Consistency in Reporting of Loss of Righting Reflex for Assessment of General Anesthesia in Rats and Mice: A Systematic Review.

General anesthesia induces a reversible loss of consciousness (LOC), a state that is characterized by the inability to feel pain. Identifying LOC in animals poses unique challenges, because the method most commonly used in humans, responding to questions, cannot be used in animals. For over a century, loss of righting reflex (LORR) has been used to assess LOC in animals. This is the only animal method that correlates directly with LOC in humans and has become the standard proxy measure used in research. However, the reporting of how LORR is assessed varies extensively. This systematic literature review examined the consistency and completeness of LORR methods used in rats and mice. The terms 'righting reflex,' 'anesthesia,' 'conscious,' 'rats,' 'mice,' and their derivatives were used to search 5 electronic databases. The abstracts of the 985 articles identified were screened for indications that the study assessed LORR in mice or rats. Full texts of selected articles were reviewed for LORR methodological completeness, with reported methods categorized by 1) animal placement method, 2) behavioral presence of righting reflex, 3) duration of LORR testing, 4) behavioral LORR, and 5) animal position for testing LORR. Only 22 papers reported on all 5 methodological categories. Of the 22 papers, 21 used unique LORR methodologies, with descriptions of LORR methods differing in at least one category as compared with all other studies. This variability indicates that even papers that included all 5 categories still had substantial differences in their methodological descriptions. These findings reveal substantial inconsistencies in LORR methodology and reporting in the biomedical literature likely compromising study replicability and data interpretation.

Assessing Susceptibility to Carbon Dioxide Gas in Three Rat Strains Using the Loss of Righting Reflex.

Overdose of carbon dioxide gas (CO2) is a common euthanasia method for rodents; however, CO2 exposure activates nociceptors in rats at concentrations equal to or greater than 37% and is reported to be painful in humans at concentrations equal to or greater than 32.5%. Exposure of rats to CO2 could cause pain before loss of consciousness. We used 2 standardized loss of righting reflex (LORR) methods to identify CO2 concentrations associated with unconsciousness in Wistar, Long-Evans, and Sprague-Dawley rats (n = 28 animals per strain). A rotating, motorized cylinder was used to test LORR while the rat was being exposed to increasing concentrations of CO2. LORR was defined based on a 15-second observation period. The 2 methods were 1) a 1-Paw assessment (the righting reflex was considered to be present if one or more paws contacted the cylinder after the rat was positioned in dorsal recumbency), and 2) a 4-Paw assessment (the righting reflex was considered to be present if all 4 paws contacted the cylinder after the rat was positioned in dorsal recumbency). Data were analyzed with Probit regression, and dose-response curves were plotted. 1-Paw EC95 values (CO2 concentration at which LORR occurred for 95% of the population) were Wistar, 27.2%; Long-Evans, 29.2%; and Sprague-Dawley, 35.0%. 4-Paw EC95 values were Wistar, 26.2%; Long-Evans, 25.9%, and Sprague-Dawley, 31.1%. Sprague-Dawley EC95 values were significantly higher in both 1- and 4-Paw tests as compared with Wistar and Long-Evans rats. No differences were detected between sexes for any strain. The 1-Paw EC95 was significantly higher than the 4-Paw EC95 only for Sprague-Dawley rats. These results suggest that a low number of individual rats from the strains studied may experience pain during CO2 euthanasia.

Use of Loss of Righting Reflex to Assess Susceptibility to Carbon Dioxide Gas in Three Mouse Strains.

Exposure to CO2 gas is a common rodent euthanasia method. CO2 activates nociceptors in rats and is painful to humans at concentrations equal to or greater than 32.5% The concentration of CO2 at which rodents become unconsciousness is inadequately defined. We used loss of righting reflex (LORR) to identify the concentration at which CO2 caused loss of consciousness in C57Bl/6, CD1 and 129P3J mice (16 females and 16 males per strain). We used a custom built, rotating, motorized cylinder to determine LORR as CO2 concentrations were increased. Two LORR assessment methods were used: 1) a 1-Paw assessment in which the righting reflex was considered to be present if one or more paws contacted the cylinder after rotation into dorsal recumbency and 2) a 4-Paw assessment in which the righting reflex was considered to be present only if all 4 paws contacted the cylinder. LORR test data were analyzed with Probit regression and dose response curves were plotted. 1-Paw EC95 values (CO2 concentration at which LORR occurred for 95% of the population) were: C57Bl/6; 30.7%, CD1; 26.2%, 129P3J; 20.1%. The EC95 for C57Bl/6 was significantly higher than that of the 129P3J mice, with no significant differences between other strains. Four-Paw EC95 values were: C57Bl/6; 22.8%, CD1; 25.3%, 129P3J; 20.1%. Values for 129P3J mice were significantly lower than those of CD1 mice), with no significant difference between other strains. The EC95 varied significantly between 1-Paw and 4-Paw methods only for C57Bl/6 mice. These results suggest a potential for nociception and pain to occur in some individuals of some mouse strains during CO2 euthanasia.