Supplementary Material for: 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 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.

引言：超声（Ultrasound, US）成像可实现高空间分辨率的组织可视化，且检查耗时较短，因此常应用于临床前研究领域。尽管尚无系统性研究证实该结论，但包括对比增强超声（contrast-enhanced ultrasound, CEUS）在内的诊断超声被认为在实验动物中具有良好耐受性。因此本研究旨在筛查超声及对比增强超声检查对健康小鼠动物福利的潜在影响。此外，本研究还探讨了对比增强超声及分子对比增强超声对乳腺癌荷瘤小鼠的健康状况及瑞戈非尼（regorafenib）治疗应答的潜在影响。
材料与方法：将40只健康Balb/c小鼠随机分组，接受超声或对比增强超声检查（每周3次），持续4周。未接受任何处理的健康小鼠及仅接受异氟烷麻醉的小鼠作为对照组（每组n=10）。将94只4T1肿瘤荷瘤Balb/c小鼠随机分为以下组别：未行成像检查组、异氟烷麻醉组、对比增强超声组及分子对比增强超声组。所有小鼠每日通过口服灌胃给予10 mg/kg瑞戈非尼或赋形剂溶液。实验期间于两周内对小鼠进行3次检查。对比增强超声检查采用磷脂微泡完成，分子对比增强超声则使用靶向血管内皮生长因子受体-2（vascular endothelial growth factor receptor-2, VEGFR2）的磷脂微泡。动物福利评估采用每日观察评分表，每周两次测量心率、转棒实验（Rotarod）表现及粪便皮质酮代谢物水平。实验末期采集血清样本检测皮质酮浓度、完成血常规检测、称量器官重量，并观察各器官的病理改变。此外，本研究还评估了异氟烷麻醉、对比增强超声及分子对比增强超声对荷瘤小鼠瑞戈非尼治疗应答的潜在影响。统计分析采用方差分析及Dunnett事后检验。
结果：对健康小鼠重复进行超声及对比增强超声检查后，严重性相关参数未出现明显改变，但仅接受异氟烷麻醉的小鼠脾脏尺寸显著减小。在荷瘤小鼠中，重复接受对比增强超声及分子对比增强超声检查未对动物福利状态产生可观测到的影响。然而，与对照组相比，接受对比增强超声及分子对比增强超声检查的荷瘤小鼠白细胞计数及脾脏重量显著降低，该效应在接受瑞戈非尼治疗的小鼠中未被观察到。
结论：重复超声及（分子）对比增强超声检查对健康小鼠及荷瘤小鼠的动物福利状态无可检测到的影响。然而，对比增强超声及分子对比增强超声联合异氟烷麻醉可能会减弱荷瘤动物的免疫过程，进而可能对抗肿瘤治疗应答产生影响。