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.
ObjectivesComputed 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 MethodsMice 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.ResultsAccording 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.ConclusionRepeated (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.
Riboflavin carrier protein (RCP) and riboflavin transporters (RFVTs) have been reported to be highly overexpressed in various cancer cells. Hence, targeting RCP and RFVTs using riboflavin may enhance tumor accumulation and internalization of drug delivery systems. To test this hypothesis, butyl-based 3-arm peptostar polymers were synthesized consisting of a lysine core (10 units per arm) and a sarcosine shell (100 units per arm). The end groups of the arms and the core were successfully modified with riboflavin and the Cy5.5 fluorescent dye, respectively. While in phosphate buffered saline the functionalized peptostars showed a bimodal behavior and formed supramolecular structures over time, they were stable in the serum maintaining their hydrodynamic diameter of 12 nm. Moreover, the polymers were biocompatible and the uptake of riboflavin targeted peptostars in A431 and PC3 cells was higher than in nontargeted controls and could be blocked competitively. In vivo, the polymers showed a moderate passive tumor accumulation, which was not significantly different between targeted and nontargeted peptostars. Nonetheless, at the histological level, internalization into tumor cells was strongly enhanced for the riboflavin-targeted peptostars. Based on these results, we conclude that passive accumulation is dominating the accumulation of peptostars, while tumor cell internalization is strongly promoted by riboflavin targeting.
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 ultrasound (CEUS), are 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: 40 healthy Balb/c mice were randomly assigned for examination with US or CEUS (3x/week) for four 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 two 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 per week. On the last day, pathological changes of 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 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 anti-tumor therapy.
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