquid nitrogen and stored in -80OC. This method of dissecting the brain along its neuroanatomical landmarks is suitable for culturing neurons after separation of specific tissues in various brain regions of the mice. Animals (Scientific Procedures) Act 1986 The use of animal models in research is undoubtedly paramount to scientific advances and medical breakthrough treatment for human diseases. The issue of animal testing for medicinal or scientific purposes has been under scrutiny and has been an ethical dilemma since its inception. While ensuring the demand for experiments, the rational and efficient use of animals should be a primary concern for scientists and researchers who study experimental animals. As such, legal regulations have been enacted to ensure that proposed animal experiments or projects are cruelty-free and are performed under ethical grounds. The use of laboratory animals in research is tightly regulated in the UK. Research regulation dates back to 1875 with the “Cruelty to Animals Act”, which initially required that special certificates be acquired before authorizing work on animal use in research. Legislation has since evolved to include more stringent measures to control animal experimentation in order to ensure animal welfare, as well as more humane experiments involving animals. Most biomedical research in the UK should now be conducted in observance of the provisions under Animals (Scientific Procedures) Act 1986 or ASPA 1986. The act serves as the cornerstone of ethical consideration before performing biomedical research wherein the expected benefits or the experimental results should outweigh any potential animal suffering. In accordance with the Animals (Scientific Procedures) Act 1986, the protection of animals used for experimental or other scientific purposes has become a requisite. It upholds the prohibition of the use of animals in institutions where that animal can be expected to suffer adverse effects. The Act mandates researchers and scientists to exhaust all non-animal methods and alternatives before proceeding with animal use in research. ASPA 1986 has once again been amended to align the UK Law with the European Directive 2010/63/EU on the protection of animals used for scientific purposes which came into force on 1 January 2013.4 Specific directives under the legislation must be observed before animal research can be conducted. The Act particularly specifies the regulation of the use of “protected animals”, as defined under the ASPA 1986, in any scientific procedure which may cause undue harm or suffering. The protection from the legislation extends to any living vertebrate (embryonal and fetal form of mammals, birds and reptiles, fish and amphibians who can feed independently).5 Recommendations are stated for researchers to follow such as applications for licenses and certificates. No biomedical research or scientific experiments can be done that directly involves animals without first the approval from the Home Office.6 Compliance with the legislation entails that individuals and facilities that perform animal experimentation apply for licenses when using animals in experiments. ASPA license authorisation is three-tiered. The licensing system requires acquisition of an establishment license, a project license and a personal license. The personal license is specifically granted to individuals with adequate training, skills, and experience. This ensures that the procedures for the research are regulated and would be performed by qualified individuals in an establishment that has been evaluated. Additionally, no research project is exempted from undergoing an ethical review process set by the Home Office, if indeed, the potential benefits of the research outweigh any suffering. Upon issuance of the licenses, another checkpoint, in the form of a governing body would persist while conducting the approved animal research. A governing body in charge of reviewing and scrutinising all research work is appointed to the Animal Welfare and Ethical Review Body (AWERB). This is to ensure that license holders authorized to perform animal research have approved animal care practices and follow protocol while conducting research using “protected animals”. Any procedure performed to “protected animals” expected to undergo undue distress for scientific purposes are similarly regulated by the ASPA 1986.7 Proposed projects or researches should be assessed before it becomes morally permissible. Under this pretense, ASPA 1986 mandated the ethical framework of harm-benefit analysis (HBA). This framework focuses on the assessment of the harm that could possibly be inflicted on the animal, the objective of which is to reduce or minimize harm while optimizing benefits of the proposed research to the general population, animals and the environment. These measures are undertaken to ensure compliance with laws, regulations and guidelines protecting animal welfare and improving research practice. Efforts must be made to reduce suffering and the means by which this can be achieved is through the principles of Replacement, Reduction, Refinement or the three R’s. Animal Welfare and the Principles of Replacement, Reduction, Refinement (3Rs) The Principles of Replacement, Reduction, Refinement (3R’s) provides an essential framework of principles to observe in animal