MP4 shows an obvious tendency to reduce the area of no perfusion compared to the dextran and control group, but this is not significant. A reason for the absence of significance could be the surgical alteration of the anatomy due to incision of the ear. Compared to published data of other groups, we measured quite a small area of no perfusion. This could be explained by the use of young mice, which show a higher functional capillary density and a higher rate of anastomosis than older ones [20, 21].
In addition, repetitive application and a higher concentration of MP4 could be necessary. An interesting observation was the fact that the area of no perfusion in the MP4 group showed no increase after 48 h, whereas the areas in the other two groups extended between 48 and 72 h. An explanation could be the improvement in oxygenation due to MP4. The tissue in the area of no perfusion died in all three groups, but the tissue that received a minimum of perfusion survived in the MP4 group due to adequate oxygenation, whereas the oxygenation in the two other groups was insufficient and resulted in additional NPA 72 hours post induction of ischemia.
So far the influence of MP4 was investigated only in systemic ischemia as an alternative substitute for blood, which will probably be the main target in the future [13, 22–24]. In our model we investigated a locally induced ischemia in addition to the hemodilution. The values of less NPA and no further NPA after 48 h could possibly be improved by higher MP4 concentrations and repetitive applications; studies with a modified design are planned.
We could not find any morphologic changes in the kidneys in the sense of necrotic tubuli or sedimentation of proteins, which are often caused by free hemoglobin . This probably shows the positive effect of the chemical modification of free hemoglobin with polyethylene glycol, which enables the normovolemic hemodilution of about 1/3 of the blood volume in our model without causing any severe damage.
The significant increase in leukocytes in the MP4 and dextran group after 72 h could not be explained. The animals showed no alterations in behavior and had the same weight compared to the control group. The free hemoglobin could cause reactions of the immune system, but we would wonder about the low variance. Leukocytosis due to dextran is described in rats (colitis after drinking dextran sulphate sodium), in mice and rats after giving dextran sulphate [26–28]. In rhesus monkeys only a transient elevation of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase (LDH) was found 3 days after application of MP4, no elevation of leukocytes was detected . In humans a slight elevation of amylase and lipase could be detected [29, 30].
The intracardiac access reduced the mortality compared to central intravenous catheters in our laboratory and was easy to handle. One advantage of our model was the lack of hairs, which allowed a direct view of the xyphoid and facilitated orientation.
In summary, the oxygen carrier MP4 shows a clear tendency to reduce the extent of NPA and therefore necrosis compared to the dextran and control groups, and, in contrast to the other groups, the area of no perfusion in the MP4 group showed no increase after 48 h.
Further studies with different MP4 concentrations and repetitive applications should be conducted to find out whether giving MP4 instead of other commonly used substitutes definitely represents an advantage for flap survival.