Evidence-Based Severity Assessment of Animal Models for Pancreatic Cancer.

Animal models are crucial to preclinical oncological research and drug development. Animal experiments must be performed in accordance with the 3R principles of replacement and reduction, if possible, and refinement where these procedures remain crucial. In addition, European Union legislations demand a continuous refinement approach, as well as pro- and retrospective severity assessment. In this study, an objective databased severity assessment was performed in murine models for pancreatic cancer induced by orthotopic, subcutaneous, or intravenous injection of Panc02 cells. Parameters such as body weight change, distress score, perianal temperature, mouse grimace scale, burrowing, nesting behavior, and the concentration of corticosterone in plasma and its metabolites in feces were monitored during tumor progression. The most important parameters were combined into a score and mapped against a reference data set by the Relative Severity Assessment procedure (RELSA) to obtain the maximum achieved severity for each animal (RELSAmax). This scoring revealed a significantly higher RELSAmax for the orthotopic model than for the subcutaneous and intravenous models. However, compared to animal models such as pancreatitis and bile duct ligation, the pancreatic cancer models are shown to be less severe. Data-based animal welfare assessment proved to be a valuable tool for comparing the severity of differently induced cancer models.

Sex Matters-Insights from Testing Drug Efficacy in an Animal Model of Pancreatic Cancer.

Preclinical studies rarely test the efficacy of therapies in both sexes. The field of oncology is no exception in this regard. In a model of syngeneic, orthotopic, metastasized pancreatic ductal adenocarcinoma we evaluated the impact of sex on pathological features of this disease as well as on the efficacy and possible adverse side effects of a novel, small molecule-based therapy inhibiting KRAS:SOS1, MEK1/2 and PI3K signaling in male and female C57BL/6J mice. Male mice had less tumor infiltration of CD8-positive cells, developed bigger tumors, had more lung metastasis and a lower probability of survival compared to female mice. These more severe pathological features in male animals were accompanied by higher distress at the end of the experiment. The evaluated inhibitors BI-3406, trametinib and BKM120 showed synergistic effects in vitro. This combinatorial therapy reduced tumor weight more efficiently in male animals, although the drug concentrations were similar in the tumors of both sexes. These results underline the importance of sex-specific preclinical research and at the same time provide a solid basis for future studies with the tested compounds.

Repeated social defeat stress differently affects arthritis-associated hypersensitivity in male and female mice.

Chronic stress enhances the risk of neuropsychiatric disorders and contributes to the aggravation and chronicity of pain. The development of stress-associated diseases, including pain, is affected by individual vulnerability or resilience to stress, although the mechanisms remain elusive. We used the repeated social defeat stress model promoting susceptible and resilient phenotypes in male and female mice and induced knee mono-arthritis to investigate the impact of stress vulnerability on pain and immune system regulation. We analyzed different pain-related behaviors, measured blood cytokine and immune cell levels, and performed histological analyses at the knee joints and pain/stress-related brain areas. Stress susceptible male and female mice showed prolonged arthritis-associated hypersensitivity. Interestingly, hypersensitivity was exacerbated in male but not female mice. In males, stress promoted transiently increased neutrophils and Ly6Chigh monocytes, lasting longer in susceptible than resilient mice. While resilient male mice displayed persistently increased levels of the anti-inflammatory interleukin (IL)-10, susceptible mice showed increased levels of the pro-inflammatory IL-6 at the early- and IL-12 at the late arthritis stage. Although joint inflammation levels were comparable among groups, macrophage and neutrophil infiltration was higher in the synovium of susceptible mice. Notably, only susceptible male mice, but not females, presented microgliosis and monocyte infiltration in the prefrontal cortex at the late arthritis stage. Blood Ly6Chigh monocyte depletion during the early inflammatory phase abrogated late-stage hypersensitivity and the associated histological alterations in susceptible male mice. Thus, recruitment of blood Ly6Chigh monocytes during the early arthritis phase might be a key factor mediating the persistence of arthritis pain in susceptible male mice. Alternative neuro-immune pathways that remain to be explored might be involved in females.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

OBJECTIVES: Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. MATERIAL AND METHODS: Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. RESULTS: According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. CONCLUSION: Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.

