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Introduction
For centuries, animals are used to study multitudes of phenomena for furthering scientific knowledge. According to UK Home Office, 3.79 million procedures were conducted on animals for research in 2017 (Speaking of Research, 2018). These animals include mice, fish, rats, birds, dogs, among others. The use of massive number of animals in research pushed forth regulations for animal welfare. Animal welfare came a long way from public discourse of vivisection to the foundation of five freedoms. It was only on the past few decades that animal welfare started to change its perspective from the absence of disease and pain to promoting positive and natural behavior (Broom, 2011).
One of the main challenges in animal welfare is alleviating pain not only due to a lesser pharmacological armamentarium for analgesia per species but also due to the lack of an absolute effective method to assess pain among different animals. Historically, pain assessment method have been very subjective like the Visual Analog Scale (VAS). In a survey conducted by Hawkins (2002), they have concluded that although there is a basic understanding that procedures will introduce pain or suffering; each institution applied varying techniques for pain assessment and a team approach is the best way to guarantee consistency and efficiency. The great discrepancies in subjective assessment of pain have resulted to more research on objective quantification system with the use of pain behaviors (Roughan and Flecknell, 2006). Different pain assessment models have been developed in large animals mostly companion animals, cattle and horses to study pain and efficiency of intervention therapy after procedures (Gigliuto et al., 2014)
This study was done to evaluate the Visual Analog Scale in comparison to Quantitative Behavior Analysis for pain assessment in laboratory rats. It was conducted to prove the hypothesis that short-term training for objective detection and quantification of pain behavior would improve the pain severity assessment in post-operative rats among students.
Materials and Methods
The study participants were 126 master degree (MSc) students with different background experience on animal handling and behavior from 2017 to 2019. This study was conducted in Roslin Building, University of Edinburgh, Scotland.
The students were given instructions to watch 5-minute video clips. Each clip presented a rat that underwent laparotomy for surgical implantation of tumor cells in the bladder. The rats were categorized to four treatment groups: one control and three experimental groups. The control group was given saline solution (T1) 1 hour prior to procedure while the experimental groups were given a non-steroidal anti-inflammatory drug, meloxicam, of varying dosage namely 0.5mg/kg (T2), 1mg/kg (T3), 2mg/kg (T4). The study was performed in two phases with Phase 1 (Visual Analog Scale) done prior to Phase 2 (Quantitative Behavior Analysis).
In Phase 1, the participants were given a sheet that contains a 10-cm line, with 0 as no pain and 10 as severe pain, of the Visual Analog Scale. They were presented with four video clips of the rats in random order and were blinded to the treatment groups. However, they were informed that all of the animals underwent laparotomy. After watching each clip, the participant made a mark in the scale that indicated the severity of pain the rat experienced.
In Phase 2, participants underwent training as a group using video clips for detecting and noting pain behaviors in rats. The pain behaviors emphasized in these clips were back arching, falling/staggering, and twitching. This training lasted for approximately 10 minutes. Thereafter, the participants were handed a sheet with boxes to score the occurrence of each pain behavior per animal. The four video clips were shown in random order to the participants in the same manner as phase 1. They were also blinded to the treatment groups. The participants were asked to tally the number of times they saw the pain behavior for each animal and made a total score for each clip. The total score indicated the severity of pain the rat experienced in the clip.
Data analysis
Due to the different nature of the scores produced using the VAS and Behaviour quantification, direct comparison between the two scoring systems was not possible. In order to assess the relative agreement of the observers with the hypothesised pattern of pain severity, success rates for both VAS and Behavioural Quantification were calculated as the proportion of individual scores that conformed to predictions, based on Roughan and Flecknell (2006) (i.e. salineM2 and M1>M2). VAS scores and behaviour scores for these specific pairs of treatments were compared using the Wilcoxon test (two-tailed).
Results
Success rates are described in Table 1. Overall, when using VAS scores success rates were lower than when using behavioural quantification (e.g. using VAS, 61.9% of observers were able to identify a higher perceived pain level in the saline-treated rat compared to the rat given 2 mg/kg meloxicam, whilst 95.5% of observers were able to discriminate between those treatments using behavioural quantification). No statistically significant difference was identified between VAS scores obtained for the saline treatment and those for 0.5 mg/kg meloxicam (p=0.63; Table 1) and observers also failed to identify a difference between 1 and 2 mg/kg meloxicam (p=0.11). However, VAS scores for saline were significantly different than those for 2mg/kg meloxicam (p
Table 1: VAS and cumulative behaviour scores generating by MSc students (2017-2019, n=126) observing video clips of rats treated with varying levels of Meloxicam (0.5, 1, 2, mg/kg) or with saline following laparotomy.