experimentation and serves as the most crucial responsibility of any establishment or institution license holders. The amended ASPA 1986 references the three R’s throughout the legislation. The guiding principles for the ethical practice of animal use in research and other scientific purposes (animal-based research) was a concept proposed by British scientist William M.S. Russell and microbiologist Rex L.Burch. The principles of Replacement, Reduction, Refinement (3Rs) highlight the rational and efficient use of experimental animals. The 3R’s reflects the provisions stated by ASPA 1986. The 2010 EU Directive or European Directive 2010/63/EU has concomitantly supported the principle of 3R’s as part of the humane experimental technique using animals. It is meant to be a guideline for ethically sustainable animal models in research. The three R’s, replacement, reduction and refinement are defined by Russel and Burch as, “Replacement means the substitution for conscious living higher animals of insentient material. Reduction means reduction in the numbers of animals used to obtain information of a given amount and precision. Refinement means any decrease in the incidence or severity of inhumane procedures applied to those animals which still have to be used.”8. The three R’s emphasise the utilitarian philosophy by Jeremey Bentham of minimizing harm and maximizing benefits. Experiments on animals must remain humane because researchers have the moral responsibility for the well-being of the animals. The contention of the principle of replacement is that non-animal methods, such as computer modelling and human cell lines, should be explored and exhausted before considering animal use.9 Animal studies are only permitted when absolutely no alternatives can be done. If there are no known non-animal methods available, reducing the number of animals to a minimum is required. A minimum that could nonetheless attain sound experimental results; the right number of animals for the right amount of data. This is the direct application of the principle of reduction. In vivo experimentation should observe the principle of refinement the, such that the animal would not undergo unnecessary suffering or harm. Harm can mean any form of injury or discomfort implicated on the animal during the procedure, this covers lasting effects that the animal may endure. The key consideration in conducting or performing animal experiment, therefore, is whether researchers can establish animal models without causing any form of distress to the animal while also using as few animals as possible to attain the intended and expected positive results. Animal models help in our understanding the mechanism of human diseases. The purpose of conducting animal research should always be subjected to regulations to ensure that the animals used for scientific purposes get the best care and welfare. The implementation and application of 3R’s – replacement with other possible experiments, reduction of number of animals, and refinement of experimental protocols – can significantly improve welfare of animals used in research as well as the research practice. The biomedical field still necessitates the use of animal models in research until such time that alternative or viable ethical non-animal systems would begin to exist in order to replace the contributions from animal experimentation in shaping the future of medicine. Mice, in particular, make efficient research models since their anatomy, physiology and genetics are better understood by scientists. Biological and physiological behaviors of mice closely resemble those in humans which make them the perfect candidates for biomedical studies. Outcomes and results gathered from approved projects are expected to physiologically be replicable to humans. While viable ethical non-animal systems are currently under development, the biomedical field will still necessitate and rely on the use of animal models in research. Where computer simulation and in vitro or tissue cultures would fail to yield results, animal research prevails. Results from non-animal or in vitro techniques are of limited value compared to those of in vivo studies. Ultimately, compliance to legal regulations will continue to protect the welfare of the animals. And the ethical strategy on animal experimentation remains to be continuously working towards the implementation of the three R’s. References: An efficient way in mouse brain dissection. (2014). 生理学报 Acta Physiologica Sinica, 66(2), 210–214. doi:-/j.aps- Animal testing and research. (n.d.). Retrieved from https://www.gov.uk/guidance/research-and-testing-using-animals. Festing, S., & Wilkinson, R. (2007). The ethics of animal research. Talking Point on the use of animals in scientific research. EMBO Reports, 8(6), 526–530. doi: 10.1038/sj.embor- Home Office. (2014). Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. London. Mammal Brain Dissection Guide. (2005). Retrieved November 7, 2019, from https://www.carolina.com/teacher-resources/Document/mammal-brain-dissection-guide/tr10991.tr. Russell WMS, Burch RL. 1959. (as reprinted 1992). The principles of humane experimental technique. Wheathampstead (UK): Universities Federation for Animal Welfare. Spijker, S. (2011). Dissection of Rodent Brain Regions. Neuromethods Neuroproteomics, 13–26. doi: 10.1007/-_2