Affective state determination in a mouse model of colitis-associated colorectal cancer

Lauren C Chartier; Michelle L Hebart; Gordon S Howarth; Alexandra L Whittaker; Suzanne Mashtoub

doi:10.1371/journal.pone.0228413

. 2020 Jan 27;15(1):e0228413. doi: 10.1371/journal.pone.0228413

Affective state determination in a mouse model of colitis-associated colorectal cancer

Lauren C Chartier ^1,², Michelle L Hebart ³, Gordon S Howarth ^2,³, Alexandra L Whittaker ^3,^#, Suzanne Mashtoub ^1,^2,^4,^*,^#

Editor: Mathilde Body-Malapel⁵

PMCID: PMC6984705 PMID: 31986185

Abstract

Behavioural indicators of affective state, including burrowing, clinical scores and the Mouse Grimace Score have not yet been validated in mouse models of chronic gastrointestinal disease. Additionally, a comparison of these methods has not been characterised. This study aimed to determine which behavioural assessment was the optimal indicator of disease, evidenced by correlation with clinically-assessed measures, in an azoxymethane (AOM)/dextran sulphate sodium (DSS) mouse model of colitis-associated colorectal cancer. C57BL/6 mice were allocated to four groups (n = 10/group); 1) saline control, 2) saline+buprenorphine, 3) AOM+DSS+water, 4) AOM+DSS+buprenorphine. Mice were gavaged thrice weekly with water or buprenorphine (0.5mg/kg; 80μL) for 9 weeks. Disease activity index (DAI) was measured daily; burrowing and grimace analyses occurred on days -1, 5, 19, 26, 40, 47 and 61. Colonoscopies were performed on days 20, 41 and 62. All animals were euthanized on day 63. Burrowing activity and retrospective grimace analyses were unaffected (P>0.05), whilst DAI was significantly increased (P<0.05) in mice with colitis-associated colorectal cancer compared to normal controls. In addition, DAI was positively correlated with colonoscopically-assessed severity and tumour number (P<0.05). We conclude that traditional measures of DAI or clinical scoring provide the most reliable assessment of wellbeing in mice with colitis-associated colorectal cancer.

Introduction

Pain, as defined by the Oxford dictionary, refers to a ‘highly unpleasant physical sensation caused by illness or injury’. In biomedical research, rodents are the most widely used species and it is estimated that globally approximately 4.6 million will experience procedure-related pain [1]. Prevention and alleviation of pain through accurate pain assessment and appropriate analgesic use should be a priority for researchers working with laboratory animals [2]. However, assessment of pain is challenging in all animal species, and is particularly problematic in rodent-prey species that mask pain as part of a survival mechanism [3].

Although directly measuring pain in animals is near impossible, it can be presumed that animals are in pain when they display pain-like behaviours [4]. Such behaviours include reduced ambulation, agitation and increased grooming of an affected area [4]. A number of techniques have been established to measure pain-like behaviour in animal models. The first included stimulus-evoked measures such as the Von Frey, Randall-Selitto and Hargreaves techniques. These methods are now used less widely due to a growing concern over clinical translatability [4], since these methods are regarded as not measuring the affective pain response. In response to this concern, scientists developed a range of behavioural assessment methods proposed to measure the affective or emotional component of the pain response. A method that has received much attention is the characterisation of facial expression.

The first standardised system for facial expression scoring in rodents, ‘The Mouse Grimace Scale’ (MGS) was developed by Langford et al. (2010). The MGS scores five facial features or ‘action units’ from 0–2 (not present to severe). These features are: orbital tightening, nose bulge, cheek bulge, ear position and whisker change. A higher MGS score is indicative of increased levels of pain [5]. Whilst this system represents a considerable advancement in pain assessment of rodents, validation studies have typically involved retrospective assessment through analysis of stored video behavioural data in models of acute pain. Consequently, refinement possibilities are limited, since humane endpoints and analgesic provision are not able to be immediately implemented. Therefore, an alternative live-scoring method should be considered to allow ‘cage-side’ analysis, whereby interventions can be applied by researchers to rapidly improve animal welfare as needed. Leung et al. (2016) determined that a real-time grimace scoring method was reliable in rats [6]. Miller and Leach (2016) investigated the validity of baseline grimace scoring in various cohorts, strains and sexes of mice [7]; however, the effectiveness of real-time scoring in mice with chronic disease is yet to be determined. Furthermore, there have been relatively few investigations into the validity of MGS in mice expected to be experiencing chronic visceral pain, as opposed to acute pain, initiated via a non-surgical insult.

In addition to pain, animals may also experience distress or sickness leading to a negative affective state and potentially compromising their welfare. Negative affective state has traditionally been assessed in biomedical research using general clinical scoring, for example the Morton and Griffiths (1985) schema. This scheme describes appearance, food/water intake, behaviour, digestive and cardiovascular signs on a scale of normal to severe for rodents, guinea pigs, rabbits, cats and dogs [8]. This method remains the predominant method for laboratory rodent welfare assessment globally, as prescribed by animal ethics committees and regulatory documents. More recently, deterioration in activities of daily living (ADL) has been proposed to indicate decreased welfare in mice [9]. The most common measurable ADL in mice are burrowing, nesting and hoarding. These techniques are inexpensive, simple to run and also provide environmental enrichment for laboratory mice.

The current study sought to address these deficiencies in knowledge by determining the effectiveness of a range of measures of pain and well-being in a pre-clinical setting of colitis-associated colorectal cancer, using the azoxymethane (AOM)/dextran sulphate sodium (DSS) mouse model. Methods examined were the MGS, burrowing activity and clinical scoring and we aimed to determine which method was the most reliable in this pre-clinical model of colitis-associated colorectal cancer. Buprenorphine, a long-lasting opioid analgesic (up to 8 hours), has few effects on the immune system and has displayed efficacy in reducing the acute and chronic pain experience of mice and rats [10–12]. Therefore, buprenorphine was administered to validate the tests, especially the pain-specific MGS, to determine if pain was a contributing factor in behavioural response. The current study represents the first validation of a live-scoring method of the MGS, compared to the traditional retrospective scoring, in a mouse model of chronic disease. Finally, this study aimed to determine the most reliable behavioural assessment technique (MGS, clinical scoring or burrowing) for indication of disease and its progression in experimentally-induced colitis-associated colorectal cancer, as evidenced by correlation with clinically-assessed disease measures in mice.

Materials and methods

Animal studies

All animal studies were conducted in compliance with the Australian Code for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Ethics Committee of the Children, Youth and Women’s Health Service (AE1095/7/21). This study was conducted as part of another study evaluating naturally-sourced therapies in colitis-associated colorectal cancer with control groups being utilised in the current study (AE1079/3/21). Female C57BL/6 mice (C57BL/6JArc, n = 40; average weight 18.36g) at 8 weeks of age were sourced from a SPF production facility, the Animal Resource Centre (ARC; Perth, Western Australia) and group-housed in standard open-top cages (polypropylene; 470mm x 175mm x 120mm; Crestware Industries) with pelleted paper bedding materials (>99% recycled paper product; Fibrecycle PtyLtd, Helensvale, QLD, Australia). The ARC undertakes a quarterly health screening, covering various bacterial, viral and parasitic organisms, all of which the obtained colony screened negative for. Only female mice were used to remain consistent with data obtained from previous studies [13, 14]. Environment was regulated at 21-24°C with 42–44% humidity and a light/dark cycle of 14:10 h. Mice were fed standard mouse chow (meat free mouse diet; Specialty Feeds, Glen Forrest, Western Australia) and provided with enrichment items including shredded paper, polycarbonate ‘houses’ and cardboard toilet paper rolls for the duration of the trial. All mice received ad libitum access to plain drinking water during the experimental period (except where group allocation precluded it).

