The high lethality and post treatment neurological sequalae in patients with cerebral malaria demand new adjuvant therapies. The main finding of the present study is that whole blood transfusion resulted in substantial improvement of survival of mice with late-stage ECM treated with artemether.
Mice with ECM showed only mild to moderate (∼10%) decrease in hematocrit. However, artemether treatment resulted in further marked drops in hematocrit after 6 and 24 hours, a phenomenon also reported in human malaria 26, especially in non-immune travelers with hyperparasitemia 27. The fact that mice with ECM show parasitemia over 10% and parasites are rapidly killed by artemether may help to explain the rapid decrease in hematocrit following treatment.
Transfusion of 200µL of whole blood to mice with ECM resulted in improved hematocrit at both 6 and 24 hours. Maintenance of the hematocrit strengthens the oxygen-carrying capacity of the blood, which in a setting of cerebral ischemia may be a critical advantage for the patient. Fresh RBCs also improve the hemorheological properties, which is known to be deteriorated in severe malaria infections 28,29. And finally, lower hematocrit results in decreased vascular wall shear stress 30, with decreased eNOS activity, which leads to worsened endothelial function 31. Therefore, increasing hematocrit through whole blood transfusion should help restore endothelial function 32,33, help clear vessels blocked by parasitized erythrocytes and inflammatory cells, increasing tissue perfusion and decreasing acidosis 19,34,35 as well as immune cell-mediated endothelial damage. The effects on endothelial function are supported by the observation that blood transfusion prevented worsening of BBB breakdown and restored Ang-2 levels. Indeed, decreased levels of Ang-1 and increased levels of Ang-2, disturbing endothelial quiescence with loss of vascular health 8 7, have been associated with pediatric severe malaria 10,36,37. Since Ang-2 has been proposed as a risk factor for cognitive injury in pediatric CM, this finding is of critical importance, indicating that fresh blood rapidly counteracts the ECM-related inflammation and vascular insult. These findings are in line with data showing that interventions that counteract vascular dysfunction and inflammation are beneficial in ECM 25,32,33,38−40.
Thrombocytopenia is also one of the risk factors for mortality in African children with falciparum malaria 41. Whole blood transfusion induced significant upturn in circulating platelets in mice with ECM. The availability of fresh, quiescent platelets, could help restoring the normality of the coagulation system without the deleterious inflammatory actions of activated platelets 42. This effect of partially restoring the platelet counts in 24 hours cannot be ascribed only to a passive, repository effect due to the platelets present in the limited amount of transfused blood. Therefore, it is apparent that blood transfusion stimulates the body to actively respond, increasing platelet production. Other effects such as improved BBB response and decreased splenic congestion also support an active modulatory, rather than just repository, effect of blood transfusion.
Acute and severe hemolysis usually leads to a consumption of haptoglobin, as seen in severe malaria 43. In mice with ECM, anemia was only mild to moderate whereas inflammation is overwhelming, and this might help to explain why haptoglobin levels were high, since haptoglobin is an acute phase protein that increases with conditions such as inflammation and infection 44. On the other hand, a hemolytic event might also help to explain the decrease in haptoglobin levels following artemether treatment. Blood transfusion did not seem to interfere with haptoglobin levels following artemether treatment.
In this study, 10mL/kg (200µL) of blood was administered, which is half the usual amount given for severe anemia, because mice with ECM showed only mild to moderate anemia and also because malaria induces splenic congestion and a sudden increase in hematocrit might exacerbate this condition. Interestingly, blood transfusion actually helped to decrease the weight of enlarged spleen in mice with ECM, suggesting that it helped to decrease congestion. It is possible that fresh red blood cells improved blood flow throughout the body, improving overall hemodynamics and decreasing the burden at the spleen. Indeed, in sickle cell disease acute splenic sequestration is treated by RBC transfusion 45. The increase in spleen weight in mice with ECM was paralleled by an increase in the total splenocyte population. However, the magnitude of the increase in total splenocyte population was much lower than the increase in spleen weight (20% versus 200%), indicating that congestion with accumulation of blood-circulating cells was the major reason for the increased spleen weight. The vast majority of the increase in splenocyte numbers was in the B cell compartment, in line with previous findings 46. Although there was a change in the dynamics of different splenocyte populations in mice with ECM and after treatment with artemether, blood transfusion had no effect on the outcomes of each cell population.
The benefit of blood transfusion, however, on hematological and vascular parameters in mice with ECM treated with artemether was associated with a marked improvement in survival. These findings are in line with a recent prospective multicenter observational study showing that blood transfusion improved survival of children hospitalized with severe falciparum malaria 19,20. Blood transfusion is currently recommended by the WHO in severe malaria when hemoglobin is below 4.0g/dL, or below 6.0g/dL when associated with complications such as acidosis and coma. However, the authors showed that when signs of vital organ hypoperfusion are present, blood transfusion can benefit patients even at higher hemoglobin thresholds (7.7g/dL or even higher in case of impaired consciousness or severely elevated lactate concentration) 19. For patients with higher hemoglobin levels, our study in mice indicates that even the transfusion of half the usual blood volume can be of great benefit.
In conclusion, the transfusion of whole blood as an adjuvant therapy in ECM showed promising results and conduction of additional studies to prove the potential of this strategy as a viable, cheap and effective adjunctive therapy for cerebral malaria is therefore worth pursuing.