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

Analgesic and anesthetic drugs may have an impact on the results achieved from animal experiments. In the study presented here, we try to enlighten whether anesthesia with fentanyl/fluniasone and midazolam (Hypnorm and Dormicum) has an influence on measurements of hemoglobin in mice. In a cross-over study, we have compared hemoglobin levels in two groups of mice: anesthetized versus non-anesthetized and found significant decrease in hemoglobin levels in the anesthetized group (p  < 0.05) unrelated to which group received the anesthesia. The mean hemoglobin levels after intraperitoneal administration of Hypnorm and Dormicum was 8.7 mmol/L compared to mean hemoglobin 9.9 mmol/L before anesthesia (p  < 0.001), and the decrease lasted for more than 30 min. These results show that anesthesia can be an important confounder in studies involving measurements of hemoglobin, and this should be taken into account when planning studies and analyzing data. Key Words: anesthesia - hemoglobin - in vivo - fentanyl - midazolam Introduction

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.

Material and Methods Animals

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.

Anesthesia

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.

Measurement of the Hemoglobin Levels

Hemoglobin was analyzed in millimole per liter from blood samples using a HemoCue Hb201+ (HemoCue, AB, Sweden). A hematocrit straw was inserted in the medial angle of the eye, directed along the bony wall and the plexus retro-orbitalis was punctured. A drop of blood was placed on a HemoCue slide and analyzed immediately.

Design of Cross-Over Study The design of the study is visualized in Table 1. Twelve mice were divided in two groups A and B. Blood samples were collected at day 0 and from then once a week. The first four blood samples were collected during anesthesia in group A and without anesthesia in group B. From the fifth blood sample and to termination of the study, blood samples were collected without anesthesia in group A but during anesthesia in group B.