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Evaluating the Effect of Chloroform Inhalation As a Method of Euthanasia on the Cerebellum and Hippocampus of Adult Wistar Rats

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Evaluating the Effect of Chloroform Inhalation As a Method of Euthanasia on the Cerebellum and Hippocampus of Adult Wistar Rats Journal of Advances in Medical and Pharmaceutical Sciences 22(6): 14-25, 2020; Article no.JAMPS.58750 ISSN: 2394-1111 Evaluating the Effect of Chloroform Inhalation as a Method of Euthanasia on the Cerebellum and Hippocampus of Adult Wistar Rats Ugochukwu Samuel Aguwa1*, Okeke Somadina Nnamdi1, Ezejindu Darmian Nnabuihe1, Eze Chinyere Elizabeth1, Azurunwa Ogechi2, Obinwa Benedict Nzube1, Ovie Ogbo. Felix2, Obi Kelvin Chukwuemeka3, Onwuelingo Sopuru2, Okonkwo David2, Doris Ogbuokiri1 and Okeke Chijioke1 1Anatomy Department, Faculty of Basic Medical Sciences, Nnamdi Azikiwe University, Nnewi, Nigeria. 2Anatomy Department, Faculty of Basic Medical Sciences, Madonna University Nigeria, Nigeria. 3Anatomy Department, Faculty of Basic Medical Sciences, University of Abuja, Nigeria. Authors’ contributions This research work was carried out in collaboration among all authors. Author USA designed and coordinated the research and also drafted the first manuscript. Authors OSN and EDN corrected the first manuscript and drafted the final manuscript. Authors ECE, AO, OBN, OS and OD carried out the bench work. Authors OOF and OKC oversaw the laboratory analysis while authors DO and OC oversaw the statistical analysis. All authors read and approved the final manuscript. Article Information DOI: 10.9734/JAMPS/2020/v22i630176 Editor(s): (1) Dr. Sadaf Jamal Gilani, The Glocal University, India. Reviewers: (1) Sinisa Franjic, University of Osijek, Republic of Croatia. (2) Khalid Mohammad Mahmoud Mohany, Assiut University, Egypt. Complete Peer review History: http://www.sdiarticle4.com/review-history/58750 Received 07 May 2020 Accepted 12 July 2020 Original Research Article Published 27 July 2020 ABSTRACT Chloroform inhalation is a common method of rodent euthanasia in Nigeria for research purposes. This work is designed to evaluate the consequence of this method of sacrifice on the cerebellum and hippocampus of Wistar rats. Twenty male Wistar rats weighing between 160 and 180 were divided into 4 groups of 5 rats each. Group A served as control and was sacrificed using cervical dislocation (a widely acceptable non-inhalation method of sacrifice). Groups B, C and D were exposed to chloroform for 5 minutes once a day (group B), once a day for 2 days and once a day _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected]; Aguwa et al.; JAMPS, 22(6): 14-25, 2020; Article no.JAMPS.58750 for 3 days. The brains were removed; four from each group was processed for antioxidant assay while one from each group was fixed in Bouin’s fluid for histological studies. Our results show that chloroform inhalation adversely affected the results of the antioxidant parameters studied in a dose- dependent fashion. That means that the adverse effect worsened as the number of days increased. This was also the case with the histology results as there was evidence of cell necrosis in the cerebellar and hippocampal tissues. This also showed dose dependence. We therefore conclude from our results that when studying the brain tissues or carrying out brain related researches, chloroform inhalation is not the method of choice for rat euthanasia. Keywords: Euthanasia; chloroform; inhalation; cerebellum; hippocampus. 1. INTRODUCTION classified chloroform as a Group B2, probable human carcinogen [2]. Chloroform is a colorless liquid that is not very soluble in water and is very volatile. It has a Chloroform, which is toxic to the central nervous pleasant, nonirritating odor with an odor system, can cause a person to become threshold is 85 ppm. The chemical formula for unconsciousness and can be fatal at high doses. chloroform is CHCl3, a molecular weight of It has not been found to harm a fetus. Effects 119.38 g/mol, a vapor pressure of 159 mm Hg at noted in humans exposed to chloroform via 20°C, and a log octanol/water partition coefficient anesthesia include changes in respiratory rate, (log Kow) of 1.97 [1,2]. Chloroform is a cardiac effects, gastrointestinal effects, such as commonly used chemical in biological nausea and vomiting, and effects on the liver and laboratories and for industrial processes. It is kidney [5,2]. In humans, a fatal oral dose of nonflammable. Most of the population is exposed chloroform may be as low as 10 mL (14.8 g), with to very low levels of chloroform every day in the death due to respiratory or cardiac arrest [5,2]. air, food and water we take in. Low levels of Inhalation exposures of animals have also chloroform are found in the air and in coastal resulted in effects on the kidney [5,2]. waters, inland rivers, lakes and