Clinical, Physiologic, and Behavioral Evaluation of Permanently Catheterized NMRI Mice.

Vascular catheterization is becoming a popular technique in laboratory rodents, facilitating repetitive blood sampling and infusion in individual animals. In mice, catheterization is complicated by their small body size, which may increase the risk of postoperative complications that may both threaten catheter longevity and animal welfare. Less obvious complications to a permanent catheter may include subclinical infection, visceral tissue damage from disseminating microthrombi released from the catheter, and distress from being isolated from conspecifics and other experimental stressors. Such complications may go unnoticed and may affect animal welfare as well as confound research outcomes. This study investigated the implications of long-term arterial catheterization in NMRI mice by evaluating clinical, physiologic and behavioral parameters. Body weight and food and water consumptions were monitored during the study period. Fecal corticosterone metabolites were quantified as biomarkers of stress, and nucleic acid metabolites (8-oxo-7,8-dihydro-2'-deoxyguanisine and 8-oxo-7,8-dihydroguanosine) as biomarkers of oxidative damage. Behavioral dysfunction was studied by scoring animal welfare and nest building. Catheters were placed the right common carotid artery of mice; catheterized mice were compared with sham-operated and nonsurgical control mice. Except for an increase in the body weight of catheterized mice during the experimental period, clinical parameters (body weight and food and water consumptions) did not differ between groups. Physiologic parameters (oxidized nucleic acid metabolites and fecal corticosterone metabolites) were higher in control mice during the first week of experimentation compared with the end of study but did not differ between groups. Likewise, catheterization had no effect on behavioral parameters (nest building and animal welfare assessment). Long-term arterial catheterization of mice had no detectable implications on animal welfare in this study.

Applicability of Commercially Available ELISA Kits for the Quantification of Faecal Immunoreactive Corticosterone Metabolites in Mice.

BACKGROUND: Commercially available ELISA kits are popular among investigators that quantify faecal corticosterone or cortisol metabolites (FCM) for stress assessment in animals. However, in faeces, these assays mainly detect immunoreactive glucocorticoid metabolites. Since different assays contain antibodies of different origin, the detection level and cross-reactivity towards different metabolites and other steroids differ considerably between assays. Thus, the validity of one assay for FCM quantification in stress assessment is not necessarily the same for another assay. MATERIALS AND METHODS: The present study was designed to investigate corticosterone (CORT) in serum and FCM levels in faeces of laboratory mice, as quantified in four different ELISA kits (DRG EIA-4164, Demeditec DEV9922, Enzo ADI-900-097 and Cayman EIA kit 500655). Assay kits were chosen based on the origin of the antibody, detection level and variation in cross-reactivity. RESULTS: As expected, all four assay kits could detect higher serum CORT levels in mice treated with adrenocorticotropic hormone (ACTH), compared to untreated mice. Unexpectedly though, the measured concentration of serum CORT differed significantly between assays, in both groups of mice. In faecal samples, there was no consistent positive correlation between the levels detected in the four assays and the measured concentration of FCM also differed between assays. CONCLUSION: Whereas commercially available CORT ELISAs are frequently successfully used for FCM quantification, validation of the assays is necessary as all assays do not work well under all circumstances. In this study, the ELISAs could determine relative differences in serum CORT levels and FCM levels between samples; however, the fidelity of the measurements to the true concentrations was low.

Carotid Catheterization and Automated Blood Sampling Induce Systemic IL-6 Secretion and Local Tissue Damage and Inflammation in the Heart, Kidneys, Liver and Salivary Glands in NMRI Mice.

Automated blood sampling through a vascular catheter is a frequently utilized technique in laboratory mice. The potential immunological and physiological implications associated with this technique have, however, not been investigated in detail. The present study compared plasma levels of the cytokines IL-1ß, IL-2, IL-6, IL-10, IL-17A, GM-CSF, IFN-γ and TNF-α in male NMRI mice that had been subjected to carotid artery catheterization and subsequent automated blood sampling with age-matched control mice. Body weight and histopathological changes in the surgical area, including the salivary glands, the heart, brain, spleen, liver, kidneys and lungs were compared. Catheterized mice had higher levels of IL-6 than did control mice, but other cytokine levels did not differ between the groups. No significant difference in body weight was found. The histology revealed inflammatory and regenerative (healing) changes at surgical sites of all catheterized mice, with mild inflammatory changes extending into the salivary glands. Several catheterized mice had multifocal degenerative to necrotic changes with inflammation in the heart, kidneys and livers, suggesting that thrombi had detached from the catheter tip and embolized to distant sites. Thus, catheterization and subsequent automated blood sampling may have physiological impact. Possible confounding effects of visceral damage should be assessed and considered, when using catheterized mouse models.

Physiological and pathological impact of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy in laboratory mice.