Does Sex Matter in Liver Surgery? Comparison of Severity Assessments between Female and Male Rats after Partial Hepatectomy: A Pilot Study.

INTRODUCTION: Current animal-based biomedical research, including studies on liver function and disease, is conducted almost exclusively on male animals to mitigate confounding effects of the estrous cycle. However, liver diseases afflict both men and women, so translational research findings should also be applicable to female patients. This pilot study investigated sex differences in objective and subjective severity assessment parameters in rats following 50% partial hepatectomy. MATERIALS AND METHODS: This study was performed using Wistar Han rats, in which measurements of body weight, spontaneous motor activity in the open field (OF) (movement distance, movement velocity, rearing frequency), and fecal corticosterone metabolites were conducted at baseline and at multiple times after partial hepatectomy. Subjective postsurgical severity assessments were conducted using modified score sheets. Blood parameters such as leukocyte count and serum aspartate aminotransferase, as well as estrogens and testosterone were measured from samples obtained during partial hepatectomy and at sacrifice. In addition, the amount of resected liver tissue was measured at partial hepatectomy, and the proliferated liver was weighed at sacrifice. RESULTS: Fecal corticosterone metabolite concentrations differed significantly between males and females at baseline and following hepatectomy. Also, leukocyte counts and estrogen concentrations were significantly different between sexes before partial hepatectomy. Alternatively, there were no sex differences in severity assessments, body weight changes, and behavior in the OF at any measurement time point. Liver weight was significantly different in males and females at the time point of partial hepatectomy and sacrifice. CONCLUSION: The results of this pilot study suggest that males and females respond similarly following partial hepatectomy. Examination of both sexes is very important for translation to humans, where both men and women suffer from liver disease. Furthermore, the use of both sexes in animal-based research would improve the utilization of the animal breeding in terms of the 3 Rs. However, due to some limitations, larger scale investigations including a broader spectrum of pathophysiolological, behavioral, and pharmacokinetic measures are planned.

Welfare Assessment on Healthy and Tumor-Bearing Mice after Repeated Ultrasound Imaging.

INTRODUCTION: Ultrasound (US) imaging enables tissue visualization in high spatial resolution with short examination times. Thus, it is often applied in preclinical research. Diagnostic US, including contrast-enhanced US (CEUS), is considered to be well-tolerated by laboratory animals although no systematic study has been performed to confirm this claim. Therefore, the aim of this study was to screen for possible effects of US and CEUS examinations on welfare of healthy mice. Additionally, the potential influence of CEUS and molecular CEUS on well-being and therapy response to regorafenib was investigated in breast cancer-bearing mice. MATERIAL AND METHODS: Forty healthy Balb/c mice were randomly assigned for examination with US or CEUS (3×/week) for 4 weeks. Untreated healthy mice and mice receiving only isoflurane anesthesia served as controls (n = 10/group). Ninety-four 4T1 tumor-bearing Balb/c mice were allocated randomly to the following groups: no imaging, isoflurane anesthesia, CEUS, and molecular CEUS. They either received 10 mg/kg regorafenib or vehicle solution daily by oral gavage. Animals were examined three times within 2 weeks. CEUS measurements were performed using phospholipid microbubbles, and phospholipid microbubbles targeting the vascular endothelial growth factor receptor-2 were applied for molecular CEUS. Welfare evaluation was performed by daily observational score sheets, measuring the heart rate, Rotarod performance, and fecal corticosterone metabolites twice a week. On the last day, pathological changes in serum corticosterone concentrations, hemograms, and organ weights were obtained. Moreover, a potential influence of isoflurane anesthesia, CEUS, and molecular CEUS on regorafenib response in tumor-bearing mice was examined. Analysis of variance and Dunnett's post hoc test were performed as statistical analyses. RESULTS: Severity parameters were not altered after repeated US and CEUS examinations of healthy mice, but spleen sizes were significantly lower after isoflurane anesthesia. In tumor-bearing mice, no effect on animal welfare after repeated CEUS and molecular CEUS could be observed. However, leukocyte counts and spleen weights of tumor-bearing mice were significantly lower in animals examined with CEUS and molecular CEUS compared to the control groups. This effect was not visible in regorafenib-treated animals. CONCLUSIONS: Repeated US and (molecular) CEUS have no detectable impact on animal welfare in healthy and tumor-bearing mice. However, CEUS and molecular CEUS in combination with isoflurane anesthesia might attenuate immunological processes in tumor-bearing animals and may consequently affect responses to antitumor therapy.

