Change in Hemoglobin Levels due to Anesthesia in Mice: An Important Confounder in Studies on Hematopoietic Drugs

When performing studies involving laboratory animals, appropriate anesthesia is warranted although certain drugs may influence on the results.It has been shown that anesthetized mice have a decreased respiratory rate and may develop hypoxia, hypercapnia, and acidosis [1], all of which theoretically can affect the data depending of aim and design of the study.In our unit, we use the combination of Hypnorm (fentanyl/fluniasone), which is a neuroleptanalgesia, and a sedative Dormicum (midazolam). This combination provides sufficient degree of anesthesia and analgesia but has side effects as shown by Whelan et al. [1].During studies with gene electrotransfer to muscle [2,3] and skin (Gothelf et al, submitted for publication) with erythropoietin (EPO) involving sequential blood sampling over time, we have discovered some inconsistency regarding the blood samples. At the end of the study, an unexpected decrease in hemoglobin levels compared to earlier levels was observed, the only obvious difference being that the animals were anesthetized.We thus planned two studies to enlighten whether the anesthesia has an influence on the measurement of hemoglobin.Female NMRI mice (own breed, Copenhagen University Hospital Herlev), 15 weeks old, were used in this study. The animals were kept in a pathogen-free environment with 12 h/12 h light/darkness cycle and food and water ad libitum. The study was performed with permission from the Danish Animal Experiments Inspectorate and in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimentations.All mice were humanely euthanized with quick cervical dislocation at the termination of the study.When applied, the anesthesia was Hypnorm (0.4 ml/kg, Janssen Saunderton, Buckinghamshire, England) and Dormicum (2 mg/kg, Roche, Basel, Switzerland) diluted in sterile water. The anesthesia was administered as intraperitoneal injections.Hemoglobin was analyzed in millimole per liter from blood samples using