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained.

Comparison of silicone and polyurethane catheters for the catheterization of small vessels in mice.

Vascular catheterization is increasingly carried out in laboratory mice, but the long-term patency of catheters implanted in mice is limited owing to their small size. The authors compared mice that were catheterized in their right common carotid artery either with a silicone catheter with a polyurethane tip or with a 100% polyurethane catheter to determine which catheter type was better suited for long-term studies in mice. The catheters were inspected daily and were flushed if blood was visible in the catheters; if no blood was visible, they were flushed every 3 d. Silicone catheters were patent for a shorter period of time than polyurethane catheters, which were patent for a median of 6 d and up to 25 d. The authors identify the principal causes of catheter failure and discuss how they can be prevented.

The effect of isoflurane anaesthesia and vasectomy on circulating corticosterone and ACTH in BALB/c mice.

The use of blood corticosterone and faecal corticosterone metabolites as biomarkers of post-surgical stress and pain in laboratory animals has increased during the last decade. However, many aspects of their reliability in laboratory mice remain uninvestigated. This study investigated serum corticosterone and adrenocorticotropic hormone (ACTH) in mice subjected to isoflurane anaesthesia and vasectomy, and mice subjected to isoflurane anaesthesia without surgery. Serum levels of corticosterone and ACTH after pre-treatment with dexamethasone were analysed to provide further information about the stress hormone profiles. Vasectomy resulted in an increase in corticosterone for at least four hours after surgery with a peak 30min after the mice regained righting reflex. Mice subjected to isoflurane anaesthesia without surgery had the highest level of serum corticosterone 5min after regained righting reflex and the level returned to baseline levels four hours after the procedure. In vasectomised mice, treated with dexamethasone, high levels of corticosterone remained 30min after the procedure, whereas the anaesthetised mice, treated with dexamethasone, had significantly lower levels of corticosterone compared to anaesthetised mice not treated with dexamethasone. Thus, dexamethasone effectively inhibited the corticosterone response in the anaesthetised-only mice, but not in the mice subjected to surgery. In conclusion, both isoflurane anaesthesia and vasectomy during isoflurane anaesthesia resulted in an increase in serum glucocorticoids, but the negative feedback mechanism of newly operated mice, was altered. This may have consequences for the interpretation of glucocorticoids measurements as a biomarker of post-surgical stress in mice.

The effect of automated blood sampling on corticosterone levels, body weight and daily food intake in permanently catheterized male BALB/c mice.

BACKGROUND/AIM: Automated blood sampling has the benefit of sampling without human intervention, thus minimizing the associated stress response. Since this technique has not been thoroughly investigated in mice, the present study was designed to evaluate this technology in mice. MATERIALS AND METHODS: Male catheterized BALB/c mice were subjected to automated blood sampling, fecal sampling and daily recording of body weight and food intake for three days post-surgery. Corticosterone levels in blood and feces were investigated as biomarkers of stress. RESULTS: Plasma corticosterone levels were elevated, and the circadian rhythm was disrupted as reflected in both plasma and feces. The body weight and daily food intake declined for the first two days post-surgery and increased at day three. DISCUSSION: These results demonstrate that surgery and subsequent automated blood sampling induce a stress response for up to three days post-surgery, and it is concluded that further refinement of this technique is essential.

Quantitative effects of diet on fecal corticosterone metabolites in two strains of laboratory mice.

The analysis of glucocorticoids excreted in feces is becoming a widespread technique for determining animal wellbeing in a wide variety of settings. In the present study an extraction protocol and an ELISA assay for quantifying fecal corticosterone metabolites (FCM) in BALB/c and C57bl/6 mice were validated. Lower ratios of solvent (ethanol) to mass of fecal sample were found to be sufficient in extracting FCM compared to what has been reported previously. Feeding mice a high energy diet, high in fat content (60% of calories from fat), significantly lowered the FCM excretion, approximately halving the FCM output. This diet also reduced the fecal mass voided to approximately a third of that of the regular diet. The two reductions were not correlated. A difference in defecation pattern was seen between the two strains, with the BALB/c mice having a more pronounced diurnal rhythm compared to the C57bl/6 mice. Furthermore, throughout the experiment, the C57bl/6 mice excreted significantly higher levels of FCM compared to the BALB/c mice. The mice were also challenged with synthetic adrenocorticotropic hormone (ACTH) and dexamethasone (DEX). The effect of the challenges could readily be detected, but had a considerably lesser impact on data than did the difference in diet. The study demonstrates some problematic consequences of expressing FCM excretion as a measure of fecal dry mass. The study also serves to emphasize the caution that must be exercised when interpreting FCM excretion in conjunction with an uncontrolled or varied diet, or perturbations of gastro-intestinal functioning.