Severity Assessment in Rats Undergoing Subarachnoid Hemorrhage Induction by Endovascular Perforation or Corresponding Sham Surgery.

INTRODUCTION: Animal models for preclinical research of subarachnoid hemorrhage (SAH) are widely used as much of the pathophysiology remains unknown. However, the burden of these models inflicted on the animals is not well characterized. The European directive requires severity assessment-based allocation to categories. Up to now, the classification into predefined categories is rather subjective and often without underlying scientific knowledge. We therefore aimed at assessing the burden of rats after SAH or the corresponding sham surgery to provide a scientific assessment. METHODS: We performed a multimodal approach, using different behavior tests, clinical and neurological scoring, and biochemical markers using the common model for SAH of intracranial endovascular filament perforation in male Wistar rats. Up to 7 days after surgery, animals with SAH were compared to sham surgery and to a group receiving only anesthesia and analgesia. RESULTS: Sham surgery (n = 15) and SAH (n = 16) animals showed an increase in the clinical score the first days after surgery, indicating clinical deterioration, while animals receiving only anesthesia without surgery (n = 5) remained unaffected. Body weight loss occurred in all groups but was more pronounced and statistically significant only after surgery. The analysis of burrowing, open field (total distance, erections), balance beam, and neuroscore showed primarily an effect of the surgery itself in sham surgery and SAH animals. Only concerning balance beam and neuroscore, a difference was visible between sham surgery and SAH. The outcome of the analysis of systemic and local inflammatory parameters and of corticosterone in blood and its metabolites in feces was only robust in animals suffering from larger bleedings. Application of principal component analysis resulted in a clear separation of sham surgery and SAH animals from their respective baseline as well as from the anesthesia-only group at days 1 and 3, with the difference between sham surgery and SAH being not significant. DISCUSSION/CONCLUSION: To our knowledge, we are the first to publish detailed clinical score sheet data combined with advanced behavioral assessment in the endovascular perforation model for SAH in rats. The tests chosen here clearly depict an impairment of the animals within the first days after surgery and are consequently well suited for assessment of the animals' suffering in the model. A definitive classification into one of the severity categories named by the EU directive is yet pending and has to be performed in the future by including the assessment data from different neurological and nonneurological disease models.

Toward Evidence-Based Severity Assessment in Mouse Models with Repeated Seizures: (II.) Impact of Surgery and Intrahippocampal Kainate.

INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.

Effect of Different Ambient Temperatures on Reproductive Outcome and Stress Level of Lactating Females in Two Mouse Strains.

Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21 °C represents a chronic cold stress or the 30 °C of the thermoneutral zone constitutes heat stress for the animals. In this study, we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20 °C, 25 °C, and 30 °C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that B6D2F1 hybrid mothers weaned more pups compared to C57BL/6N mothers, and that the number of weaned pups was reduced when mothers of both strains were kept at 30 °C. Furthermore, at 30 °C, mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any temperature effects on adrenocortical activity in breeding and control mice. Independent of the ambient temperature, however, we found that females raising pups showed elevated levels of faecal corticosterone metabolites (FCMs) compared to controls. Peak levels of stress hormone metabolites were measured around birth and during the third week of lactation. Our results provide no evidence of an advantage for keeping lactating mice in ambient temperatures near the thermoneutral zone. In contrast, we found that a 30 °C cage temperature during lactation reduced body mass in females and their offspring and declined female reproductive performance.

