Refinement: Evaluating stress and accuracy of different intraperitoneal techniques in mice

Abstract

The intraperitoneal (i.p.) injection in mice is a common, efficient and easy way to administer an animal a substance. Several restraining methods for this injection are in use. Various studies mention a frequency of misplacing an i.p. injection using a certain restraining method, however the frequency of misplacing an i.p. injection has never been compared between methods. To safely administer a substance through an i.p. injection, some form of restraint is necessary. The restraining itself causes a degree of stress in mice. However, the level of stress has never been determined when comparing different restraining methods. In the context of animal welfare and the 3R’s principle, it is relevant to determine which restraining method is most accurate and causes the least amount of stress. In this study frequency of misplacing the i.p. injection and the degree of stress, was determined comparing three restraining methods. Restraining the mouse head down (HD), restraining the mouse head up (HU) and using a method described by Baek et al.(2015)1 in which minimal restraint is required (BK). Accuracy was determined by administering a methylene solution intraperitoneally after which macroscopic pathology was conducted to determine the location. The acute stress reaction was measured by determining the level of blood plasma corticosterone (pCORT) and measuring eye, ear and tail temperature through infrared thermography (IRT). These parameters were measured after mice were restrained either HD, HU or BK (within group comparison, n=). Temperature of thermal images and level of corticosterone were compared between different restraining methods. Accuracy of the i.p. injection after all restraining methods was not significantly different, considering this all three restraining methods are equally valid to be used in practice. Corticosterone levels after HU restraint were significantly highest (HU n=18, HD n=19, BK n=18). However, this was not supported by the IRT data; temperature change patterns did not differ between procedures. When different restraints were combined with i.p. injection corticosterone results regarding HU restraint were not confirmed (group comparison, HU n=14, HD n=13, BK n=20 per group). Thermal changes were present in the eye-, tail- and in some extent ear area during an acute stress reaction. These changes could not be correlated to pCORT, further study is necessary to validate IRT as a reliable stress parameter.