Experimental design

Female C57BL/6 mice (n = 10/group) were randomly assigned to four treatment groups (n = 10/group); 1) saline + water + water, 2) saline + water + buprenorphine, 3) AOM + DSS + water and 4) AOM + DSS + buprenorphine. Mice were stratified to groups based on baseline body weight. Group size (n = 10/group) was calculated using Clin.Calc for mouse grimace scale outcomes from Rosen et al. (2017) [15]. This calculation assumed a power of 80%, and indicated that a minimum sample size of n = 9/group was necessary for statistical power. All mice were administered (oral-gastric gavage) 80μL of either water or buprenorphine (0.5mg/kg; Reckitt Benckiser Healthcare, Hull, U.K) thrice weekly for the duration of the trial. Buprenorphine was administered via oral gavage as control animals (groups 1 and 3) utilised in another study were gavaged with water and thus exposed to the same procedural distress. On day 0, mice received a single intraperitoneal injection of saline or AOM (7.4mg/kg; average injection volume 0.14ml; 27G needle;Sigma-Aldrich, Castle Hill NSW, Australia), and then underwent three DSS/water cycles comprised of 7 days DSS (ad libitum; 2%w/v, 2g/100ml distilled water; MP Biomedicals LLC, Santa Ana California, USA) followed by 14 days plain water (ad libitum). Negative control animals (groups 1 & 2) received plain water in their drinking bottles for the duration of the 9-week experimental period. All animals were euthanised on day 63 via CO₂ asphyxiation followed by cervical dislocation (experimental timeline; Fig 1).

Fig 1 — Animals were injected (i.p.) on day 0 and underwent three dextran sulphate sodium (DSS)/water cycles, comprising one week 2% DSS followed by 2 weeks of plain water. Animals were gavaged thrice weekly with water or buprenorphine. All mice were euthanised after 9-weeks via CO₂ asphyxiation, followed by cervical dislocation.

Disease activity index

DAI was calculated daily (at 8am, prior to buprenorphine administration) from general clinical signs including bodyweight loss, general condition, stool consistency and rectal bleeding during the experimental period. General condition included features such as ruffled coat and grooming, hunching, alertness and abdominal twitching. Each parameter was scored from 0–3 with increasing severity and totalled to give a final DAI value, with a maximum possible score being 12 [16]. As DAI was a part of daily monitoring and welfare measurements, the researchers were not blinded to treatment groups when obtaining DAI scores.

Colonoscopy

Colonoscopies using a high-resolution Karl Storz colonoscope (1.9mm outer diameter, Tuttlingen, Germany) were performed at the end of each DSS/water cycle (days 20, 41 and 62) to assess colitis progression and tumour development. Mice were anaesthetised using isoflurane inhalant (AbbVie Inc, Illinois, USA) in oxygen via mask for the duration of the procedure, and closely monitored on a heating pad during and immediately following the procedure. From anaesthetic induction to recovery, the colonoscopy procedure lasted approximately 10 minutes. Colitis severity was measured from recorded videos in a blinded fashion using five parameters described by Becker et al. (2005). These parameters include; thickening of the colon, vasculature pattern, presence of fibrin, granularity of mucosal surface, and stool consistency. Each parameter was scored from 0 to 3 with increasing severity and totalled, with the maximum possible severity score being 15 [17]. Additionally, colonic tumours were also counted from videos in a blinded fashion.

Burrowing analyses

Burrowing analyses were conducted as a measure of affective state or activities of daily living using a modified protocol described by Deacon [18]. At 6pm, one hour after commencement of the dark cycle and 8 hours after buprenorphine administration (from 6pm), mice were placed in individual cages with a pre-weighed (400g kitty litter ‘pebbles’; Black and Gold, Australian Asia/Pacific Wholesalers Pty Ltd) burrow attached (modified plastic Coca-Cola bottle; 6.9cm diameter, 16cm long) and left for an hour. After this time, the burrows were re-weighed and the weight difference taken to represent the amount burrowed. Burrowing analyses occurred on day -1 (baseline), at the end of each DSS week for a severe disease measure (days 5, 26 and 47) and at the end of each DSS/water cycle to assess recovery (days 19, 40 and 61).

Mouse grimace scale

The affective experience of pain was assessed using the Mouse Grimace Scale (MGS; [5] at baseline, the end of each DSS week and end of a DSS/water cycle (days -1, 5, 19, 26, 40, 47 and 61). Real-time [6] and retrospective [5] MGS scoring methods were conducted in the morning (following buprenorphine administration; approx. 9am-12pm) at all indicated time-points. Five facial features (orbital tightening, nose bulge, cheek bulge, ear position and whisker change) were scored by a treatment-blinded grimace experienced observer from 0–2 (not present to severe), with a maximum possible MGS score being 10.

Real-time MGS

Animals were removed from their home cage and placed individually in a clear plastic cage for scoring by a treatment-blinded experienced observer. The mouse remained in the scoring cage for a five minute period, where the observer assigned a score for each facial feature every 15 seconds. The animal was then returned to its home cage. A median score was calculated for each parameter per 15 second time-point and then an average was obtained of the medians per 90 second period. A final mean was then calculated from each 90 second period to produce a final grimace score for each mouse.

Retrospective MGS

Over the same time period as the real-time method, video recording of the clear cage was performed using two video cameras placed on perpendicular cage sides (Panasonic HC-V180, Osaka, Japan). Still images of the mice were extracted from video footage, cropped to show the face alone and placed into a pre-designed excel spreadsheet by an investigator blinded to treatment allocation. A random number generator was used to select three images for scoring of each mouse at each time-point. These images were then scored by a treatment-blinded scorer using the methods described by Langford et al. 2010. Scores for each parameter were totalled to give a score per photo, and then the three photo scores were averaged to give a final reportable score for each mouse per time-point [5].

Statistical analysis

Statistical analysis was completed using SPSS, version 25 for Windows (SPSS Inc. Chicago, Illinois, USA). Data were tested for normality using a Shapiro–Wilk test. DAI, burrowing activity, colitis score, and tumour number were analysed by repeated measures ANOVA with least significance difference (LSD) to compare among and within a group. MGS data were analysed non-parametrically using a Friedman test to assess temporal differences within groups and a Kruskal-Wallis with a Mann-Whitney post-hoc test to compare differences between groups within time-points. To determine any correlations between behavioural outputs and the measured clinical parameters, a non-parametric spearman-rho test was applied. P<0.05 was considered statistically significant.

Results

Disease activity index

In normal animals, buprenorphine administration did not impact DAI scores during the experimental period compared to saline controls (P>0.05; Fig 2). AOM/DSS significantly increased DAI scores on days 2, 3 and 5–63 compared to untreated saline controls (P<0.05). In AOM/DSS mice, buprenorphine administration increased DAI scores on days 16, 18–21, 25, 41 and 59 compared to AOM/DSS controls (P<0.05). Furthermore, buprenorphine administration decreased DAI scores on days 3, 8, 9, 28, 35 and 48 in AOM/DSS animals compared to AOM/DSS alone (P<0.05).

Fig 2 — Data were analysed using a repeated measures ANOVA with least significance difference (LSD) and are expressed as mean DAI score ± SEM (black line on the x axis represents a dextran sulphate sodium; DSS week). ***p<0.001, **p<0.01, *p<0.05 compared to Saline + Water + Water, ^^^p<0.001, ^^p<0.01, ^p<0.05 compared to AOM + DSS + Water.

Colitis severity and tumour number

Buprenorphine administration did not impact colitis progression in saline control animals throughout the experimental trial (P>0.05; Fig 3). AOM/DSS significantly increased colonoscopically-assessed colitis severity compared to saline controls at all three time-points (days 20, 41 and 62; P<0.05). Mice administered buprenorphine and treated with AOM/DSS presented with increased colitis severity on day 20 and decreased colitis severity scores on day 62 compared to AOM/DSS controls (P<0.05).

