Recombinant human erythropoietin increases survival and reduces neuronal apoptosis in a murine model of cerebral malaria

Abstract

Background: Cerebral malaria (CM) is an acute encephalopathy with increased pro-inflammatory cytokines, sequestration of parasitized erythrocytes and localized ischaemia. In children CM induces cognitive impairment in about 10% of the survivors. Erythropoietin (Epo) has - besides of its well known haematopoietic properties - significant anti-inflammatory, antioxidant and anti-apoptotic effects in various brain disorders. The neurobiological responses to exogenously injected Epo during murine CM were examined.

Methods: Female C57BL/6j mice (4-6 weeks), infected with Plasmodium berghei ANKA, were treated with recombinant human Epo (rhEpo; 50-5000 U/kg/OD, i.p.) at different time points. The effect on survival was measured. Brain pathology was investigated by TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labelling), as a marker of apoptosis. Gene expression in brain tissue was measured by real time PCR.

Results: Treatment with rhEpo increased survival in mice with CM in a dose- and time-dependent manner and reduced apoptotic cell death of neurons as well as the expression of pro-inflammatory cytokines in the brain. This neuroprotective effect appeared to be independent of the haematopoietic effect.

Conclusion: These results and its excellent safety profile in humans makes rhEpo a potential candidate for adjunct treatment of CM.

Figures

Figure 1

Dose depandant increase in survival in recombinant human Erythropoietin-treated mice with cerebral malaria. Dose dependant increase in survival in recombinant human erythropoietin-treated mice with CM: Cumulative survival analysis of mice ECM treated with rhEpo from day 4–7 in different doses from 1–200 U daily versus vehicle treated controls. Mice treated with a high dose (50–200 U daily, red lines) show increased survival by the end of the second week of 44.4%, 42.9% and 55.6% respectively while all saline treated controls (black dotted line) survive no longer than day 11 (p < 0.001). Mice treated with 25 U daily survive in 30% (p < 0.001 vs. vehicle). Survival of mice treated with a low dose (1–10 U daily) did not show a statistically significant increase in survival. Statistical test: log rank statistic for the survival curves with pair wise comparison using the Holm-Sidak method.

Figure 2

Packed cell volume in recombinant human erythropoietin-treated and untreated mice with cerebral malaria. Packed cell volume (PCV) measured on day 8 in animals treated on day 4–7 or 1–7 respectively and vehicle treated controls: Treatment with recombinant human erythropoietin (rhEpo) increases PCV levels in mice with cerebral malaria. 1 U daily from day 4–7 increases the PCV to 51% while 10 U or more daily given on day 4–7 lead to mean PCV levels between 58.4% and 59.7%. PCV levels in the group treated on day 1–7 with 100 U daily reached very high values of 73.0% unless these mice where bled for 10% of their total blood volume on day 5. This intervention reduced the mean PCV level to 58.6% (right plot). P < 0.001. Statistical test: One way ANOVA. The ends of the boxes define the 25th and 75th percentiles, with a line at the median and error bars defining the 10th and 90th percentiles.

Figure 3

Time depandant increase in survival in recombinant human erythropoietin-treated mice with cerebral malaria. Time dependant increase in survival in mice with CM treated with recombinant human Erythropoietin (rhEpo): The cumulative survival analysis of mice ECM treated with 100 U daily of rhEpo show increased survival at the end of the second week only in mice treated from day 4–7 (42.9%, p < 0.001 vs. vehicle). The other treatment schemes used (day 1–4; day 7–10; day 1–7) and mice that received treatment from day 1–7 and where bleed for 10% of their total blood volume to prevent excessively high levels of packed cell volume did not show a statistically significant increase in survival. Statistical test: log rank statistic for the survival curves with pair wise comparison using the Holm-Sidak method.

Figure 4

Mean parasitaemia levels on day 8. Mean parasiteaemia levels on day 8 as percentage of parasitized red blood cells in Giemsa-stained smears: The mean values for the different groups range from 6.3 – 12.6%. Only the treatment group that received recombinant human Erythropoietin 25 U/day from day 4–7 (**) was significantly different from the control group that had received normal saline (NaCl) (P < 0.05). All other differences between the groups were not significant. Statistical test: One way ANOVA. Data are presented as mean values +/- standard error.

Figure 5

Body temperature of mice with cerebral malaria. Vehicle treated mice (left graph) show a characteristic drop of body temperature on day 9. Surviving mice in the group treated with recombinant human Erythropoietin 200 U daily on day 4–7 show a similar drop on day 9–10, but recover afterwards (right graph). The data shown are representative for repeated experiments. The data presented are from one single experiment. Data for the treatment group is representative for mice that received rhEpo 50–200 U daily on day 4–5.

Figure 6

Gene expression in the brains of recombinant human erythropoietin-treated and untreated mice with cerebral malaria. Gene expression in the brains of recombinant human erythropoietin-treated and untreated mice with cerebral malaria on day 8 relative to uninfected control mice (black columns). Increased gene expression in infected mice was seen for IL-1β, TNF, INF-γ (P < 0.001) and Caspase 1 (P < 0.05), but not for LT-α, and Caspase 3. Gene expression for the IL-β, TNF and INF-γ was significantly reduced in rhEpo treated mice (P < 0.001). The treatment does not alter the expression of LT-α and the Caspases 1 and 3. Statistical test: Two way ANOVA. Values are presented as mean values relative to uninfected controls. Error bars: Standard deviation (** = P < 0.001).

Figure 7

Treatment with recombinant human erythropoietin in mice with cerebral malaria reduces neuronal apoptosis in the brain. Apoptotic neurons in the brain of mice with ECM. The micrograph shows TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labelling) positive nuclei of cells in the cortex of a terminally ill mouse with ECM. Earlier work had shown that the round shaped nuclei belong to neurons [24]. The mean number of TUNEL+ neurons in the brains of recombinant human Erythropoietin (rhEpo) treated mice with ECM is significantly lower than in vehicle treated controls (P = 0.006). Countings from TUNEL-stained sagittal sections of both hemispheres. Scale bar: 50 μm. Statistical analysis: Students t-test. The ends of the boxes define the 25th and 75th percentiles, with a line at the median and error bars defining the 5th and 95th percentiles.