groundwater. Exposure to higher levels of chloroform is very However, even with all these known negative unlikely for anyone outside industries using or effects, chloroform is still commonly used as an manufacturing chloroform. Levels can be higher agent for euthanasia of experimental animals, in industrial areas as well as in the air above especially rodents in Africa [6]. However, how swimming pools containing chlorine [3]. much damage can result from brief exposure to chloroform as in when used for animal sacrifice is Chloroform is used as a solvent for lacquers, the subject of this research work. Chloroform can floor polishes, resins, adhesives, alkaloids, fats, be detected in blood, urine, and body tissues. oils and rubber. It is also used in the building, However, these methods are not very reliable paper and board industries, in pesticide and film because chloroform is rapidly eliminated from the production. Until the mid-1900s, chloroform was body, and the tests are not specific for used as an anesthetic to reduce pain during chloroform [2]. medical procedures. Today, it is not used in this way due to its harmful effects. Breathing air with Animal sacrifice is a method of inducing humane chloroform for a short time causes headache, death in an animal by a method that results in fatigue and dizziness. Longer exposure to rapid loss of consciousness and death for chloroform damages the brain, liver and kidneys. research purposes [7,8]. The Oxford English It may also cause cancer. Respiratory injuries Dictionary reports that sacrifice was first used to from chloroform exposure include respiratory refer to killing research animals in 1903 [9]. depression, pneumonitis and pulmonary edema. Humans have been using other vertebrate Drinking water with chloroform over a long animal species as models of their anatomy and period damages the liver and kidneys. It may physiology since the dawn of medicine. Because also cause cancer. On the other hand, eating of the taboos regarding the dissection of food with chloroform in it over a long period humans, physicians in ancient Greece dissected damages the liver and kidneys and may cause animals for anatomical studies [10]. As the cancer as well [4]. Body contact with liquid experimentation on animals increased, especially chloroform causes sores and skin irritation. It the practice of vivisection, so did criticism and may also cause cancer. In fact, EPA has controversy. Objections in the use of animal for 15 Aguwa et al.; JAMPS, 22(6): 14-25, 2020; Article no.JAMPS.58750 experiment came from two main angles; animal physical methods have some limited evidence right and applicability of result [11]. Some early that they may not immediately abolish brain scientists believed that animals were inferior to activity or consciousness [21]. Barbiturates are humans and so that results from animals could used extensively and are considered the agents not be applied to humans [12]. On the other of choice for most animal research but hand, scientists in favor of animal testing held barbiturates reduce cerebral metabolism leading that experiments on animals were necessary to to decreases in cerebral blood flow and advance medical and biological knowledge. intracranial pressure. They also produce Claude Bernard, who is sometimes known as the respiratory depression and can elicit dose- "prince of vivisectors" [13] established animal dependent decreases in blood pressure [19]. experimentation and sacrifice as part of the Carbon dioxide is recommended for the standard scientific method [14]. euthanasia of both rats and mice, considering behavioral criteria. Ether is unsuitable as a Sacrificing research animals is one of the most sacrifice method as it is dangerous, slow acting challenging tasks in animal studies, and it is and an irritant [22]. This research therefore seeks imperative to do it as humanely as possible. In to shed some light in these areas as regarding the course of research, the fate of almost all rat sacrifice. experimental animals is to be sacrificed at certain stages of the study, either to gain blood, tissue The cerebellum and hippocampus are essential and other specimens or at humane endpoints to parts of the hindbrain and forebrain respectively. prevent any extension of stress or pain [15]. The cerebellum is responsible for a number of functions including motor skills such as balance, Sacrificing techniques ultimately cause death by coordination, and posture [23]. The hippocampus three basic mechanisms; direct depression of on the other hand is part of the limbic system neuronal activity necessary for life function, located in the brain's medial temporal lobe, near hypoxia and/or physical disruption of brain the center of the brain. It comprises primarily of activity [16]. Common methods of animal pyramidal cells. The hippocampus plays sacrifice include; inhalation of anesthesia gas –
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