Distress Analysis of Mice with Cervical Arteriovenous Fistulas.

The welfare of laboratory animals is a consistent concern for researchers. Its evaluation not only fosters ethical responsibility and addresses legal requirements, but also provides a solid basis for a high quality of research. Recently, a new cervical arteriovenous model was created in mice to understand the pathophysiology of arteriovenous fistula, which is the most commonly used access for hemodialysis. This study evaluates the distress caused by this new animal model. Ten male C57B6/J mice with cervical arteriovenous fistula were observed for 21 days. Non-invasive parameters, such as body weight, faecal corticosterone metabolites, burrowing activity, nesting activity and distress scores were evaluated at each time point. Six out of ten created arteriovenous fistula matured within the observation time as defined by an increased diameter. The body weight of all animals was reduced after surgery but recovered within five days. In addition, the distress score was significantly increased during the early time point but not at the late time point after arteriovenous fistula creation. Neither burrowing activity nor nesting behaviour were significantly reduced after surgical intervention. Moreover, faecal corticosterone metabolite concentrations did not significantly increase. Therefore, the cervical murine arteriovenous fistula model induced moderate distress in mice and revealed an appropriate maturation rate of the fistulas.

Diagnostic Ability of Methods Depicting Distress of Tumor-Bearing Mice.

Subcutaneous tumor models in mice are the most commonly used experimental animal models in cancer research. To improve animal welfare and the quality of scientific studies, the distress of experimental animals needs to be minimized. For this purpose, one must assess the diagnostic ability of readout parameters to evaluate distress. In this study, we evaluated different noninvasive readout parameters such as body weight change, adjusted body weight change, faecal corticosterone metabolites concentration, burrowing activity and a distress score by utilising receiver operating characteristic curves. Eighteen immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used for this study; half were subcutaneously injected with A-375 cells (human malignant melanoma cells) that resulted in large tumors. The remaining mice were inoculated with SCL-2 cells (cutaneous squamous cell carcinoma cells), which resulted in small tumors. The adjusted body weight and faecal corticosterone metabolites concentration had a high diagnostic ability in distinguishing between mice before cancer cell injection and mice bearing large tumors. All other readout parameters had a low diagnostic ability. These results suggest that adjusted body weight and faecal corticosterone metabolites are useful to depict the distress of mice bearing large subcutaneous tumors.

Lidocaine and bupivacaine as part of multimodal pain management in a C57BL/6J laparotomy mouse model.

While the use of local anesthesia as part of multimodal pain management is common practice in human and veterinarian surgery, these drugs are not applied routinely in rodent surgery. Several recommendations on the use of local anesthesia exist, but systematic studies on their efficacy and side effects are lacking. In the present study, male and female C57BL/6J mice were subjected to a sham vasectomy or a sham embryo transfer, respectively. We tested whether a mixture of subcutaneously injected Lidocaine and Bupivacaine in combination with systemic Paracetamol applied via drinking water results in superior pain relief when compared to treatment with local anesthesia or Paracetamol alone. We applied a combination of methods to assess behavioral, emotional, and physiological changes indicative of pain. Voluntary Paracetamol intake via drinking water reached the target dosage of 200 mg/kg in most animals. Local anesthesia did not lead to obvious side effects such as irregular wound healing or systemic disorders. No relevant sex differences were detected in our study. Sevoflurane anesthesia and surgery affected physiological and behavioral measurements. Surprisingly, Paracetamol treatment alone significantly increased the Mouse Grimace Scale. Taken together, mice treated with a combination of local anesthesia and systemic analgesia did not show fewer signs of post-surgical pain or improved recovery compared to animals treated with either local anesthesia or Paracetamol.

Additional Assessment of Fecal Corticosterone Metabolites Improves Visual Rating in the Evaluation of Stress Responses of Laboratory Rats.

