Inflammatory changes in the central nervous system are associated with behavioral impairment in Plasmodium berghei (strain ANKA)-infected mice

Abstract

Experimental cerebral malaria is a neuroinflammatory condition that results from the host immune response to the parasite. Using intravital microscopy, we investigated leukocyte recruitment in the brain microcirculation and the temporal relationship of this process to the behavioral changes observed in Plasmodium berghei (strain ANKA)-infected C57Bl/6 mice. We found that leukocyte recruitment was increased from day 5 post-infection (p.i.) onwards. Histopathological changes and increased levels of inflammatory cytokines in the brain were also observed. Behavioral performance evaluated by the SHIRPA protocol showed functional impairment from day 6 p.i. onwards. Thus, early leukocyte migration into the brain and associated inflammatory changes may be involved in neurological impairment in parasite-infected C57Bl/6 mice.

Fig. 1

(A) Time course of parasitemia in infected mice (n=8). (B) Time course of survival of mice infected with PbA (n=8). (C) Weight variation in control (n=4) and infected (n=8) mice. Each point of parasitemia and weight loss is expressed as mean ± SEM.

Fig. 2

Visualisation of performance of control animals and PbA -infected animals in the five distinct functional categories (neuropsychiatric state; motor behavior; autonomic function; muscle tone and strength, and reflex and sensory function) (A–E). Overall scores of the functional categories are shown in groups of at least five mice. Scores of animals at different time points of infection and scores of control animals were compared by One-way ANOVA with Newman-Keuls post-test. *p<0.05; **p<0.01; ***p<0.001.

Fig. 3

Hemorrhages and intravascular inflammatory infiltrates in the brain of PbA-infected C57Bl/6 mice (H&E). (A and B) Hematoxylin and eosin-stained sections of cerebellum and cerebrum of control mice without morphological changes. (C and D) Cerebellum and cerebrum parenchyma without inflammatory cells and a typical architecture in mice on day 3 p.i. (E) Intravascular inflammatory cells and hemorrhagic areas in the cerebellum on day 5 p.i. (F) Cerebrum parenchyma with intravascular inflammatory infiltrates in the same date. (G) Multifocal hemorrhages in the cerebellum and neuronal damage on day 7 p.i. (H) Vasculat alteration with edema and intravascular inflammatory cells on day 7 p.i. Magnification, ×200.

Fig. 4

NAG levels in cerebral tissue from control and infected mice. Infected mice on day 5 p.i. showed increased NAG levels when compared with control. Groups of at least five mice and results are expressed as mean ± SEM, where *p<0.05.

Fig. 5

The study of leukocyte-endothelium interaction was performed by intravital microscopy. The rolling (A) and adhesion (B) of leukocytes in the brain microvasculature were assessed. The protocol included control (sham) and infected animals. Mice, infected by PbA, on day 5 p.i. showed an increase in cell recruitment when compared with control. Groups of at least five mice, results are expressed as mean ± SEM, where **p<0.01; ***p<0.001. ANOVA confirmed the significance.

Fig. 6

Comparative analysis of TNF-α, CCL2, CXCL1, CXCL9, CCL5 and CCL3 concentration in control and PbA-infected mice by ELISA. Results were expressed as the mean ± SEM from at least five animals per group. Asterisk(s) indicate statistical differences where *p<0.05, **p<0.01 and ***p<0.001.