Saline control animals and those treated with buprenorphine did not develop colorectal tumours during the experimental period. AOM/DSS resulted in increased tumour number compared to saline controls (P<0.05; Fig 4). Additionally, in AOM/DSS mice, buprenorphine did not significantly impact tumour development compared with AOM/DSS controls (P>0.05).

Fig 4 — Data were analysed using a repeated measures ANOVA with least significance difference (LSD) and are expressed as mean total tumour number ± SEM. ***p<0.001, **p<0.01, *p<0.05 compared to Saline + Water + Water, ^^^p<0.001, ^^p<0.01, ^p<0.05 compared to AOM + DSS + Water.

Burrowing

Buprenorphine administration significantly increased burrowing activity in normal mice compared to saline controls on days 19, 26 and 40 (P<0.05; Fig 5). AOM/DSS did not significantly affect burrowing compared to saline controls at any time-point.

Fig 5 — Data were analysed using a repeated measures ANOVA with least significance difference (LSD) and are expressed as mean amount burrowed ± SEM (black line on the x axis represents a dextran sulphate sodium; DSS week). ***p<0.001, **p<0.01, *p<0.05 compared to Saline + Water + Water, ^^^p<0.001, ^^p<0.01, ^p<0.05 compared to AOM + DSS + Water.

Mouse grimace scale

Buprenorphine administration in normal mice resulted in no significant differences in real-time grimace scores at any time-point when compared to saline controls (Table 1; P>0.05). On day 19, AOM/DSS controls had higher real-time grimace scores compared to saline controls (Table 1; P<0.05), with no other significant differences on other days. AOM/DSS controls presented with significantly higher real-time grimace scores on day 19 compared to baseline (P<0.05). Buprenorphine administration in AOM/DSS mice resulted in significantly higher real-time grimace scores on day 19 when compared to baseline; and on day 47 compared to day 40 (P<0.05). Finally, buprenorphine administration in AOM//DSS mice significantly reduced real-time grimace scores on day 40 compared to day 19 (P<0.05). Scoring of retrospective grimace frames resulted in no significant differences within or across groups (P>0.05).

Table 1. Real-time and retrospective MGS scores (n = 10/group).

	Saline + Water + Water		Saline + Water + Bup		AOM + DSS + Water		AOM + DSS + Bup
	Retrospective	Real-time	Retrospective	Real-time	Retrospective	Real-time	Retrospective	Real-time
Baseline	0 ± 0	0 ± 0	0.133 ± 0.07	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Day 5	0.167 ± 0.06	0 ± 0	0.233 ± 0.11	0.056 ± 0.04	0.296 ± 0.12	0 ± 0	0.185 ± 0.10	0 ± 0
Day 19	0.067 ± 0.04	0 ± 0	0.167 ± 0.13	0 ± 0	0.185 ± 0.08	0.198 ± 0.08^**^#	0.444 ± 0.22	0.111 ± 0.05^#^{^}
Day 26	0.267 ± 0.10	0 ± 0	0 ± 0	0 ± 0	0.222 ± 0.10	0.062 ± 0.03	0.333 ± 0.18	0.049 ± 0.05
Day 40	0.167 ± 0.09	0 ± 0	0.100 ± 0.05	0 ± 0	0.111 ± 0.08	0 ± 0	0.037 ± 0.04	0 ± 0
Day 47	0.100 ± 0.07	0 ± 0	0.133 ± 0.07	0 ± 0	0.259 ± 0.19	0.012 ± 0.012	0.333 ± 0.14	0.086 ± 0.06^{^}
Day 61	0.267 ± 0.12	0 ± 0	0.133 ± 0.05	0 ± 0	0.259 ± 0.11	0 ± 0	0.148 ± 0.11	0 ± 0

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Data were analysed non-parametrically using a Friedman test and a Kruskal-Wallis with a Mann-Whitney post-hoc test and are expressed as mean MGS score ± SEM.

**p<0.01 compared to Saline + Water + Water at same time-point

#p<0.05 compared to baseline within a group

^p<0.05 compared to day 40 within a group.

Correlations

Real-time grimace scores were positively correlated with colitis severity and tumour number on day 19 (Table 2; P<0.05). Burrowing was negatively correlated with colitis severity and tumour number at all three time-points (days 19, 40 and 61; P<0.05). Furthermore, DAI was positively correlated with colitis severity score and tumour number at all three time-points (days 19, 40 and 61; P<0.05).

Table 2. Correlations between data sets of behavioural and clinical indicators on days 19, 40 and 61.

		Colitis Severity day 19	Tumour Number day 19	Colitis Severity day 40	Tumour Number day 40	Colitis Severity day 61	Tumour Number day 61
Real-time Grimace	Correlation Coefficient	0.517	0.668	n.e	n.e	n.e	n.e
Real-time Grimace	Significance (2-tailed)	0.001^***	0.000^***	n.e	n.e	n.e	n.e
Photo Grimace	Correlation Coefficient	0.240	0.289	-0.147	-0.194	0.080	-0.027
Photo Grimace	Significance (2-tailed)	0.146	0.078	0.379	0.244	0.633	0.872
Burrowing	Correlation Coefficient	-0.478	-0.405	-0.393	-0.408	-0.538	-0.266
Burrowing	Significance (2-tailed)	0.002^**	0.012^*	0.015^*	0.011^*	0.000^***	0.106
DAI	Correlation Coefficient	0.689	0.764	0.751	0.650	0.863	0.838
DAI	Significance (2-tailed)	0.000^***	0.000^***	0.000^***	0.000^***	0.000^***	0.000^***

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Data were analysed using a non-parametric spearman-rho test.

***p<0.001

**p<0.01 and

*p<0.05.

Note–no correlation coefficients could be derived between real-time grimace data and other measures on day 40 and 61 due to number of zero scores (n.e.–not estimable due to no variation in real-time grimace scores [all scores were 0]).

Discussion

AOM/DSS administration successfully induced colitis-associated colorectal cancer in mice, as evidenced by colonoscopically-assessed severity, tumour development and increased colon weights. However, the disease state was not reliably translated in the results of the two affective state measurement techniques utilised, namely burrowing and MGS. Clinical scores of disease such as DAI used in the current study, include scoring of non-specific mouse illness signs such as bodyweight loss, coat appearance, activity and stool consistency. Our findings suggest that the DAI is in fact the most reliable determinant of the clinical picture presented in these mice, and humane endpoint implementation in this model should continue to be based on this scoring scheme.

Analgesic administration did not affect normal animals; however, interestingly, buprenorphine increased the clinical DAI score of mice with colitis-associated colorectal cancer on selected days, attributed to bodyweight loss. This result was likely to be primarily due to in-appetence, possibly brought about by nausea, and consequent bodyweight loss as a side-effect of analgesic intervention [1, 19]. Nonetheless, this effect was not observed consistently throughout the experimental period. Overall, results were unable to conclude a significant impact of opioid analgesic (buprenorphine) intervention on pain reduction in the measures used at selected time-points, as highlighted by the minimal differences in grimace scores, burrowing behaviour and DAI in disease mice. This implied either that: 1) These animals were not experiencing pain, 2) The tests utilised were not sensitive enough to detect the type of pain experienced, or that 3) buprenorphine was ineffective in the face at the chosen time-points in mice with colitis-associated colorectal cancer.

