Lab Anim OnlineFirst, published on July 10, 2015 as doi:10.1177/0023677215595554
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ab orator y
Original Article
a
n imals
l i m i t e d
Effects of acute chemotherapy-induced mucositis on spontaneous behaviour and the grimace scale in laboratory rats
Laboratory Animals 0(0) 1–11 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677215595554 la.sagepub.com
A L Whittaker1, M C Leach2, F L Preston1, K A Lymn1 and G S Howarth1,3
Abstract Intestinal mucositis is a frequent side-effect of chemotherapy treatment. Many oncological research programs aim to identify novel treatments for this distressing condition, and these programs frequently use rat models. Little is known about the presence and progression of pain in these models and how this can best be treated by analgesic therapy. We used a number of behaviour-based methods of pain assessment to determine which tools were best suited for pain identification. Baseline measures for behavioural assessment, rat grimace score and sociability were determined through analysis of continuously recorded video data and an applied social interaction test (n ¼ 16). Mucositis was then induced by intraperitoneal injection of 5-fluorouracil (150 mg/kg) and further behavioural analyses undertaken. An assessment of enrichment interaction was also made by determining the mass of a plastic chew toy gnawed both pre- and post-chemotherapy injection. Behavioural scoring was performed 1, 6, 12, 24 and 48 h after injection, with facial expression being scored at the 12, 24 and 48 h time-points. Sociability testing was performed once during the post-injection period. No significant differences were found in grimace scores between baseline and later daily measures. Behaviours similar to those previously reported post-laparotomy were observed. Writhing, twitching and back-arching behaviours were most evident in rats affected by mucositis and were increased in frequency (respective P values: 0.002, 0.004 and 0.008) 48 h after chemotherapy injection compared with baseline, implying that pain onset occurred around this time-point. Social investigatory behaviour was also increased (P ¼ 0.002) following disease onset. Each day, rats post-5FU injection gnawed a greater percentage of their nylabone enrichment by weight than the saline-injected control rats (P ¼ 0.046). These data suggest that, of the tools tested, behavioural assessment scoring may find greatest utility in rodent models of intestinal mucositis and should be investigated further.
Keywords rat grimace score, pain assessment, mucositis, behaviour
Mucositis is an ulcerative disorder that affects the digestive system, manifesting as a frequent consequence of chemotherapy treatment; significantly impacting on quality of life in cancer patients treated with chemotherapeutic agents.1,2 The pathogenesis of mucositis involves the direct inhibition of DNA replication and mucosal cell proliferation by the chemotherapy agent, leading to gastrointestinal tract damage. Such damage includes apoptosis of cells, crypt cell hypoproliferation, villous atrophy, collagen breakdown, ulcerations and increased intestinal permeability.3 As a result, serious clinical symptoms occur which include abdominal
1
School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, South Australia, Australia 2 School of Agriculture, Food and Rural Development, Agriculture Building, Newcastle University, Newcastle-Upon-Tyne, UK 3 Gastroenterology Department, Children, Youth and Women’s Health Services, Adelaide, South Australia, Australia Corresponding author: A L Whittaker, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, Roseworthy, South Australia, 5371, Australia. Email: [email protected]
Downloaded from lan.sagepub.com at Monash University on November 14, 2015
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Laboratory Animals 0(0)
bloating, pain, nausea, diarrhoea and weight loss.4 Pain largely arises from inflammation and consequent ulceration of the gastrointestinal tract, with the mouth (oral mucositis) and small intestine (intestinal mucositis) most frequently affected.5 The distressing nature of these symptoms leads to the mucositic condition being a major dose-limiting factor in the treatment of many cancers.1 Consequently, a number of research programs are aimed at investigation of mucositis disease mechanisms, and identification of novel therapeutic targets. Animal models are widely employed in these research investigations.6 Rats are commonly used as animal models for mucositis disease investigations. Typically, mucositis is induced through an administration of a chemotherapeutic agent. Chemotherapy administration leads to the rapid-onset of