Cognitive dysfunction is sustained after rescue therapy in experimental cerebral malaria, and is reduced by additive antioxidant therapy

Abstract

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.

Figure 1. Time-course of parasitemia and survival rate of C57BL6, BALB/c and Swiss Webster (SW) mice after infection with PbA, Pch, PyNXL or PyXL (n = 12–20/group).

Date on parasitemia (A and B) are shown as mean ± SEM. Comparisons of C57BL6-PbA versus C57BL6-Pch (#), C57BL6-PbA versus BALB/c-PbA (*) and C57BL6-Pch versus BALB/c-PbA (+), were significant by Tukey's Multiple Comparison Test (A). (θ) and (∞) indicate p<0.05 in relation to day 3 post-infection during PyNXL and PyXL infection, respectively (B). Survival curves (C and D) were evaluated by Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.

Figure 2. Open field task analysis identifies mouse strains that are susceptible to CM after PbA infection.

C57BL/6 (A–D) and BALB/c (E and F) mice (n = 12–20/group) were infected with PbA (10⁶ PRBC i.p.) and treated with chloroquine (25 mg/kg) starting at day 6 post-infection. Data are expressed as mean ± S.E.M. of crossings (A, C, and E) and rearings (B, D and F) during training (gray bars) and test (black bars) sessions performed at days 15 and 16 (A,B,E, and F) or 30 and 31 (C and D) respectively; * significant difference between groups in training and test sessions (Student's T test, p<0.05 A and B, E and F; Mann Whitney test, p<0.05 to C and D).

Figure 3. Impaired performance in the open field task is associated with development of CM.

C57BL/6 mice (n = 12–20/group) were infected with PbA or Pch (10⁶ PRBC, A and B), or SW mice (n = 10–12/group) were infected with PyNXL or PyXL (10⁶ PRBC, C and D) and treated with chloroquine (25 mg/kg) starting at day 6 post-infection. Control groups were inoculated with the same number of uninfected RBC. Data are expressed as mean ± S.D. of crossings (A and C) and rearings (B and D) of training (gray bars) and test (black bars) sessions performed on days 15 and 16 post-infection respectively; *significant difference between groups in training and test studies (Student's T test, p<0.05).

Figure 4. Aversive memory is also affected by CM as detected by the inhibitory avoidance task.

C57BL/6 and BALB/c mice (n = 12–20/group) were infected with PbA (10⁶ PRBC) and treated with chloroquine starting at day 6 post-infection. Control groups were inoculated with the same number of uninfected RBC. (A) 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 on the bars. (B) 24 h later, 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 median latencies, in seconds; *Significant difference compared with uninfected controls (comparisons among groups were performed by Mann-Whitney U test; individual groups were analyzed by Wilcoxon tests, p<0.05).

Figure 5. Long term aversive memory is not affected by CM as detected by continuous multiple-trials step-down inhibitory avoidance.

C57BL/6 and BALB/c mice (n = 12–20/group) were infected with PbA (10⁶ PRBC) and treated with chloroquine starting at day 6 post-infection. Control groups were inoculated with the same number of uninfected RBC. On day 15 all the animals were submitted to a training session that consisted of a 0.3 mA 2.0 sec foot shock at the time that the animal stepped down on the grid. (A) Number of trials needed to achieve acquisition criterion (180 sec on the platform) one hour and thirty minutes after the training session. (B) Latency period on the platform 24 hours after the training session (cut-off at 180 seconds). Data are expressed as mean ± S.E.M. of the number of trials required to reach acquisition criterion (50 sec on the platform) in (A) and as individual values with median represented by a horizontal line in (B). *Significant difference between groups (comparisons among groups were performed by Mann-Whitney U test, the within individual groups were analyzed by Wilcoxon tests, p<0.05).

Figure 6. The ability to recognize new objects is impaired in mice after CM.

C57BL/6 and BALB/c mice (n = 12–20/group) were infected with PbA (10⁶ PRBC) and treated with chloroquine starting at day 6 post-infection. Control groups were inoculated with the same number of uninfected RBC. On day 15 all the animals were submitted to memory recognition task. Results are shown as mean ± S.E.M. of the object recognition index calculated as described in Materials and Methods. *p<0.05 Kruskal–Wallis analyses of variance followed by Mann–Whitney U-tests.

Figure 7. Oxidative stress is increased in the brains of mice with CM.

Oxidative damage was assayed by measuring TBARS (A, B, E and F) and conjugated diene (C and D) formation in brains on days 3 (A and C) and 6 (B, D, E and F) post-infection of C57BL/6 with PbA or Pch (10⁶ PRBC, n = 10/group, A–E) or BALB/c with PbA (10⁶ PRBC, n = 6, F). 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 according to Student's t-test.

Figure 8. Additive antioxidant treatment prevents cognitive impairment after CM.

C57BL/6 (A and B) and SW mice (C and D) (n = 12–20/group) were infected with PbA or PyXL, respectively (10⁶ PRBC). As a control, one group was inoculated with the same number of uninfected RBC (n = 6/group). Starting on day 6 post infection, infected mice were divided into 2 groups and treated orally with chloroquine (25 mg/kg b.w.), chloroquine plus desferoxamine (DFX, 20 mg/kg b.w., i.p.), chloroquine plus N-acetylcysteine (NAC, 20 mg/kg b.w., i.p.) or with the combination of chloroquine/DFX/NAC for 7 days. On day 15 and 16 post-infection all the animals were submitted to open field task training and test sessions, respectively. Data are expressed as mean ± S.E.M. of crossings (A and C) and rearings (B and D) 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). Panels E–M ilustrate histological examinations (H&E staining) of different brain regions in non-infected (RBC), infected (PRBC, PbA 10⁶) and mice treated with the combination of chloroquine/DFX/NAC (PbA+Cq+AOX). E–G) cerebral cortex; H–J) hippocampus; K–M) and cerebellum. Microvascular congestion and plugging (arrows) were detected in all analyzed regions in PbA-infected mice, but were not seen in controls or treated animals rescued with chloroquine and additive antioxidants. Scale bar, 100 µm.