With demographic changes, age-associated diseases will become challenging, and urinary incontinence is one of the ‘four giants of geriatrics’.
1 With a high prevalence of 12% in Europe and 17% in the US, overactive bladder (OAB) is a common condition with increased age.
2,3 Patients who suffer from OAB complain about urinary urgency often accompanied by increased frequency and nocturia, and sometimes with incontinence episodes.
4 The gold-standard for treatment of OAB is still the use of antimuscarinics which have good initial response rates.
5 However, due to their adverse side-effects and decreasing efficacy in the long-term, overall effectiveness is limited and alternative therapies are desirable.
5 In addition to the treatment limits, the cause of OAB is still unknown. As in other medical disciplines, the use of animal models can help answer questions about the disease pathogenesis. But the definition of OAB by itself highlights the difficulties for the development of animal models.
6 In 2006, McMurray et al. pointed out that the understanding of normal physiological control of continence and pathophysiological dysfunction is mandatory for the investigation of urinary incontinence and that, therefore, animal models should be used which closely resemble the human lower urinary tract in its anatomical and physiological functions.
7,8 For the investigation of lower urinary tract function, different animal species have been used, particularly non-human primates, dogs, pigs, cats, rabbits, rats, guinea-pigs, mice and hamsters.
7 Although rodent models are commonly used for the investigation of lower urinary function, their use has limitations in several translational aspects, for instance because of anatomical or diurnal differences.
9,10 Another aspect of the development of animal models is the difficulty of developing chronic models, especially in functional urology. Because of this, chronic models of OAB should have several features: stability for several weeks, robustness and reproducibility, permission to obtain unanesthesized in vivo measurements, and small tissue damage.
7 Especially with regard to in vivo measurements, large animal models offer advantages for continuous monitoring by telemetry.
7 In biomedical research and preclinical toxicological testing, pigs, especially Göttingen minipigs, are increasingly used in translational research as alternatives to dogs or monkeys.
11,12 In urological research only few data are available for Göttingen minipigs.
13–15
To the best of our knowledge, this study is one of the first to present reference data of micturition volume and frequency in Göttingen minipigs. We also performed a literature review to compare our data with data from different species (human, pigs, rats and mice).
Material and methods
Animals
Five female Göttingen minipigs aged 32 ± 3.2 months (mean + standard deviation of mean [SD]) from a disease-free breeding facility (University Göttingen, Göttingen, Germany) with an average weight of 54.3 ± 3 kg (mean + SD) were used in this study. The clinical health status of the animals was checked on arrival by a veterinarian. Blood samples were taken under anesthesia with prior overnight fasting with water ad libitum. For pre-sedation, minipigs received an intramuscular injection of 4 mg/kg azaperone and 0.1 mg/kg atropine. After 10 to 15 min, a second intramuscular injection of 15 mg/kg azaperone was given. Blood samples were used for standard blood counts (leukocytes, erythrocytes, platelets, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin). Fecal samples were investigated via native and flotation microscopy for endoparasites (worms and worm eggs, such as Trichuris spp., Strongylus spp. and Ascaris spp.) and via smears for Salmonella spp. All animals were acclimatized at least seven days prior to experiments.
Animal care
All procedures were performed in accordance with German legislation governing animal studies and the
Guide for the Care of Animal Use of Laboratory Animals (NIH Publication No. 85–23, revised 2011).
16 Official permission was granted by the governmental Animal Care and Use Office (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany). The five Göttingen minipigs were all housed in a certified facility (DIN ISO:9001/2008) with humidity and temperature-controlled environment and a 12:12 h light:dark cycle. Göttingen minipigs were housed singly, allowed contact with other pigs, or pair-housed on hardwood bedding (Lignocell F14; JRS, Rosenberg, Germany) in a pen of at least 3 m
2 in size and fed with energy-reduced, standardized minipig diet (ssniff Special diets, Soest, Germany). For enrichment nesting material (Voll Logging GmbH, Bad Camberg, Germany), a plastic ball and a metal chain (Bioscape GmbH, Castrop-Rauxel, Germany) were provided.
Experimental design
With metabolic cage studies, micturition frequency and volume of Göttingen minipigs were determined. We performed a literature review to establish reference data of micturition frequency and volume in humans, pigs, rats and mice.