VAS (quartiles) Behaviour (quartiles) Wilcoxon test Success Rate (%)
Treat Q1 Median Q3 Q1 Median Q3 VAS Beh. VAS Beh.
T1.
Saline 50.0 69.0 80.0 13.0 16.0 20.0 T1vs.T2
W=2869
p=0.63 T1vs.T2
W=1982 p=0.007 T1T4 =61.9 T1>T4 =95.5
T3.
M1 32.0 51.0 68.0 6.0 10.0 12.0 T3vs.T4
W=1942.5
p=0.11 T3 vs.T4
W=4173.5 p=0.002 T3>T4 =31.2 T3>T4 =76.3
T4.
M2 29.0 63.0 80.0 7.0 9.0 12.0
Figure 1: Student Behavioural Scores (Median and Inter-quartile Range, n=126) and a single expert score taken from Roughan and Flecknell (2006).
Discussion
The results of this study showed that Quantitative Behavior Analysis was more accurate than the Visual Analog Scale in the evaluation of pain. Furthermore, the short-term training in pain behaviors enabled students of varying background experiences to assess pain in these animals more effectively.
The output of this study were not directly compared to each other but rather compared to an expert opinion handed out by (Roughan and Flecknell, 2006). As indicated, this expert opinion serves as a gold standard for which the observations were compared. It is important to note that using the VAS method, observers have significantly identified (p The Visual Analog Scale is one of the most widely used subjective method for assessing pain. Nevertheless, the results vary widely among institutions, observer experience and field of study. It is a simple psychometric measurement that is widely used in human medicine to assess the effectiveness of intervention therapy and monitor chronic diseases especially in respiratory conditions (Klimek et al., 2017). However, they were highly variable when applied in veterinary practice (Welsh et al., 1993). Moreover, keeping good records of highly experienced laboratory technicians can give valuable information of the welfare of laboratory animals especially when correlated with other assessment tools (Hawkins, 2002). However, subtle differences in between experimental groups given increasing dosage of meloxicam did not differ significantly. These minimal differences would need a better-equipped personnel or a more efficient tool to evaluate pain and assess the response to therapy.
The Quantitative Behavior Analysis was introduced by (Roughan and Flecknell, 2006) for an objective behavior-based scoring system for pain evaluation. This was a product of series of studies conducted by their group in Newcastle to formulate a more efficient tool in looking at highly prevalent pain behaviors in a limited amount of time. It stemmed from the notion that the use of simpler methods will be more beneficial when considering assessments in a shorter period. In addition, detection of these predominant pain behaviors do not require extensive training and experience to be applied on a day-to-day basis. Using this method, individuals of different backgrounds can be trained in approximately 10 minutes with the use of audiovisual aids in detecting these key behaviors to improve pain assessment in rodents.
In comparison to VAS, it not only accurately identified the control group from the experimental group with the highest dose of meloxicam (p It was implicated in previous research that the major study bias was derived from the fact that the Visual Analog Scale was done prior to behavior-scoring system (Roughan and Flecknell, 2006). However, this bias cannot be addressed since training the individuals first would greatly affect the respondents subjective assessment. Also, the two methods cannot be carried out simultaneously by the participants. The study also address only acute post-operative period. As such, it should be interpreted for that duration only and duration of analgesic effects should be taken into account (National Research Council (US) Committee, 2009). Use of opioid analgesics may greatly alter the behavior in rodents even in healthy, pain free animals (Roughan and Flecknell, 2000). Also, behavioral changes caused by non-analgesic effect can be caused by some drugs like buprenorphine (Hayes et al., 2000). It is important to note that rats are basically prey species and would might also mask certain behaviors in response to threat
Newer studies have compared behavior-based scoring with Rodent Grimace Scale (RGS). In the study of (Klune et al., 2019), they have found out that these methods have different sensitivity and it has potential to discriminate the effects of different types of analgesic. On the other hand, (Leach et al., 2010) indicated that in assessing post-operative pain after laparotomy among rabbits, facial observations were of lesser value than behavior-based scoring. Behavior-based scoring were also adapted to the evaluation of post-operative efficacy of analgesia for surgical procedures in the orofacial region which showed consistent and efficient results in determining the relief of pain (Ramirez et al., 2015).
Conclusion
This study demonstrated that the Quantitative Behavior Analysis was more accurate than the Visual Analog Scale in assessing pain severity among post-operative rats who underwent laparotomy. Moreover, even untrained individuals can efficiently carry out this method after a short period of training for detecting pain behaviors. This method was also sensitive in discriminating between severe and moderate types of pain and response to analgesia. The method has been applied mainly in rodent research. Further studies in other laboratory animals, different surgical procedures and different analgesics would elucidate some of the limitations of this study. Improving pain assessment and proper pain intervention in laboratory animals would greatly improve animal welfare and the quality of animal research.
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