mucositis as shown by body weight loss, clinical scoring, and retrospective histological analysis.7,8 Clinical scoring typically records the presence or absence of clinical signs including piloerection, diarrhoea and body condition score in order to assign a value for each, which is summed to arrive at a total clinical score. However, despite these measures being objective in nature, they are typically retrospective, and therefore do not aid in humane endpoint determination or analgesic therapy modification. This inability to alleviate pain or distress poses both an animal welfare and ethical issue, and could potentially impact on the research data obtained. It is assumed that rats subjected to mucositis experience similar symptoms to those reported by human patients. Abdominal pain is one such symptom that may be amenable to amelioration through analgesic administration. As such, determining a method of evaluating both presence of, and level of pain would be of value for animal welfare reasons as well as improving the validity of therapeutic testing. Pain tends to evoke changes in behaviour which could potentially be measured quantitatively;9 however pain assessment in rodents remains challenging, since as prey species they need to mask signs of pain to avoid predator attention.10 Nevertheless, there has been significant progress in determining visceral signs of pain in rats,11 although much of this previous work has been carried out in a laparotomy model.2,12,13 More recently, changes in facial expression have been characterized as a potential tool for rodent pain assessment, the so-called ‘grimace scales’.14,15 This method may offer considerable advantages over other methods of behavioural assessment scoring since human observers tend to focus on the facial area,16 and scoring is likely to be less time-consuming than traditional behaviour-based techniques. This technique has largely been tested in the evaluation of short-lived acute pain,17,18 as opposed to longer-term pain states.
The social interaction test is generally regarded as a measure of anxiogenic effect,19 rather than pain. However, pain has been shown to impact upon social interaction and vice versa.20 Hence, the social interaction test may be a valuable adjunctive measure under controlled experimental conditions. The aim of this exploratory study was to investigate behavioural assessment scoring, facial grimace scoring, and the social interaction test, to determine which tool (if any) might be most appropriate for the assessment of pain in rats subjected to mucositis, given the unique nature of this condition and the accompanying environmental conditions (metabolism-cage housed). We also sought to identify observation time-points that allowed effective pain assessment. An assessment of enrichment interaction was also made to provide a non-specific indicator of welfare state. Specifically we were interested in methods that could show the presence of pain and chart its progression.
Materials and methods Animals This study used 16 male Hsd:Sprague Dawley (SD) rats (University of Adelaide, Laboratory Animal Services, Adelaide, Australia). Rats were housed individually in metabolism cages (Tecniplast, Exton, PA, USA) throughout the study. A standard casein-based diet21 and potable RO water were provided ad libitum. All animals were provided with a half plastic bone toy (Nylabone Products, Neptune, NJ, USA) as enrichment. A five-day period of acclimatization was provided prior to commencement of behavioural observations. The animal room was maintained at 21–23 C with a 12 h reversed light/dark cycle. Red light (approximately 650 nm wavelength) was provided during darkness to facilitate video recording. This study was conducted as part of a larger program of work investigating novel therapeutic agents for the treatment of chemotherapy-induced mucositis; the animals forming the disease control group of this larger study. The research was approved by the Animal Ethics Committee of the University of Adelaide and conducted in accordance with the provisions of the Australian code for the care and use of animals for scientific purposes.22
Experimental design Animals acted as their own controls to minimize the effects of inter-individual variability in behavioural data interpretation. Rats were orally gavaged with water once daily for nine days (between days 0 and 8). On day 6, all animals
Downloaded from lan.sagepub.com at Monash University on November 14, 2015
Whittaker et al.
3
were injected with 5-fluorouracil (5-FU) (Mayne Pharma Pty Ltd, Mulgrave, Victoria, Australia; 150 mg/kg) via the intraperitoneal route. Rats were humanely killed 72 h following chemotherapy injection by carbon dioxide asphyxiation.
