Histological Alterations in the Internal Organs of Wistar Han Rats (Rattus norvegicus) Euthanized by Five Different Methods

Selecting a method of euthanasia is an important step in designing research studies that use animals; euthanasia methods must be humane, cause minimal pain and suffering to the animal, and preserve the tissue architecture of the organs of interest. In this study, we evaluated the histomorphology of the internal organs (lung, spleen, heart, kidney, liver, brain, and adrenal gland) of rats submitted to five different methods of euthanasia, with the goal of determining which protocol caused the least alteration of histomorphology. Twenty adult Wistar Han rats ( Rattus norvegicus ) were divided into 5 groups of 4 rats each (2 females and 2 males) and were euthanized by CO 2 or isoflurane inhalation, sodium thiopental or xylazine plus ketamine overdose, or decapitation. All euthanasia was performed in accordance with published guidelines and local legal requirements. Necropsy was performed immediately after euthanasia. Specific internal organs were removed and placed in formalin and submitted for routine histologic processing. Histomorphological examination of hematoxylin and eosin-stained tissues revealed circulatory alterations in multiple organs, predominantly congestion in multiple tissues, pulmonary hemorrhage, and hepatic degeneration. The euthanasia methods that induced the most severe alterations were exposure to CO 2 and anesthetic overdose with xylazine plus ketamine or sodium thiopental. Euthanasia by overexposure to isoflurane caused less damage, and the alterations were of minimal severity. Decapitation resulted in the lowest incidence of lesions in multiple organs but due its traumatic nature, it caused the highest incidence of pulmonary hemorrhage. In selecting a method of euthanasia, factors to consider are the species of animal, the purpose of the research, and the practical ability to perform the procedure to achieve maximal animal welfare without iatrogenic changes that could compromise the outcome and reproducibility of the study.