Non-facial indicators of pain such as abdominal twitching, hunching, writhing, and belly press were identified in mice throughout the experimental period. Although these characteristics are not considered in facial grimace scores, they have been identified as validated pain associated behaviours that commonly occur following laboratory procedures [20, 21]. This highlights a key point in comparing real-time with retrospective measures especially when using personnel experienced with mice as real-time observers. Experienced observers are likely to subconsciously note general condition, and other pain-like behaviours such as hunching, writhing, belly press and immobility which may influence their scoring. These indicators are unable to be scored with a head-only photo image. Therefore, it would be advisable to use naïve observers for real-time grimace scoring in future studies.

The MGS action units have been validated in acute or moderate pain which lasts from minutes to hours. It has been reported that these action units are unable to be identified in mice days or weeks after a procedure, injury or surgical insult [5]. This is plausible since a fitness advantage would be gained by not communicating evidence of injury to predators via expression of the ‘pain face’ [22]. Consequently, the time-points selected in the current chronic study may have been too long after procedures to be able to identify pain present in the face. Furthermore, the MGS scores obtained were generally low (maximum 0.4 ± 0.2; live and retrospective analysis), implying a lack of sensitivity which may have precluded the finding of an analgesic effect. Similarly, in a study of rats with the gastrointestinal condition mucositis, Whittaker et al. (2016) reported that other behavioural measures utilised in the mucositis study including writhing, twitching, back-arching and sociability, to be more indicative of a pain status in the disease rats than facial grimace [23]. Moreover, the low MGS results in the current study may have indicated that the mice were not experiencing pain, it could also highlight the evolutionary characteristic of mice hiding pain in their face to deter predators [4].

Animal ethics committees often recommend analgesic implementation in studies when animals are induced with disease, therefore, it is crucial to understand that analgesic intervention will not affect experimental design. In the current study, a minimal effect on colitis severity and DAI was observed in buprenorphine treated animals; however, these results were not consistently represented throughout the experimental period and may have been due to the timing of DAI scoring in respect to buprenorphine-administration. Furthermore, buprenorphine was orally-administered to mice thrice weekly for 63 days and it is possible that during this time mice established a tolerance to the analgesia. Dum and Herz (1981) concluded that rats subcutaneously injected with buprenorphine twice daily developed a tolerance after just five days [24]. Additionally, in a study of DBA/2 mice with SL2 lymphoma, there was no interaction between dietary-administered buprenorphine and time, indicating that a drug tolerance may have been established during the 20 day period [25]. Furthermore, Van Loo and authors (1997) concluded that there were no clear indicators that buprenorphine impacted the pain experienced by mice with tumours, concluding that it was an undesirable analgesia in a lymphoma tumour model [25]. Hence, these data cannot confirm an action of buprenorphine in reducing pain based on the MGS scores obtained, nor any improvement in wellbeing based on DAI score or burrowing behaviour. However, this needs to be considered in light of the difficulty in teasing apart beneficial, versus side effects using the DAI, and the differences obtained in baseline burrowing score. Moreover, buprenorphine does not modify experimental outcomes which is an important finding when considering analgesic use in gastrointestinal animal models.

In normal mice, burrowing activity was increased in buprenorphine–treated groups on days 19, 26 and 40. This hyper-excitability is supported by Cowan et al. (1977), whereby the authors documented that buprenorphine-administration increased activity (walking and hopping) in non-painful mice [12]. In another study, resting behaviours were decreased in buprenorphine-treated cancerous mice compared to controls [25]. Moreover, increased levels of activity are suggested to be a side-effect of buprenorphine administration in rodents [26]. In the current study, AOM/DSS control animals displayed a higher baseline (day -1) burrowing ability compared to AOM/DSS administered together with buprenorphine, which may have impacted the burrowing results obtained at the other time-points. In future studies, it would be beneficial to allocate treatment groups based on burrowing activity and adjust these to ensure that all experimental groups display similar burrowing abilities at baseline. Furthermore, there was no significant difference in burrowing activity between colitis-associated colorectal cancer and normal control mice in the current study, suggesting that burrowing is not an effective behavioural measure in the AOM/DSS model. Interestingly, DSS-administration alone has been reported to significantly impact burrowing behaviour in mice with acute [27] and chronic [28] colitis.

Conclusions

Although the MGS scores obtained through real-time and retrospective analyses were unable to be validated in regards to pain alleviation in this chronic study, we were able to conclude that real-time grimace scores and daily clinical scores were correlated with increased colitis severity and tumour number across treatment groups. However, retrospective grimace scores were not correlated with other data sets. This indicated that real-time grimace may be a more accurate technique to complement other measures of disease in animal studies. However, as previously discussed, there are limitations with the use of this method as a practical tool. Importantly, burrowing activity was negatively correlated with colitis severity and tumour number, indicating that as chronic disease develops, mouse behaviour will decrease as wellbeing is impacted. Given the lack of statistically-significant differences between groups we cannot recommend this measure in the colitis-associated colorectal cancer model. We conclude that the traditional disease activity index, or clinical score, presents the most comprehensive welfare assessment tool in the colitis-associated colorectal cancer mouse model, having a degree of sensitivity and comprising of both objective and subjective measures to constitute a final score.

In the current study, use of the MGS was unable to identify pain in the mouse model of colitis-associated colorectal cancer. Furthermore, the live-scoring MGS method was unable to be validated in this model of chronic gastrointestinal disease. Nonetheless, this study is the first to use the MGS in a chronic model of colorectal pain and it is the first to discuss the correlation between live and retrospective scoring methods with other study measurements. Further investigation of the MGS in this model is necessary to validate its reliability in chronic disease Consideration should be given to use of other methods for measuring ADL or affective state, for example nest making or judgement biasing in models of chronic disease. Moreover, DAI or clinical scores may be the most reliable method for affective state assessment in mouse models of chronic gastrointestinal diseases.

Supporting information

S1 Dataset. Disease activity index data files.

(XLSX)

Click here for additional data file.^{(1.2MB, xlsx)}

S2 Dataset. Colonoscopically-Assessed colitis severity and tumour scores data files.

(XLSX)

Click here for additional data file.^{(56.7KB, xlsx)}

S3 Dataset. Burrowing data files.

(XLSX)

Click here for additional data file.^{(60.3KB, xlsx)}

S4 Dataset. Live mouse grimace scale data files.

(XLSX)

Click here for additional data file.^{(14.4KB, xlsx)}

S5 Dataset. Retrospective mouse grimace scale data files.

(XLSX)

Click here for additional data file.^{(29.8KB, xlsx)}

S6 Dataset. Correlation output data.

(DOCX)

Click here for additional data file.^{(44.8KB, docx)}

Acknowledgments

The authors would like to acknowledge Rebecca George and Chloe Mitchell for assisting with real-time grimace scoring.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

LCC received partial funding from The Australian Veterinary Association for the current study (Animal Welfare Trust Grant; www.ava.com.au). LCC is also supported by an Australian Government Research Training Program Stipend and a PhD Top-Up Scholarship from AgriFutures Australia (www.agrifutures.com.au). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

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PLoS One. doi: 10.1371/journal.pone.0228413.r001

Decision Letter 0

Mathilde Body-Malapel

18 Oct 2019

PONE-D-19-22786

Affective state determination in a mouse model of colitis-associated colorectal cancer

PLOS ONE

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Reviewer #2: Partly

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Reviewer #2: I Don't Know

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Reviewer #1: In the present study, the AA evaluated behavioral assessment, mouse grimace score (MGS) and burrowing, as indicators of affective state in a mouse model of colitis-associated colorectal cancer. Furthermore, this study compares real-time MGS with retrospective MGS.

The purpose of the study is to implement and evaluate affective state in the disease model to better optimize analgesia protocols which is important for the welfare of laboratory animals.

Control groups receiving analgesia, buprenorphine, were used to assess any pain-associated effect on the used behavior tests (MGS and burrowing).