Behavioural measures The rats were video-recorded continuously in their home cages by closed-circuit television (CCTV) cameras (OzSpy CLOC 600IR, Brisbane, Australia). Cameras were mounted on a rack directly behind the metabolic cages (one camera per cage) and at approximately 20 cm distance from the cage. These data were used for later rat behavioural analysis and facial image collection. A social interaction test (modified from Sams-Dodd23) was performed on one occasion preand post-mucositis induction (days 2 and 7). This test used a circular homogeneously illuminated arena with the base and sides made of plastic sheeting and hessian (50 cm radius). The floor was covered with a grey bedding material (Animal Bedding; FibreCycle Pty Ltd, Yatala, Queensland, Australia) which had been exposed to other rats’ odour prior to testing. Behaviour during the test was recorded using a video camera (Aiptek ADD T2 Pro; Aiptek International Inc, Hsinchu, Taiwan) from above the arena. The tests were performed in a separate room free from personnel entry.
To perform the test, the experimental rat was placed into the arena simultaneously, and approximately 40 cm apart, from an unfamiliar SD male rat which was not part of this study. The experimental rat was marked with a non-toxic black marker pen to enable recognition from video footage. Behaviour was recorded for 10 min. In order to assess enrichment use, the method described by Turner et al. was used.24 The NylaboneTM toy was weighed at the start of the study, just prior to the 5-FU injection and at the completion of the study.
Behavioural data analysis Home-cage observations. Behaviour data were collected by one treatment-blinded observer using analysis software (CowLog, University of Helsinki, Helsinki, Finland).25 Due to a technical failure with a CCTV camera unit, data from one rat was lost, hence only n ¼ 15 rats for these observations. The occurrences of a number of behaviours previously shown to be associated with abdominal pain were scored.2,26 Behaviours were scored over a 20 min period at each of the seven time-points in the dark and light phases of the circadian cycle; 18 and 12 h prior to the 5-FU injection and 1, 6, 12, 24 and 48 h post-injection. Frequency only, or frequency and duration, was scored based on the characteristics of the behaviour being observed. The ethogram used is illustrated in Table 1.
Table 1. Ethogram for in-cage behavioural analysis (adapted from Roughan and Flecknell2). Behaviour
Description
Behavioural qualifiers
Attend
Movement vibrissae accompanying head lifting Arching of back (‘cat stretch’) Drinking from bottle Feeding in cage area or inside hopper Grooming Standing on hindlegs Inactivity for >90 s Movement of vibrissae Loss of balance while walking or falling while grooming Transient inactivity
l
ab orator y
Original Article
a
n imals
l i m i t e d
Effects of acute chemotherapy-induced mucositis on spontaneous behaviour and the grimace scale in laboratory rats
Laboratory Animals 0(0) 1–11 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677215595554 la.sagepub.com
A L Whittaker1, M C Leach2, F L Preston1, K A Lymn1 and G S Howarth1,3
Abstract Intestinal mucositis is a frequent side-effect of chemotherapy treatment. Many oncological research programs aim to identify novel treatments for this distressing condition, and these programs frequently use rat models. Little is known about the presence and progression of pain in these models and how this can best be treated by analgesic therapy. We used a number of behaviour-based methods of pain assessment to determine which tools were best suited for pain identification. Baseline measures for behavioural assessment, rat grimace score and sociability were determined through analysis of continuously recorded video data and an applied social interaction test (n ¼ 16). Mucositis was then induced by intraperitoneal injection of 5-fluorouracil (150 mg/kg) and further behavioural analyses undertaken. An assessment of enrichment interaction was also made by determining the mass of a plastic chew toy gnawed both pre- and post-chemotherapy injection. Behavioural scoring was performed 1, 6, 12, 24 and 48 h after injection, with facial expression being scored at the 12, 24 and 48 h time-points. Sociability testing was performed once during the post-injection period. No significant differences were found in grimace scores between baseline and later daily measures. Behaviours similar to those previously reported post-laparotomy were observed. Writhing, twitching and back-arching behaviours were most evident in rats affected by mucositis and were increased in frequency (respective P values: 0.002, 0.004 and 0.008) 48 h after chemotherapy injection compared with baseline, implying that pain onset occurred around this time-point. Social investigatory behaviour was also increased (P ¼ 0.002) following disease onset. Each day, rats post-5FU injection gnawed a greater percentage of their nylabone enrichment by weight than the saline-injected control rats (P ¼ 0.046). These data suggest that, of the tools tested, behavioural assessment scoring may find greatest utility in rodent models of intestinal mucositis and should be investigated further.