Since animal experiments cannot be completely avoided, the pain, suffering, and distress of laboratory animals must be minimized. To this end, a major prerequisite is reliable assessment of pain and distress. Usually, evaluation of animal welfare is done by visual inspection and score sheets. However, relatively little is known about whether standardized, but subjective, score sheets are able to reliably reflect the status of the animals. The current study aimed to compare visual assessment scores and changes in body weight with concentrations of fecal corticosterone metabolites (FCMs) in a neuroscientific experimental setup. Additionally, effects of refinement procedures were investigated. Eight male adult Sprague-Dawley rats underwent several experimental interventions, including electroencephalograph electrode implantation and subsequent recording, positron emission tomography (PET), and sleep deprivation (SD) by motorized activity wheels. Additional 16 rats were either used as controls without any treatment or to evaluate refinement strategies. Stress responses were determined on a daily basis by means of measuring FCMs, body weight, and evaluation of the animals' welfare by standardized score sheets. Surgery provoked a significant elevation of FCM levels for up to five days. Increases in FCMs due to PET procedures or SD in activity wheels were also highly significant, while visual assessment scores did not indicate elevated stress levels and body weights remained constant. Visual assessment scores correlate with neither changes in body weight nor increases in FCM levels. Habituation procedures to activity wheels used for SD had no impact on corticosterone release. Our results revealed that actual score sheets for visual assessment of animal welfare did not mirror physiological stress responses assessed by FCM measurements. Moreover, small changes in body weight did not correlate with FCM concentration either. In conclusion, as visual assessment is a method allowing immediate interventions on suffering animals to alleviate burden, timely stress assessment in experimental rodents via score sheets should be ideally complemented by validated objective measures (e.g., fecal FCM measured by well-established assays for reliable detection of FCMs). This will complete a comprehensive appraisal of the animals' welfare status in a retrospective manner and refine stressor procedures in the long run.

A mechanism for population self-regulation: Social density suppresses GnRH expression and reduces reproductivity in voles.

Nearly 100 years ago, Charles Elton described lemming and vole population cycles as ecological models for understanding population regulation in nature. Yet, the mechanisms driving these cycles are still not fully understood. These rodent populations can continue to cycle in the absence of predation and with food supplementation, and represent a major unsolved problem in population ecology. It has been hypothesized that the social environment at high population density can drive selection for a low-reproduction phenotype, resulting in population self-regulation as an intrinsic mechanism driving the cycles. However, a physiological mechanism for this self-regulation has not been demonstrated. We manipulated population density in wild meadow voles Microtus pennsylvanicus using large-scale field enclosures over 3 years and examined reproductive performance and physiology. Within the field enclosures, we assessed the proportion of breeding animals, mass at sexual maturation, and faecal androgen and oestrogen metabolites. We then collected brain tissue from juvenile voles born at high or low density, quantified mRNA expression of gonadotropin-releasing hormone (GnRH) and oestrogen receptor alpha (ERα) and measured DNA methylation at six CpG sites in a region that was highly conserved with the mouse GnRH promoter. At high density, there was a lower proportion of reproductive animals. Juvenile voles born at high densities had reduced expression of GnRH in the hypothalamus, accompanied by marginally lower faecal sex hormone metabolites. Female juvenile voles born at high density also had higher methylation levels at two CpG sites while males did not, aligning with prior observations that females (but not males) from high-density environments retain reduced reproduction long term. Our results support a physiological basis for population self-regulation in vole cycles, as altering population density alone induced reproductive downregulation at the hypothalamic level. Our results demonstrate that altering the early-life social environment can fundamentally impact reproductive function in the brain. This, in turn, can drive population demography changes in wild animals.

Relationship between exploratory activity and adrenocortical activity in the black rat (Rattus rattus).