In the present study the AA show that burrowing and MSG (both real-time and retrospective) was not affected in the colitis-associated colorectal cancer models, while the traditional measures DAI score and colonoscopy severity was increased. No consistent effect of buprenorphine was present when comparison with control groups, which might be due to insensitiveness of the behavior test or simply that the mice did not experienced any pain.

The real-time and retrospective MSG correlated to each other, but only real-time MSG correlated with colitis severity at day 19.

The study concludes that behavioral testing with MGS and burrowing should not be used for pain assessment in this disease model where traditional methods like DAI and colonoscopy is still preferable.

The study is well-written although I think the general purpose of the study could be stated more clear in the introduction. Should these behavioral tests replace traditional measurements or be done in addition to?

Minor Points:

- Aim should definitely be stated more clearly in the discussion

- Specify DAI and colonoscopy parameters – what is the maximum possible score?

- Statistical method used should be stated in all figure legends

- Table 2: correlation coefficients from all real-time MSG points should be stated even though they do not correlate to colitis severity. All other coefficients is stated (also the not significant ones) for retrospective and burrowing.

- Organ weights and length. These results are not well integrated in the manuscript. The results are not evaluated in any figure or table. They are not discussed and they do not contribute to the conclusion.

- Figures and legends: Abbreviation should only be used when writing out in the figure legend (WCH, ARC, i.p, DSS, scope)

Reviewer #2: The manuscript titled “Affective state determination in a mouse model of colitis-associated colorectal cancer” by Chartier et al. investigated whether the Mouse Grimace Scale and the burrowing test are a useful tool to assess the well-being of mice with colitis-associated colorectal cancer when compared to disease activity index. The results of the study indicated that neither of these two methods reliably measured the well-being while the clinical scores were increased in diseased mice and were positively correlated with colonoscopically-assessed severity and tumor number. The content of the manuscript is of interest for researchers using the AOM/DSS mouse model of colitis-associated colorectal cancer as well as laboratory animal veterinarians and can contribute to a better understanding of welfare indicators that are sensible to detect compromised well-being in this animal model. Overall, the manuscript is well-written, it is clear and nicely organized.

Issues that should be addressed in the revision of the manuscript:

- The half-life of buprenorphine is quite short (lasting effects: approx. 6-8 hours) and therefore it is currently discussed to use sustained release formulation of buprenorphine providing consistent, long-lasting analgesia. I was wondering how the authors made sure that the serum concentrations of buprenorphine were consistent until mice received the next treatment. Did the effect of a single dosage last for 3 days? It would be important to know the exact interval (in hours) between the last buprenorphine treatment and the analysis of MGS, burrowing tests, and clinical scoring.

- In the present study, water or buprenorphine were administered by oral gavage although there are several refinement methods for this procedure that prevent the mice from the distress caused by oral gavage. Buprenorphine can be administered via the drinking water or using flavored gelatin or Nutella etc. I would appreciate if you could explain in the section "experimental design" why these refinement methods were not applied.

- Data availability: The authors state that all relevant data are within the manuscript and its supporting information files. Since I have not received the supporting information files, I cannot check whether this statement is true.

- P5L95 Introduction: Miller and Leach also compared live versus retrospective MGS scores and found that in general live scores were lower than those obtained from images, that contradicts the results of the present study. Miller and Leach did not use the same mouse model as the authors of the present study. However, I would like to encourage the authors to discuss the discrepancy in their MGS results. Miller, Amy L., and Matthew C. Leach. "The mouse grimace scale: a clinically useful tool?." PloS one 10.9 (2015): e0136000.

- P5-6L104-114 Animal studies: Please explain why female mice were used only and provide more details of relative humidity, weights of animals, cage enrichment, type of bedding material, and number of cage companions. Explain how the number of animals was arrived at and provide details of sample size calculation used. How were animals allocated to experimental groups (details of randomization).

- P6L120-125: Please indicate the administration volumes for water, buprenorphine, saline, and AOM, injection site and size of cannula used for the ip injection, and the route of administration for DSS/water (L123-124).

- P7LL136-141 DAI: Were the experimenters blinded when scoring the parameters?

- P7L143-153 Colonoscopy: How long did the procedure last? Provide details of the compound isoflurane (supplier etc.).

- P8L155-163: Was the same burrow used as described by Deacon? Please add more information on the pebbles used for this test (approximate size, supplier etc.) and the time of day when the test was conducted.

- P8L165ff MGS: How many persons generated live and retrospective scores? It is advisable that more than one scorer is deployed in MGS scoring and the interrater reliability is calculated. Could you provide more details of the experience of experimenters in using the MGS and the time of day when live MGS scores were obtained?

- P9LL174-180: I cannot follow the explanation of calculation. Could you provide an example calculation.

- P9-10 Statistical analysis: Did you check for normal distribution of data?

- P10-18 Results: Provide details of the statistical methods used for each analysis and test statistics (not p value only).

- P12-13L251ff MGS: Did you examine whether live and retrospective MGS score correlate?

- P18L295 Discussion: While the MGS was originally developed to assess pain, it is currently discussed that changes in facial expression described in the MGS are not present in pain only but also in other affective states. Moreover, the weights of the facial action units seem to vary between the different states (Langford et al. 2010, . Could the authors determine which of the facial features were most affected? I would kindly ask the authors to adapt their statement that the MGS is specific to pain (it is not).

Dalla Costa, Emanuela, et al. "Can grimace scales estimate the pain status in horses and mice? A statistical approach to identify a classifier." PloS one 13.8 (2018): e0200339.

Langford, Dale J., et al. "Coding of facial expressions of pain in the laboratory mouse." Nature methods 7.6 (2010): 447.

- P19L309-311 Discussion: Depending on the interval between the last administration of buprenorphine and the assessment of well-being using the MGS or the burrowing test, the three hypotheses should be rethought. With regard to hypothesis #2) the authors should also consider the issues raised above (number of MGS scorers, experience of MGS scorers)

- P20L322: I see your point in using naïve scorers not being familiar with mouse behavior, though I think it is crucial that they are properly trained in using the MGS.

- P22L373: How do you explain the discrepancy between your findings and results of Safaeian et al.?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Jan 27;15(1):e0228413. doi: 10.1371/journal.pone.0228413.r002

Author response to Decision Letter 0

12 Nov 2019

Responses to Reviewers

Title: Affective state determination in a mouse model of colitis-associated colorectal cancer

Authors: Lauren C Chartier, Michelle L Hebart, Gordon S Howarth, Alexandra L Whittaker and Suzanne Mashtoub

Manuscript ID: PONE-D-19-22786

The authors would kindly like to thank the two reviewers for their comments and questions regarding the current manuscript. All answers to comments and revisions have been documented in the manuscript file labelled ‘Revised Manuscript with Track Changes. Page/line number references for amendments are listed on this document for your convenience. Additionally, a copy of the completed reviewed manuscript has been uploaded with the revision and is labelled ‘Manuscript’.

Reviewer #1: In the present study, the AA evaluated behavioural assessment, mouse grimace score (MGS) and burrowing, as indicators of affective state in a mouse model of colitis-associated colorectal cancer. Furthermore, this study compares real-time MGS with retrospective MGS. The purpose of the study is to implement and evaluate affective state in the disease model to better optimize analgesia protocols which is important for the welfare of laboratory animals. Control groups receiving analgesia, buprenorphine, were used to assess any pain-associated effect on the used behaviour tests (MGS and burrowing). In the present study the AA show that burrowing and MSG (both real-time and retrospective) was not affected in the colitis-associated colorectal cancer models, while the traditional measures DAI score and colonoscopy severity was increased. No consistent effect of buprenorphine was present when comparison with control groups, which might be due to insensitiveness of the behaviour test or simply that the mice did not experienced any pain. The real-time and retrospective MSG correlated to each other, but only real-time MSG correlated with colitis severity at day 19. The study concludes that behavioural testing with MGS and burrowing should not be used for pain assessment in this disease model where traditional methods like DAI and colonoscopy is still preferable. The study is well-written although I think the general purpose of the study could be stated more clear in the introduction. Should these behavioural tests replace traditional measurements or be done in addition to?