Keywords rat grimace score, pain assessment, mucositis, behaviour
Mucositis is an ulcerative disorder that affects the digestive system, manifesting as a frequent consequence of chemotherapy treatment; significantly impacting on quality of life in cancer patients treated with chemotherapeutic agents.1,2 The pathogenesis of mucositis involves the direct inhibition of DNA replication and mucosal cell proliferation by the chemotherapy agent, leading to gastrointestinal tract damage. Such damage includes apoptosis of cells, crypt cell hypoproliferation, villous atrophy, collagen breakdown, ulcerations and increased intestinal permeability.3 As a result, serious clinical symptoms occur which include abdominal
1
School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, South Australia, Australia 2 School of Agriculture, Food and Rural Development, Agriculture Building, Newcastle University, Newcastle-Upon-Tyne, UK 3 Gastroenterology Department, Children, Youth and Women’s Health Services, Adelaide, South Australia, Australia Corresponding author: A L Whittaker, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, Roseworthy, South Australia, 5371, Australia. Email: [email protected]
Downloaded from lan.sagepub.com at Monash University on November 14, 2015
2
Laboratory Animals 0(0)
bloating, pain, nausea, diarrhoea and weight loss.4 Pain largely arises from inflammation and consequent ulceration of the gastrointestinal tract, with the mouth (oral mucositis) and small intestine (intestinal mucositis) most frequently affected.5 The distressing nature of these symptoms leads to the mucositic condition being a major dose-limiting factor in the treatment of many cancers.1 Consequently, a number of research programs are aimed at investigation of mucositis disease mechanisms, and identification of novel therapeutic targets. Animal models are widely employed in these research investigations.6 Rats are commonly used as animal models for mucositis disease investigations. Typically, mucositis is induced through an administration of a chemotherapeutic agent. Chemotherapy administration leads to the rapid-onset of mucositis as shown by body weight loss, clinical scoring, and retrospective histological analysis.7,8 Clinical scoring typically records the presence or absence of clinical signs including piloerection, diarrhoea and body condition score in order to assign a value for each, which is summed to arrive at a total clinical score. However, despite these measures being objective in nature, they are typically retrospective, and therefore do not aid in humane endpoint determination or analgesic therapy modification. This inability to alleviate pain or distress poses both an animal welfare and ethical issue, and could potentially impact on the research data obtained. It is assumed that rats subjected to mucositis experience similar symptoms to those reported by human patients. Abdominal pain is one such symptom that may be amenable to amelioration through analgesic administration. As such, determining a method of evaluating both presence of, and level of pain would be of value for animal welfare reasons as well as improving the validity of therapeutic testing. Pain tends to evoke changes in behaviour which could potentially be measured quantitatively;9 however pain assessment in rodents remains challenging, since as prey species they need to mask signs of pain to avoid predator attention.10 Nevertheless, there has been significant progress in determining visceral signs of pain in rats,11 although much of this previous work has been carried out in a laparotomy model.2,12,13 More recently, changes in facial expression have been characterized as a potential tool for rodent pain assessment, the so-called ‘grimace scales’.14,15 This method may offer considerable advantages over other methods of behavioural assessment scoring since human observers tend to focus on the facial area,16 and scoring is likely to be less time-consuming than traditional behaviour-based techniques. This technique has largely been tested in the evaluation of short-lived acute pain,17,18 as opposed to longer-term pain states.
The social interaction test is generally regarded as a measure of anxiogenic effect,19 rather than pain. However, pain has been shown to impact upon social interaction and vice versa.20 Hence, the social interaction test may be a valuable adjunctive measure under controlled experimental conditions. The aim of this exploratory study was to investigate behavioural assessment scoring, facial grimace scoring, and the social interaction test, to determine which tool (if any) might be most appropriate for the assessment of pain in rats subjected to mucositis, given the unique nature of this condition and the accompanying environmental conditions (metabolism-cage housed). We also sought to identify observation time-points that allowed effective pain assessment. An assessment of enrichment interaction was also made to provide a non-specific indicator of welfare state. Specifically we were interested in methods that could show the presence of pain and chart its progression.