The relationship between physiological and behavioral stress markers is documented in several rodent species. However, there is no information regarding the role of adrenocortical activity in behavior of the black rat (Rattus rattus). Therefore, we hypothesize that the adrenocortical activity of black rats varies between individuals and is related to some of the behaviors in a novel environment. To test this hypothesis, we (i) validated a method for quantifying glucocorticoid metabolites from feces (fGCMs) with an enzyme immunoassay (EIA); (ii) examined variation and diurnal rhythms of feces and GCM production; and (iii) examined the relationship between GCM levels and exploratory behavioral traits. We fulfilled the first aim (i) by successfully performing an ACTH challenge test to validate the use of a 5α-pregnane-3ß,11ß,21-triol-20-one EIA for measuring fGCMs. Second (ii) we detected considerable consistent interindividual variability in production of both feces and glucocorticoids. The peak production of feces occurred in the first hour of the dark cycle, the peak of fGCMs occurred approximately 3 h later. Lastly, (iii) there was no clear relationship between behavior in the hole board test and GCMs. Grooming, a typical behavioral stress marker, was negatively associated with stress reactivity, while head-dipping in the hole-board test (traditionally considered an exploratory behavior independent of stress) was not correlated with the GCMs. This study offers a first look at GCMs in the black rat, successfully validates a method for their measurement and opens possibilities for future research of the relationship between glucocorticoids and exploratory behavior in this species.

MicroRNAs as systemic biomarkers to assess distress in animal models for gastrointestinal diseases.

Severity assessment of animal experiments is mainly conducted by using subjective parameters. A widely applicable biomarker to assess animal distress could contribute to an objective severity assessment in different animal models. Here, the distress of three murine animal models for gastrointestinal diseases was assessed by multiple behavioral and physiological parameters. To identify possible new biomarkers for distress 750 highly conserved microRNAs were measured in the blood plasma of mice before and after the induction of pancreatitis. Deregulated miRNA candidates were identified and further quantified in additional animal models for pancreatic cancer and cholestasis. MiR-375 and miR-203 were upregulated during pancreatitis and down regulated during cholestasis, whereas miR-132 was upregulated in all models. Correlation between miR-132 and plasma corticosterone concentrations resulted in the highest correlation coefficient, when compared to the analysis of miR-375, miR-203 and miR-30b. These results indicate that miR-132 might function as a general biomarker for distress, whereas the other miRNAs were altered in a disease specific manner. In conclusion, plasma miRNA profiling may help to better characterize the level of distress in mouse models for gastrointestinal diseases.

Severity assessment in mice subjected to carbon tetrachloride.

The Directive 2010/63 EU requires classifying burden and severity in all procedures using laboratory animals. This study evaluated the severity of liver fibrosis induction by intraperitoneal carbon tetrachloride (CCl4) injections in mice. 29 male C57BL/6N mice were treated three times per week for 4 weeks with an intraperitoneal injection (50 µl) of either 0.6 ml/kg body weight CCl4-vehicle solution, germ oil (vehicle-control) or handling only. Severity assessment was performed using serum analysis, behavioral tests (open field test, rotarod, burrowing and nesting behavior), fecal corticosterone metabolite (FCM) measurement, and survival. The most significant group differences were noticed in the second week of treatment when the highest AST (1463 ± 1404 vs. 123.8 ± 93 U/L, p < 0.0001) and nesting values were measured. In addition, respective animals showed lower moving distances (4622 ± 1577 vs. 6157 ± 2060 cm, p < 0.01) and velocity in the Open field, identified as main factors in principal component analysis (PCA). Overall, a 50% survival rate was observed within the treatment group, in which the open field performance was a good tracer parameter for survival. In summary, this study demonstrates the feasibility of assessing severity in mice using behavioral tests and highlight the open field test as a possible threshold parameter for risk assessment of mortality.

Influence of MRI Examinations on Animal Welfare and Study Results.