Minor Points:

- Aim should definitely be stated more clearly in the introduction

The final paragraph of the introduction has been amended to ensure that the aim of the study is clear. We aimed to assess all behavioural/pain assessments (clinical scoring, MGS and burrowing) in order to identify the most reliable method for pre-clinical models of colitis-associated colorectal cancer. Furthermore, pain assessment using the real/retrospective MGS had not yet been validated in a model of colitis-associated colorectal cancer prior to this manuscript (P5L92-95).

- Specify DAI and colonoscopy parameters – what is the maximum possible score?

We have included the maximum possible scores for these parameters in the methods section of the manuscript. The maximum score for DAI is 12, and for colonoscopically-assessed severity is 15.

- Statistical method used should be stated in all figure legends

We have amended and included the statistical method used in all figure legends.

Correlation coefficients for real-time MGS were originally not stated in Table 2 for days 40 and 61 as the correlation coefficient cannot be calculated due to the fact that real-time grimace scores were all zero for these time-points. We have amended Table 2 and placed not estimable (n.e) in these time-points and a note in the legend to clarify that there was no variation in grimace scores on days 40 and 61.

We agree with your observation and have thus removed the ‘Organ weights and length’ data and section from the manuscript.

- Figures and legends: Abbreviation should only be used when writing out in the figure legend (WCH, ARC, i.p, DSS, scope)

All abbreviations have been stated in full prior to being abbreviated throughout the manuscript. Moreover, figure legends have been amended to include the full word before being abbreviated (e.g. dextran sulphate sodium; DSS).

Issues that should be addressed in the revision of the manuscript:

Mice were administered thrice weekly with buprenorphine. These administrations occurred in the morning, 2 hours prior to MGS scoring and 8 hours prior to burrowing analyses. Due to the timings of these analyses we expected that we would be able to observe the effect of buprenorphine as the analyses occurred within the estimated half-life. However, as clinical scoring was measured first thing in the morning for initial monitoring of all mice, these scores were obtained prior to buprenorphine administration. As a result we understand that we may not have picked up the effect of buprenorphine in the clinical scoring analyses. We have now indicated specific time-frames following buprenorphine administration for each behavioural test in the methods section of the manuscript.

Buprenorphine was administered via oral gavage as the control groups utilised in this study were also used as part of another study and thus were receiving water oral gavages. Although we acknowledge that some minor distress is caused through the gavage procedure, we administered buprenorphine in this manner to ensure that all mice were exposed to the same procedures and therefore significant results (e.g body weight loss) could not be attributed to the procedures alone. We have included an extra sentence justifying the administration route of buprenorphine in the experimental design section (P7L133-134).

All supporting raw data files have been uploaded with the revised versions of this manuscript on Figshare.

Further details on housing humidity, average animal weight at the beginning of the experiment and enrichment items have now been included in the Animal studies section of the manuscript (P5P6L105-122). Only female mice were utilised in this study to remain consistent with data obtained from previous trials using the AOM/DSS model. A statement to highlight this reasoning has been included in the ‘Animal studies’ section of the manuscript with appropriate referencing. However, we understand that an initial study with male mice needs to be investigated in the future. Mice were stratified to groups based on day 0 body weight and baseline burrowing activity to ensure a spread of weight and burrowing ability across all treatment groups. Group size was calculated using Clin.Calc for mouse grimace scale outcomes from Rosen et al. (2017). This calculation assumed a power of 80%, and indicated that a minimum sample size of n=9/group was necessary. Therefore, as we included a n=10/group we ensured that statistical power would be reached. The details of this power calculation have now also been included in the Animal studies/ experimental design section of the manuscript for clarification (P6L124-138).

The oral-gavage administration volumes of water and buprenorphine are already stated in the manuscript as 80µL (P6L123). The volumes of saline and AOM injections were calculated based on individual mouse body weight (7.4mg/kg; P6L125). However, for clarification we have included an average injection volume (0.14mL) in this section of the manuscript, along with the needle gauge used (27G) for the intraperitoneal injections. DSS/water was provided in drinking bottles for ad libitum access, these details have also been included in the manuscript (P6L127).

- P7LL136-141 DAI: Were the experimenters blinded when scoring the parameters?

DAI scoring was not performed in a blinded-fashion as it was measured routinely by the researchers when conducting initial monitoring of all mice first thing in the morning. This is because DAI/ clinical scoring is required for ethical purposes and is crucial for determining humane end-points. Furthermore, the euthanasia criteria accepted by the animal ethics committees that approved this study is based upon the DAI scoring system, hence it was important to provide un-biased scores. Moreover, the researchers that monitor and obtain DAI scores in this study are experienced and adequately trained to provide standardised scores for each parameter.

- P7L143-153 Colonoscopy: How long did the procedure last? Provide details of the compound isoflurane (supplier etc.).

Isoflurane was sourced from AbbVie Inc., and the colonoscopy procedure lasted approximately 10 minutes from anaesthetic induction to recovery. This has now been detailed in the revised manuscript (P8L156-167).

The burrows used in the current study were modified from those detailed by Deacon. We used Coca-Cola bottles (diameter and length provided) and kitty litter as pebbles. These burrowing analyses were conducted at 6pm, one hour after commencement of the dark cycle and 8 hours following buprenorphine administration. These specific details have been included in the revised manuscript (P8L170-179).

Live and retrospective MGS scores were obtained from one experiment-blinded observer at each time-point throughout. Although it is advisable to have more than one scorer, previous studies have also been published with one observed (Cho C, Michailidis, V., Lecker, I. et al. Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale. Sci Rep 9, 359 (2019); Akintola, T., Raver C, Studlack P. et al. The grimace scale reliably assesses chronic pain in a rodent model of trigeminal neuropathic pain. Neurobiol Pain 2, 13-17 (2017). Furthermore, live MGS scores/ videos for retrospective scoring were obtained in the morning (9-12pm; 2 hours following buprenorphine administration), as detailed in the manuscript (P9L200). The scorers conducting were experienced in grimace identification but naïve to experimental conditions. Dr Alexandra Whittaker and Miss Rebecca George have published studies utilising the grimace scale (George RP, Howarth GS, Whittaker AL. Use of the Rat Grimace Scale to Evaluate Visceral Pain in a Model of Chemotherapy-Induced Mucositis. Animals (Basel). 2019 Sep 12;9(9); Whittaker AL, Leach MC, Preston FL, Lymn KA, Howarth GS. Effects of acute chemotherapy-induced mucositis on spontaneous behaviour and the grimace scale in laboratory rats. Lab Anim. 2016 Apr;50(2):108-18.) and Miss Lauren Chartier has performed grimace analyses in two studies (unpublished). Furthermore, training in the scoring techniques was performed using the grimace posters provided in the publication by Langford et al. (2010) and using old video data.

- P9LL174-180: I cannot follow the explanation of calculation. Could you provide an example calculation?

We have edited the written explanation to be more clear of the real-time MGS score calculation (P9L214-228) and have included a table with a sample calculation below.

