ChangeinHemoglobinLevelsduetoAnesthesiainMice2

Table 1 The design of the cross-over study

Group A had the first four blood samples collected during anesthesia, whereas group B served as controls. From sample number 5 and to the end of the study group B received anesthesia and group A served as controls

Design of the Time-Course Study

To investigate the levels of hemoglobin over time in anesthetized and non-anesthetized animals, a time-course study was performed. Mice were randomly divided in two groups and had pre-study values of hemoglobin measured at time t?=??10 min. Group 1 (N?=?8) was subsequently anesthetized as previously described, whereas group 2 (N?=?6) received no anesthesia. The injection of anesthesia was considered as t?=?0 min, and blood samples were collected from both groups at t?=?10, 20, and 30 min.

An additional study with the same set-up but with continued blood sampling to t?=?105 min after injection of anesthesia was carried out to validate the results (N?=?6).

Statistical Methods

Data from the cross-over study and from the time-course study was analyzed using a repeated measurements model (statistical software SAS version 9.1). In the time-course study, pre-study hemoglobin level was regarded as baseline. P values < 0.05 were considered significant.

Results

Cross-Over Study

In Fig. 1, the results from the cross-over study are depicted. At the first four measurements of hemoglobin, group A received anesthesia prior to the blood sampling and had significantly lower hemoglobin levels compared to group B, which received no anesthesia (p?<?0.05, and p?<?0.001 at blood samples 1 and 3, respectively). From blood sample 5 and to the termination of the study, the treatments were crossed over, and group B received anesthesia prior to the blood sampling. At blood samples 5–9, group B had significantly lower hemoglobin compared to group A (p?<?0.001, 0.05, 0.001, 0.05, and 0.001, respectively). The drop in hemoglobin in group B?+?anesthesia at blood sample 5 is mainly due to a single mouse with hemoglobin of 6.4 mmol/L (mean, 8.15 mmol/L).

Fig. 1 Illustration of the hemoglobin measurements (cross-over study). At blood samples 1–4, group A received anesthesia and group B served as control. At blood samples 5–9, group A served as control and group B received anesthesia. The mean values?+?SD are presented. *P?<?0.05, **P?<?0.01, ***P?<?0.001.

Time-Course Study

At baseline (pre-study blood sample, t?=??10 min), there was no significant difference between the anesthetized (group 1) and the control group (group 2; Fig. 2). At the time point t?=?10, 20, and 30 min, the difference between the groups was significant with significance levels of p?<?0.001.

Fig. 2 Repeated blood samples over time (time-course study). Initially, both groups had blood samples collected without anesthesia (time?=??10 min), and no significant difference was observed. At time?=?0, group 1 received intraperitoneal injection of Hypnom/Dormicum. Subsequently, hemoglobin was measured at different time points, and the anesthetized group (group 1) had hemoglobin levels significantly lower than the non-anesthetized mice (group 2). *P?<?0.05, **P?<?0.01, ***P?<?0.001.