OBJECTIVES: Magnetic resonance imaging (MRI) is considered to be well tolerated by laboratory animals. However, no systematic study has been performed yet, proving this assumption. Therefore, the aim of this study was to investigate the possible effects of longitudinal native and contrast-enhanced (CE) 1-T and 7-T MRI examinations on mouse welfare as well as 4T1 breast cancers progression and therapy response. MATERIAL AND METHODS: Forty-seven healthy and 72 breast cancer-bearing mice (4T1) were investigated. One-Tesla (ICON) and 7-T (Biospec) MRI measurements were performed thrice per week under isoflurane anesthesia in healthy BALB/c mice for 4 weeks and 3 times within 2 weeks in tumor-bearing animals. Animal welfare was examined by an observational score sheet, rotarod performance, heart rate measurements, and assessment of fecal corticosterone metabolites. Furthermore, we investigated whether CE-MRI influences the study outcome. Therefore, hemograms and organ weights were obtained, and 4T1 tumor growth, perfusion, immune cell infiltration, as well as response to the multikinase inhibitor regorafenib were investigated. Statistical comparisons between groups were performed using analysis of variance and Tukey or Bonferroni post hoc tests. RESULTS: Mice showed no alterations in the observational score sheet rating, rotarod performance, heart rate, and fecal corticosterone metabolites (P > 0.05) after repeated MRI at both field strengths. However, spleen weights were reduced in all healthy mouse groups that received isoflurane anesthesia (P < 0.001) including the groups investigated by 1-T and 7-T MRI (P = 0.02). Neither tumor progression nor response to the regorafenib treatment was affected by isoflurane anesthesia or CE-MRI monitoring. Furthermore, immunohistological tumor analysis did not indicate an effect of isoflurane and MRI on macrophage infiltration of tumors, perfusion of tumor vessels, and apoptotic cell rate (P > 0.05). CONCLUSIONS: Repeated MRI did not influence the welfare of mice and did not affect tumor growth and therapy response of 4T1 tumors. However, systemic immunological effects of isoflurane anesthesia need to be considered to prevent potential bias.

Severity assessment using three common behavioral or locomotor tests after laparotomy in rats: a pilot study.

The aim of this pilot study was to evaluate whether behavioral or locomotor tests (Open Field (OF), rotarod (RR), and CatWalk (CW)) can help assess the severity of laparotomy in rats.The new EU Directive (2010/63/EU) mandates severity assessment in experiments involving animals. However, validated and objective methods are needed to relate trial-specific monitoring results to the degree of distress caused to individual animals. Therefore, we focused on non-invasive or minimally invasive, simple, and convenient severity assessment methods in a surgical model.To evaluate surgical severity in this model, we compared moving velocity among three commonly used behavioral test methods (OF, RR, and CW) after midline laparotomy within postoperative 7 days.In this study, 30 adult male Wistar Han rats (n = 10 per test) were trained in their assigned test method and subsequently subjected to surgery. Severity scoring was performed daily using a modified score sheet developed previously. In addition, blood and fecal samples were collected to analyze surgical and postoperative corticosterone metabolite levels. We found significant differences among the experimental groups in terms of the analyzed parameters. In this context, the OF test was found to be the most suitable method for severity assessment after laparotomy in rats.

Grading animal distress and side effects of therapies.

In order to combine high-quality research with minimal harm to animals, a prospective severity assessment for animal experiments is legally required in many countries. In addition, an assessment of the evidence-based severity level might allow realistic harm-benefit analysis and the appraisal of refinement methods. However, only a few examples describe the distress of animals by simple, cost-efficient, and noninvasive methods. We, therefore, evaluated the severity of an orthotopic mouse model for pancreatic cancer using C57BL/6J mice when pursuing two different chemotherapies. We assessed fecal corticosterone metabolites, body weight, distress score, and burrowing, as well as nesting activity. Moreover, we established a multifactorial model using multivariate logistic regression to describe animal distress. This multifactorial analysis revealed that metformin + galloflavin treatment caused higher distress than metformin + α-cyano-4-hydroxycinnamate therapy. Similar results were obtained by using the best cutoff calculated by Youden's J index when using only single parameters, such as burrowing activity or fecal corticosterone metabolite concentration. Thus, the present study revealed that single readout parameters, as well as multivariate analysis, can help to assess the severity of animal experiments and detect side effects of therapies.