Time Orbital tightening Ear positon Whisker change Cheek bulge Nose bulge Median per 15 sec Average per 90 sec interval

15-30sec 1 0 1 2 1 1 0.333333333

45-1:00 0 0 0 0 0 0

1:15-1:30 1 0 0 0 0 0

1:45-2:00 0 1 1 2 1 1 1

2:15-2:30 1 1 1 1 1 1

2:45-3:00 0 2 1 1 1 1

3:15-3:30 0 2 1 0 0 0 0.25

3:45-4:00 0 0 0 0 0 0

4:15-4:30 1 0 0 0 0 0

4:45-5:00 0 1 1 2 1 1

Final Score (mean of score per 90 sec) 0.527777778

- P9-10 Statistical analysis: Did you check for normal distribution of data?

Yes, as mentioned in the statistical analysis section of the manuscript, data was tested for normality using a Shapiro–Wilk test and then analysed accordingly (P10L199). Furthermore, a Levene’s test for homogeneity of variance was used and no outliers were identified.

- P10-18 Results: Provide details of the statistical methods used for each analysis and test statistics (not p value only).

The details of the statistical method used for each analysis is highlighted in the statistical analysis section of the manuscript. Additionally, these details have been included in the figure legends.

- P12-13L251ff MGS: Did you examine whether live and retrospective MGS score correlate?

All data from real-time and retrospective MGS scores had a correlation coefficient of 0.064, and thus are not strongly correlated. Furthermore, at all other time-points, the correlation coefficient for real-time and retrospective MGS are as follows; -0.017 (day 5), 0.163 (day 19), -0.05 (day 26), not estimable (day 40), 0,156 (day 47) and not estimable (day 61).

In the current study ‘orbital tightening’ and ‘ear position’ were the features of the MGS that were most affected. Other features such as ‘whisker change’ can be quite difficult to score on C57BL/6 mice as their dark coat makes it difficult to distinguish the whiskers in photographs. Moreover, we have amended the statement in the discussion as per this comment that the MGS is not specific to pain (P19L330).

As mentioned in previous responses to comments and in the manuscript, MGS scoring occurred 2 hours following buprenorphine administration, and burrowing analyses at 8 hours post buprenorphine administration. The number of MGS scorers and relative experience has been clarified in the manuscript and in an above comment to the reviewer responses, therefore, no further issues have been raised in regards to hypothesis (2) The tests utilised were not sensitive enough to detect the type of pain experienced.

- P20L322: I see your point in using naïve scorers not being familiar with mouse behavior, though I think it is crucial that they are properly trained in using the MGS.

We agree that proper training in the MGS criteria and features are necessary for reliable results, however, as mentioned in the discussion, experienced observers may subconsciously score based on other bodily measures of pain identified during live scoring. Future investigations could compare naïve scorers of live MGS and trained/experienced observers for retrospective MGS is used, as experienced observes will not be able to score non-facial measures of pain if only using facial photographs.

- P22L373: How do you explain the discrepancy between your findings and results of Safaeian et al.?

Safaeian et al. reported that DSS-treated mice presented with significantly reduced burrowing activity compared to normal controls. However, these results were founded in a model of chronic colitis, not colitis-associated colorectal cancer. Although the experimental models for chronic colitis and colitis-associated colorectal cancer share some similarities, the time-points at which burrowing was assessed in the Safaeian study were different to that used in the current study, as the timelines differ. Therefore, although we would hypothesise that the impact of burrowing ability would be similar in both disease models, the varying time-points for the measurement of burrowing between the studies is potentially what accounts for the discrepancy in our outcomes.

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PLoS One. doi: 10.1371/journal.pone.0228413.r003

Decision Letter 1

Mathilde Body-Malapel

5 Dec 2019

PONE-D-19-22786R1

Affective state determination in a mouse model of colitis-associated colorectal cancer

PLOS ONE

Dear Dr Mashtoub,

Thank you for submitting your manuscript to PLOS ONE and for revising your manuscript. It has been substantially improved but there are still a few issues that should be addressed. Therefore, we invite you to submit a revised version of the manuscript that addresses the few points raised during the second review process.

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Mathilde Body-Malapel

Academic Editor

PLOS ONE

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: All comments have been satisfactory and thoroughly addressed. I recommend the current manuscript for publication in PLOS ONE.

Reviewer #2: I thank the authors for their response to my questions and comments. However, there are still a few points that have not been addressed and revised in the manuscript:

- Although you mentioned that “mice were stratified to groups based on baseline body weight and burrowing activity” (P6LL128-130), there were significant differences in burrowing activity at baseline and you stated in the discussion part that “in future studies, it would be beneficial to allocate treatment groups based on the burrowing activity”. Please check the statement given in the part “experimental design” in LL128-130.

- Please provide information on the health status of the mice (see ARRIVE guidelines).

- Material and Methods, DAI (P8LL157-162): Please indicate that scorer(s) were not blinded and that DAI scores were obtained before administration of buprenorphine.

- Material and Methods, MGS: Time of day was not added to this section. Moreover, the authors described that there was one MGS scorer only, but in the response to my comments three scorers are listed (Alexandra Whittaker, Rebecca George, and Lauren Chartier). Did each of them score a third of the mouse faces? If this is true please add this information to the MGS methods section.

- The authors stated in their responses: “Mice were administered thrice weekly with buprenorphine. These administrations occurred in the morning, 2 hours prior to MGS scoring and 8 hours prior to burrowing analyses.”, “Furthermore, live MGS scores/videos for retrospective scoring were obtained in the morning (9-12pm; 2 hours following

buprenorphine administration)”

I assume that PM is a typing error (AM?) and DAI was obtained at 9 AM, buprenorphine was administered at 9 AM, mouse faces were scored at noon (MGS) and burrowing was monitored at 6 PM. According to this schedule, DAI could not asses the analgesic effects of buprenorphine (in contrast to the MGS and the burrowing test). Therefore, I would recommend emphasizing this issue and make it clearer for the reader in the discussion (especially in P20LL335-346; in this passage the reader has the impression that DAI was considered to assess the analgesic effect of buprenorphine administered on the respective days). Due to the short lasting analgesic effects of buprenorphine, it is unlikely that you have “picked up the effect of buprenorphine in the clinical scoring analysis”, as you also stated in your response, but of course you may have measured side effects.

Moreover, results of the burrowing test should be interpreted with more caution in the discussion part as baseline scores were significantly different – this makes it very tricky and should be pointed out for the reader.

The statement “Overall, results were unable to conclude a significant impact of opioid analgesic (buprenorphine) intervention on the measures used, highlighted by the minimal differences in grimace scores, burrowing behaviour and DAI in disease mice.” (P20LL340-343) does not consider, 1) that DAI also includes pain-specific behaviors and was actually increased in AOM+DSS groups, 2) that the time of DAI scoring did not allow for the assessment of analgesic effects of buprenorphine, 3) and that burrowing data are difficult to interpret due to baseline differences.

Based on the difficulties stated above, I would also recommend rethinking the three hypotheses given in LL343-346.

Moreover, the following phrase needs to be reformulated with regard to the above-mentioned concerns: “Hence, taken together the results suggest that buprenorphine is ineffective in improving wellbeing in mice with colitis associated colorectal cancer.” (P22LL388-390)

**********

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Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Jan 27;15(1):e0228413. doi: 10.1371/journal.pone.0228413.r004

Author response to Decision Letter 1

12 Jan 2020

Responses to Reviewers – 13th January 2020

Title: Affective state determination in a mouse model of colitis-associated colorectal cancer

Authors: Lauren C Chartier, Michelle L Hebart, Gordon S Howarth, Alexandra L Whittaker and Suzanne Mashtoub

Manuscript ID: PONE-D-19-22786

The authors would kindly like to thank the two reviewers for their comments and questions regarding the current manuscript. All answers to comments and revisions have been documented in the manuscript file labelled ‘Revised Manuscript with Track Changes’. Page/line number references for amendments are listed in this document for your convenience. Additionally, a copy of the completed reviewed manuscript has been uploaded with the revision and is labelled ‘Manuscript’.

