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. 2020 Sep 3;15(9):e0238123.
doi: 10.1371/journal.pone.0238123. eCollection 2020.

Intraperitoneal injection of sodium pentobarbital has the potential to elicit pain in adult rats (Rattus norvegicus)

J N Reimer  1 , C J Schuster  1 , C G Knight  1 , D S J Pang  1   2   3 , V S Y Leung  2   3
Affiliations

Intraperitoneal injection of sodium pentobarbital has the potential to elicit pain in adult rats (Rattus norvegicus)

J N Reimer et al. PLoS One. .

Abstract

An effective and pain-free killing method is required to achieve the goal of euthanasia, a "good death". Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely accepted technique in laboratory rats, but questions remain regarding pain associated with administration. As PB rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence of pain. The objective of this study was to assess pain associated with IP PB using an appropriate vehicle control. Adult male and female Sprague Dawley (SD) and female Wistar rats (N = 84) were block randomised by sex and strain to receive one of three treatments: 1) 800 mg/kg PB (pH 11), 2) saline or 3) vehicle controls (pH 11 or 12.5). Behavior (Rat Grimace Scale (RGS), writhing, back arching) was evaluated at baseline, before loss of righting reflex (LORR, PB group), and at 80s, 151s and 10 min post-injection (PI; saline and vehicle control groups). In the PB group, mean time to LORR was 78 ± 7.9 seconds. In the vehicle control groups, RGS scores were increased at 151s PI (SD: p = 0.0002, 95%CI 0.73 to 0.20) from baseline, as was relative frequency of writhing (SD: p < 0.0001; Wistar; p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0005, 95%CI 0.71 to 0.18; Wistar: p = 0.0234, 95%CI 0.91 to 0.07) but the relative frequency of writhing did not (p > 0.999). The RGS scores and the relative frequency of writhing remained low in the PB and saline groups (p > 0.05). These results show that, vehicle controls for IP PB result in signs associated with pain, pain may not be experienced following IP PB when LORR occurs quickly, and that the effects of PB limit behavioral pain assessments.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. RGS scores of rats that received sodium pentobarbital (PB), saline controls or vehicle controls (pH 11 or 12.5).
(a) In the female and male Sprague Dawley group, significant increases from baseline were observed in the vehicle control group at the 151s and 10 min post-injection (PI) timepoints (p < 0.01). Differences between saline and vehicle control groups were observed at the 151s PI timepoint (p < 0.05). (b) In the female Wistar group, a significant increase from baseline was only observed at 10 min PI timepoint in the vehicle control group (p < 0.05). Differences between groups were observed at the 10 min PI timepoint (p < 0.05). The horizontal dotted line represents a previously established intervention threshold of 0.67. [19] Data presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2
Fig 2. The relative frequency of writhing displayed by rats treated with sodium pentobarbital (PB), saline controls or vehicle controls (pH 11 or 12.5).
Significant increases from baseline (BL) were only observed at the 151s post-injection (PI) timepoint in both female and male Sprague Dawley rats (p < 0.0001, a) and female Wistar rats (p < 0.001, b). Increased writhing occurred between saline and vehicle control groups at the 80s and 151s PI timepoints in both female and male Sprague Dawley and Wistar rats (p < 0.01). Data presented as median ± IQR. **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3
Fig 3. The relative frequency of back arching displayed by rats treated with sodium pentobarbital (PB), saline control or vehicle controls (pH 11 or 12.5).
(a) With the female and male Sprague Dawley rats, significant increases from baseline (BL) were only observed in the vehicle control group at the 151s PI timepoint (p < 0.05). Differences between saline and vehicle control groups were not observed (p > 0.05). (b) With female Wistar rats, there were no significant differences between BL and the different timepoints (p > 0.05). A significant increase in back arching occurred between saline and vehicle control groups at 151s PI (p < 0.01). Data presented as median ± IQR. *p < 0.05, **p < 0.01.
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Publication types

Grants and funding

DJSP: Natural Sciences and Engineering Research Council (NSERC) of Canada (Discovery Grant 424022-20313) and Fondation Lévesque. JNR: University of Calgary Faculty of Veterinary Medicine Summer Undergraduate Research Experience Program and NSERC Undergraduate Stuent Research Award. CS: University of Calgary Markin USRP. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.