Materials and methods Animals This study used 16 male Hsd:Sprague Dawley (SD) rats (University of Adelaide, Laboratory Animal Services, Adelaide, Australia). Rats were housed individually in metabolism cages (Tecniplast, Exton, PA, USA) throughout the study. A standard casein-based diet21 and potable RO water were provided ad libitum. All animals were provided with a half plastic bone toy (Nylabone Products, Neptune, NJ, USA) as enrichment. A five-day period of acclimatization was provided prior to commencement of behavioural observations. The animal room was maintained at 21–23 C with a 12 h reversed light/dark cycle. Red light (approximately 650 nm wavelength) was provided during darkness to facilitate video recording. This study was conducted as part of a larger program of work investigating novel therapeutic agents for the treatment of chemotherapy-induced mucositis; the animals forming the disease control group of this larger study. The research was approved by the Animal Ethics Committee of the University of Adelaide and conducted in accordance with the provisions of the Australian code for the care and use of animals for scientific purposes.22
Experimental design Animals acted as their own controls to minimize the effects of inter-individual variability in behavioural data interpretation. Rats were orally gavaged with water once daily for nine days (between days 0 and 8). On day 6, all animals
Downloaded from lan.sagepub.com at Monash University on November 14, 2015
Whittaker et al.
3
were injected with 5-fluorouracil (5-FU) (Mayne Pharma Pty Ltd, Mulgrave, Victoria, Australia; 150 mg/kg) via the intraperitoneal route. Rats were humanely killed 72 h following chemotherapy injection by carbon dioxide asphyxiation.
Behavioural measures The rats were video-recorded continuously in their home cages by closed-circuit television (CCTV) cameras (OzSpy CLOC 600IR, Brisbane, Australia). Cameras were mounted on a rack directly behind the metabolic cages (one camera per cage) and at approximately 20 cm distance from the cage. These data were used for later rat behavioural analysis and facial image collection. A social interaction test (modified from Sams-Dodd23) was performed on one occasion preand post-mucositis induction (days 2 and 7). This test used a circular homogeneously illuminated arena with the base and sides made of plastic sheeting and hessian (50 cm radius). The floor was covered with a grey bedding material (Animal Bedding; FibreCycle Pty Ltd, Yatala, Queensland, Australia) which had been exposed to other rats’ odour prior to testing. Behaviour during the test was recorded using a video camera (Aiptek ADD T2 Pro; Aiptek International Inc, Hsinchu, Taiwan) from above the arena. The tests were performed in a separate room free from personnel entry.
To perform the test, the experimental rat was placed into the arena simultaneously, and approximately 40 cm apart, from an unfamiliar SD male rat which was not part of this study. The experimental rat was marked with a non-toxic black marker pen to enable recognition from video footage. Behaviour was recorded for 10 min. In order to assess enrichment use, the method described by Turner et al. was used.24 The NylaboneTM toy was weighed at the start of the study, just prior to the 5-FU injection and at the completion of the study.
Behavioural data analysis Home-cage observations. Behaviour data were collected by one treatment-blinded observer using analysis software (CowLog, University of Helsinki, Helsinki, Finland).25 Due to a technical failure with a CCTV camera unit, data from one rat was lost, hence only n ¼ 15 rats for these observations. The occurrences of a number of behaviours previously shown to be associated with abdominal pain were scored.2,26 Behaviours were scored over a 20 min period at each of the seven time-points in the dark and light phases of the circadian cycle; 18 and 12 h prior to the 5-FU injection and 1, 6, 12, 24 and 48 h post-injection. Frequency only, or frequency and duration, was scored based on the characteristics of the behaviour being observed. The ethogram used is illustrated in Table 1.
Table 1. Ethogram for in-cage behavioural analysis (adapted from Roughan and Flecknell2). Behaviour
Description
Behavioural qualifiers
Attend
Movement vibrissae accompanying head lifting Arching of back (‘cat stretch’) Drinking from bottle Feeding in cage area or inside hopper Grooming Standing on hindlegs Inactivity for >90 s Movement of vibrissae Loss of balance while walking or falling while grooming Transient inactivity
