Statins decrease neuroinflammation and prevent cognitive impairment after cerebral malaria

Abstract

Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Lovastatin treatment prevents contextual and aversive memory impairment after CM.

C57BL/6 mice (n = 12–20/group) were infected with PbA (106 PRBC). As a control, one group was inoculated with the same number of uninfected RBC (n = 6–12/group). Starting on day 6-post infection, uninfected and PbA-infected mice were divided into 2 groups and treated orally with chloroquine (25 mg/kg b.w.), or with the combination of chloroquine/lovastatin for 7 days. On days 15 and 16 post-infection all the animals were submitted to open field training and test sessions (A–B), and to inhibitory avoidance sessions tasks (C–D). Data are expressed as mean ± SEM of crossings (A) and rearings (B) in training (gray bars) and test (black bars) sessions. *Significant difference between groups in training and test sessions (p<0.05, Student's t test). C–D: animals were subjected to a training session in which the latency time on the platform is recorded when an electrical shock is given immediately after the mice step down onto the bars. (C) 1.5 (Short-term memory) and (D) 24 h (long-term memory) aversive memory was tested by recording the latency time on the platform (with a cut-off of 180 sec). Data are expressed as individual values and horizontal lines represent the mean of latency, in seconds; *Significant difference compared with uninfected controls and between infected groups (comparisons among groups were performed by Mann-Whitney U test, p<0.05).

Figure 2. Effect of lovastatin on parasitemia and survival rate after infection with PbA (n = 12–20/group).

Data on parasitemia (A) are shown as mean ± SEM. On day 6 post-infection mice were separated in two groups and one received lovastatin (10–40 mg/kg b.w.) daily for 7 days. Comparisons of C57BL6-PbA versus C57BL6-PbA treated animals (*) were significant by Mann-Withney Comparison test. There was no difference in parasitaemia when PbA and PbA-lovastatin treatment were compared (Mann-Withney Comparison test). (B) No differences in survival were observed among the groups (Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests).

Figure 3. Lovastatin treatment prevents impairment of short and long-term aversive memory after sepsis.

C57BL/6 mice (n = 5–10/group) were inoculated with fecal supernatant (2.5 mg/g b.w.). For control, one group was inoculated with saline (0.9%). Control and mice challenged with fecal supernatant were divided and one group was treated with lovastatin (20 mg/kg b.w.) 1 hour prior to feces or saline injection respectively, and every 24 h for 3 days. All the animals received imipenem (10 mg/kg b.w.) 6 hours after inoculation and every 24 h for 3 days. On day 15 all animals were subjected to a training session of inhibitory avoidance task, where the latency time on the platform is recorded and an electrical shock is given immediately after the mice step down onto the bars. (A) 1.5 (Short-term memory) and (B) 24 h (long-term memory) aversive memory was then tested by recording the latency time on the platform (with a cut-off of 180 sec). Data are expressed as individual values and horizontal lines represent the mean of latency, in seconds; significant difference compared between saline versus feces injected mice (comparisons among groups were performed by Mann-Whitney U test, *p

Figure 4. Lovastatin improves microvascular function and decreases leukocyte rolling and adhesion during PbA infection.

A–J: representative images of fluorescence intravital microscopy of pial vessels of uninfected mice (A and B; Magnification 100×) or infected mice (C and D) treated with vehicle or lovastatin, respectively (Magnification 40×). Venules with adherent and rolling rhodamine-labeled leukocytes in uninfected mice (E and F) or infected mice (G and H) treated with vehicle or lovastatin, respectively (Magnification 200×). Rhodamine-labeled leukocytes were associated with the venular endothelium in animals treated with vehicle (G) whereas they were largely free in the blood stream in lovastatin-treated mice (H). Fluorescent Plasmodium berghei (GFP)-infected RBC in venules of mice treated with vehicle (I) or lovastatin (J) (Magnification 200×). Mean ± SEM of functional capillary density (K), of rolling-rhodamine labeled leukocytes (L), and of number of adherent leukocytes (M) in pial venules (n = 6/group); *p<0.05 in relation to control (RBC) and vehicle-treated groups (Bonferroni's Multiple Comparison Test) and between PbA and PbA-lovastatin group (Bonferroni's Multiple Comparison Test; Scale bar, 100 µm).

Figure 5. Lovastatin treatment decreases ICAM-1 and CD11b expression and vascular permeability and induce HMOX-1 expression in the brains of mice with CM.

Panels illustrate histological examinations of cerebral cortex on day 6 post-infection, immunostained for ICAM-1 (brown and arrow) and counterstained with hematoxylin-eosin. Brain histology was examined using tissue from the following groups of animals: uninfected (A), uninfected treated with lovastatin (B), PbA-infected (C), and PbA-infected treated with lovastatin (D). Vascular congestion and edema (*) were observed in all PbA-infected mice, but were not seen in controls or treated animals. Scale bar: 50 µm. E: Dose-response relationships for effects of lovastatin on edema formation measured by Evans Blue Dye accumulation in the brain tissue on day 7 post-infection. F–H: ICAM-1, CD11b, and HMOX-1 expression in brain samples evaluated by semi-quantitative PCR on day 6 post-infection; *p

Figure 6. Lovastatin treatment reduces pro-inflammatory cytokine levels in the brains of animals with CM.

IL-1β (A), TNF-α (B), IL-12 (C) levels were determined by ELISA in brains of mice 6 days after infection with PbA (106 PRBC, n = 6–10/group). Lovastatin (10–40 mg/kg) was orally administered on day 6 post-infection together with chloroquine (25 mg/kg). *p<0.05 or less (Tukey's Multiple Comparison test) in relation to non-infected group and #p<0.05 or less in relation to PbA-infected group (Mann-Withney U Test).

Figure 7. Lovastatin treatment ameliorates oxidative stress in the brains of mice with CM.

Oxidative stress was assayed by measuring MDA formation (A) and free thiols levels (B) in brains 6 days post-infection of mice with PbA (106 PRBC, n = 10–15/group). Control groups received the same number of uninfected RBC (106). Results are expressed as mean ± S.E.M. and * represents p<0.05 compared to RBC group and # in relation to PbA-infected group, according to Tukey's Multiple Comparison Test.