Metabolic cage studies
Experimental set-up
Metabolic cages (Bioscape GmbH, Castrop-Rauxel, Germany) of 1 m × 1.15 m × 1.15 m (width × height × depth) in size were used. At the outlet, a collection reservoir was placed which was connected to a scale and corresponding software (Flow; Laborie, Montreal, Quebec, Canada) (
Figure 1). Additionally, a video camera (Mobotix, Langmeil, Germany) with two motion sensor fields on the metabolic cage outlet was used to monitor micturition frequency and volume. Video analysis was performed with the corresponding software (sm-player portable version 0.6.9, sourceforge.net; Dice Holdings Inc, New York, NY, USA).
Animal care during measurements
In line with animal welfare guidelines, the experiments were performed for no longer than 24 h in the metabolic cage, with regular diet and controlled water ad libitum. Thereafter, the minipigs were again transferred to a regular pen.
Literature review: reference data for micturition frequency and volume in humans, rodents and pigs
Literature research was performed in March 2014 using the Medical Literature Analysis and Retrieval System Online (US National Library of Medicine’s life science database; MEDLINE; PubMed), Thomson-Reuters’ Web of Science, and Google Scholar. MEDLINE and Web of Science searches were performed using the following combined terms: ‘frequency AND volume AND chart AND urology’ and ‘urology AND healthy AND micturition’ for literature concerning humans. MEDLINE search for literature concerning animals consisted of comparable filters, excluding references to human literature and following the combined term: ‘micturition AND healthy’. ‘Micturition AND animals not human AND healthy’ was the search term used with the Web of Science database. In Google Scholar, the search term used was ‘in title: micturition AND healthy’.
Statistical analysis
Results are shown as mean ± SD or median with range. Descriptive statistics and creation of figures were performed with GraphPad Prism5 software (GraphPad Software Inc, La Jolla, CA, USA).
Discussion
For the investigation of lower urinary tract disorders like OAB, many animal models with different species have been used over time.
7 The limits of rodent models have been discussed by different research groups, as well as the differences with humans, such as anatomy, volume or diurnal rhythm.
9,10,28 To the best of our knowledge, this study is one of the first to provide physiological reference data of micturition in the Göttingen minipig. Advantages of this species for scientific research are for example their physiological similarity to humans and the fact that they are of a smaller size than domestic pigs.
29 Therefore in biomedical research and preclinical toxicology testing, especially, Göttingen minipigs are increasingly being used in translational research as alternatives to dogs or monkeys.
11,12 Other reasons are the increasing resistance in society against the use of pet animals and non-human primates and the acceptance by society of the use of agricultural animals with less emotional attachment to humans.
29,30
Intra-individual data of the five pigs presented showed significant differences leading to the conclusion that in longitudinal or chronic studies each animal should function as its own control (
Figures 3 and
4). In addition individual micturition patterns can be used for crossover study design.
The comparison of our own data with literature review data showed that the volumes and frequency of micturition in Göttingen minipigs are more similar to human micturition values than are those of rodents (
Figures 6 and
7). These findings indicate that, for certain urological animal studies, Göttingen minipigs would be the better choice for translational research. In the future, the reference data provided may be used for planning new animal studies in different research fields such as urology, neurology or nephrology. With this study we have addressed the 3R concept of Russell and Burch (1959), stating a responsibility for ‘reduction, refinement and replacement’ in laboratory animal experiments. Because of the use of reliable reference values, a comprehensive statistical power calculation is possible, leading to fewer animal numbers (reduction). Göttingen minipigs in urological research can be used with telemetric approaches involving free-moving animals, which allows higher accuracy and less infection risk (refinement) in monitoring. In the future, the data provided can be used to generate computer-based modulations of drug release and enable the development of intravesical drug-delivery systems, and satisfy the replacement principle.
31,32
We are aware of the limitations of our study. Briefly, only a small number of female-only Göttingen minipigs were used, but the data presented consisted of 35 monitoring sessions with 221 single micturitions. We chose very restricted inclusion/exclusion criteria. The rationale of this paper was the discussion of translational comparability of different species with a focus on physiological function. Diagrams from 20 or more different studies would be really confusing, and yet the overall mean value would be nearly the same, so comparison of mean values among species would be the same even with less restrictive review criteria.