Reviewer #1: All comments have been satisfactory and thoroughly addressed. I recommend the current manuscript for publication in PLOS ONE.

Reviewer #2: I thank the authors for their response to my questions and comments. However, there are still a few points that have not been addressed and revised in the manuscript:

It was originally stated that bodyweight and burrowing ability was used to assign groups as mice were firstly stratified to treatment groups based on their baseline bodyweight, then baseline burrowing data was used to ensure that burrowing ability was equally distributed across groups. The significant differences in baseline burrowing ability was unavoidable due to control groups being utilised in another study of the same nature running at the same time, as stated in the ‘animal studies’ section of the Material and Methods. However, to avoid confusion for the reader, the authors have revised the ‘experimental design’ section and now reads ‘Mice were stratified to groups based on baseline body weight.’ (P6L129). The statement in the discussion remains as “in future studies, it would be beneficial to allocate treatment groups based on the burrowing activity”.

- Please provide information on the health status of the mice (see ARRIVE guidelines).

The health status of mice has now been included in the ‘Animal Studies’ section of the Methods. The included statement (P6L117-119) reads; ‘The ARC undertakes a quarterly health screening, covering various bacterial, viral and parasitic organisms, all of which the obtained colony screened negative for.’

- Material and Methods, DAI (P8LL157-162): Please indicate that scorer(s) were not blinded and that DAI scores were obtained before administration of buprenorphine.

We have now included that scorers were not blinded to treatment groups when obtaining DAI measurements and that DAI was calculated prior to buprenorphine administration (P7L153-P8L160).

In the MGS section of the experimental design, we stated that MGS scoring occurred in the morning following buprenorphine administration; however, to clarify this we have now included a time (approximately 9am-12pm) on P9L215. There was only one MGS scorer per mouse, however, due to the large number of animals and time restraints, live scoring was shared amongst the three grimace experienced researchers (Alexandra Whittaker, Rebecca George, and Lauren Chartier), whereby one researcher scored one mouse. Retrospective blinded scoring was all performed by Miss Lauren Chartier.

buprenorphine administration)”. I assume that PM is a typing error (AM?) and DAI was obtained at 9 AM, buprenorphine was administered at 9 AM, mouse faces were scored at noon (MGS) and burrowing was monitored at 6 PM. According to this schedule, DAI could not asses the analgesic effects of buprenorphine (in contrast to the MGS and the burrowing test). Therefore, I would recommend emphasizing this issue and make it clearer for the reader in the discussion (especially in P20LL335-346; in this passage the reader has the impression that DAI was considered to assess the analgesic effect of buprenorphine administered on the respective days). Due to the short lasting analgesic effects of buprenorphine, it is unlikely that you have “picked up the effect of buprenorphine in the clinical scoring analysis”, as you also stated in your response, but of course you may have measured side effects.

To clarify the timeline of events for analyses for all mice - DAI was measured first thing in the morning (7-8am) prior to buprenorphine administration (9am). Then MGS scoring/video recordings occurred between 9am-12pm, and finally burrowing analyses were performed from 6pm on selected days. We have included the approximate time of each analysis in the ‘Materials and Methods’ section of the manuscript as well as time following buprenorphine administration to avoid confusion. It is correct that at this timeline we were unlikely to pick up the analgesic effect of buprenorphine in DAI as the effects would not have lasted 24 hours, however this has been stated in the methods that DAI was measured prior to administration (P7L156). Furthermore, MGS was performed at a time where the effects of buprenorphine should have been measureable.

We have now added to the final paragraph of the discussion (P22L387-388) to highlight that burrowing was significantly different at baseline in AOM/DSS controls and therefore may have impacted the results obtained at other time-points within the study.

1) P20L326 in the discussion section has been reworded to highlight that buprenorphine did not have an effect in terms of reducing pain (as this was the purpose of administering analgesia) in AOM/DSS mice, as buprenorphine did impact DAI scores at some time-points.

2) In the current study, DAI was measured daily in the morning prior to buprenorphine administration. This time-point was selected as DAI was routinely incorporated into the daily monitoring of all mice in the study. Furthermore, the effect of buprenorphine on DAI parameters (including bodyweight loss and diarrhoea) did not confound the study as these are retrospective and slow pathological changes (P22L380-382). For example, if DAI was measured 30 mins follow buprenorphine administration, changes in bodyweight and stool consistency would not be identifiable in this short time-frame.

3) The authors acknowledge that the discrepancies in baseline burrowing data mean that these results are difficult to interpret. This issue has been identified in the discussion (P22L390-392) and that future studies should aim to have groups with relatively similar baseline burrowing ability where possible. However, based on the burrowing results from other time-points in the study it is likely that buprenorphine does not have an effect on burrowing behaviour.

In light of these responses, the authors still believe that the hypothesis mentioned on P19L328-331 of the discussion are reasonable.

We agree that the statement highlighted is a little too strong and have modified it. The statement now reads ‘Hence, these data cannot confirm an action of buprenorphine in reducing pain based on the MGS scores obtained, nor any improvement in wellbeing based on DAI score or burrowing behaviour. However, this needs to be considered in light of the difficulty in teasing apart beneficial, versus side effects using the DAI, and the differences obtained in baseline burrowing score. (P21L378-383).

Furthermore, earlier in the same paragraph we have added more clarification that the timing of DAI measurements in respect to buprenorphine-administration is likely why minimal effects were observed (P21L367-368).

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PLoS One. doi: 10.1371/journal.pone.0228413.r005

Decision Letter 2

Mathilde Body-Malapel

15 Jan 2020

Affective state determination in a mouse model of colitis-associated colorectal cancer

PONE-D-19-22786R2

Dear Dr. Mashtoub,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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Mathilde Body-Malapel

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0228413.r006

Acceptance letter

Mathilde Body-Malapel

17 Jan 2020

PONE-D-19-22786R2

Affective state determination in a mouse model of colitis-associated colorectal cancer

Dear Dr. Mashtoub:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Dataset. Disease activity index data files.

(XLSX)

Click here for additional data file.^{(1.2MB, xlsx)}

S2 Dataset. Colonoscopically-Assessed colitis severity and tumour scores data files.

(XLSX)

Click here for additional data file.^{(56.7KB, xlsx)}

S3 Dataset. Burrowing data files.

(XLSX)

Click here for additional data file.^{(60.3KB, xlsx)}

S4 Dataset. Live mouse grimace scale data files.

(XLSX)

Click here for additional data file.^{(14.4KB, xlsx)}

S5 Dataset. Retrospective mouse grimace scale data files.

(XLSX)

Click here for additional data file.^{(29.8KB, xlsx)}

S6 Dataset. Correlation output data.

(DOCX)

Click here for additional data file.^{(44.8KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(29.9KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(30.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Affective state determination in a mouse model of colitis-associated colorectal cancer

Lauren C Chartier

Michelle L Hebart

Gordon S Howarth

Alexandra L Whittaker

Suzanne Mashtoub

Roles

Abstract

Introduction

Materials and methods

Animal studies

Experimental design

Fig 1. Experimental timeline.

Disease activity index

Colonoscopy

Burrowing analyses

Mouse grimace scale

Real-time MGS

Retrospective MGS

Statistical analysis

Results

Disease activity index

Fig 2. Daily disease activity index (DAI) score (n = 10/group).

Colitis severity and tumour number

Fig 3. Colonoscopically-assessed colitis severity (n = 10/group).

Fig 4. Total tumour number measured from colonoscopy (n = 10/group).

Burrowing

Fig 5. Burrowing activity (n = 10/group).

Mouse grimace scale

Table 1. Real-time and retrospective MGS scores (n = 10/group).

Correlations

Table 2. Correlations between data sets of behavioural and clinical indicators on days 19, 40 and 61.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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