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.

Quality of reporting of prospective in vivo and ex vivo studies published in the Journal of Veterinary Emergency and Critical Care over a 10-year period (2009-2019).

OBJECTIVE: To evaluate the reporting of key items associated with risk of bias and weak study design over a 10-year period. DESIGN: Literature survey. SETTING: Not applicable. ANIMALS: Not applicable. INTERVENTIONS: Papers published in the Journal of Veterinary Emergency and Critical Care between 2009 and 2019 were screened for inclusion. Inclusion criteria consisted of prospective experimental studies describing in vivo or ex vivo research (or both), containing at least 2 comparison groups. Identified papers had identifying information (publication date, volume and issue, authors, affiliations) redacted by an individual not involved with paper selection or review. Two reviewers independently reviewed all papers and applied an operationalized checklist to categorize item reporting as fully reported, partially reported, not reported, or not applicable. Items assessed included randomization, blinding, data handling (inclusions and exclusions), and sample size estimation. Differences in assessment between reviewers were resolved by consensus with a third reviewer. A secondary aim was to document availability of data used to generate study results. Papers were screened for links to access data in the text and supporting information. MEASUREMENTS AND MAIN RESULTS: After screening, 109 papers were included. Eleven papers were excluded during full-text review, with 98 papers included in the final analysis. Randomization was fully reported in 31.6% of papers (31/98). Blinding was fully reported in 31.6% of papers (31/98). Inclusion criteria were fully reported in all papers. Exclusion criteria were fully reported in 60.2% of papers (59/98). Sample size estimation was fully reported in 8.0% of papers (6/75). No papers (0/99) made data freely available without a requirement to contact study authors. CONCLUSIONS: There is substantial room for improvement in reporting of randomization, blinding, data exclusions, and sample size estimations. Evaluation of study quality by readers is limited by the low reporting levels identified, and the risk of bias present indicates a potential for inflated effect sizes.

Randomization, blinding, data handling and sample size estimation in papers published in Veterinary Anaesthesia and Analgesia in 2009 and 2019.

OBJECTIVE: To evaluate reporting of items indicative of bias and weak study design. STUDY DESIGN: Literature survey. POPULATION: Papers published in Veterinary Anaesthesia and Analgesia. METHODS: Reporting of randomization, blinding, sample size estimation and data exclusion were compared for papers published separated by a 10 year interval. A reporting rate of more than 95% was considered ideal. The availability of data supporting results in a publicly accessible repository was also assessed. Selected papers were randomized and identifiers removed for review, with data from 59 (57 in 2009, two in 2008) and 56 (52 in 2019, four in 2018) papers analyzed. Items were categorized for completeness of reporting using a previously published operationalized checklist. Two reviewers reviewed all papers independently. RESULTS: Full reporting of randomization increased over time from 13.6% to 85.7% [95% confidence interval (CI), 57.8-86.6%; p < 0.0001], as did sample size estimation (from 0% to 20%; 95% CI, 7.6-32.4%; p = 0.002). Reporting of blinding (49.2% and 50.0%; 95% CI, -18.3% to 20.0%; p = 1.0) and exclusions of samples/animals (39.0% and 50.0%; 95% CI, -8.8% to 30.8%; p = 0.3) did not change significantly. Data availability was low (2008/2009, zero papers; 2018/2019, two papers). None of the items studied exceeded the predetermined ideal reporting rate. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that reporting quality remains low, with a risk of bias.

The short-term effects of recorded ocean sound with and without alpha frequency binaural beats on tinnitus perception.

BACKGROUND: The dichotic presentation of two auditory stimuli with a small frequency difference in each ear produces the perception of a third, fused auditory image called a binaural beat. Neural activity has been found to synchronize with these perceived beats. It is for this reason that binaural beats are often used in an attempt to induce certain psychological states that are associated with particular cortical rhythms. The aim of this proof-of-concept study was to explore the short-term effects of a binaural beat on tinnitus, to ascertain if further trials are justified. METHOD: Twenty adults with constant tinnitus and symmetrical hearing underwent audiological assessment and psychophysical measures of tinnitus. Participants were presented with two auditory stimuli: ocean waves with and without alpha frequency (8 Hz) binaural beats. Arousal and tinnitus perception were measured prior to and following each sound stimuli using the Perceived Arousal Scale and tinnitus rating scales. RESULTS: Small improvements in tinnitus rating scores occurred with sound. Some individuals showed more improvement with the binaural beats than ocean waves alone. CONCLUSION: The addition of binaural beats at 8 Hz to an ocean sound showed no significant group benefits above the ocean sound alone.