Reactive changes of retinal microglia during fatal murine cerebral malaria: effects of dexamethasone and experimental permeabilization of the blood-brain barrier

Abstract

Microglial activation and redistribution toward blood vessels are some of the earliest observable events occurring within the central nervous system (CNS) during fatal murine cerebral malaria (FMCM). To investigate stimuli that might modulate microglial reactivity during FMCM we have performed two experimental manipulations and observed microglial responses in retinal whole mounts. First, to determine whether increased blood-brain barrier (BBB) permeability in the absence of the malaria parasite initiates the microglial changes, BBB function was compromised experimentally by intracarotid injection of arabinose and retinae were examined 12, 24, or 36 hours later. Second, to determine whether the immune response against the malaria parasite modulates microglial reactivity, infected mice were treated with dexamethasone before day 4 postinoculation. This treatment regime ameliorates cerebral complications without affecting parasite growth. We observed that increased BBB permeability was sufficient to elicit thickening of microglial processes and redistribution of microglia toward the vasculature, characteristic of the early stages of FMCM. However, despite the presence of plasma constituents in the CNS for up to 36 hours, microglia with amoeboid and vacuolated morphology were not observed. Dexamethasone treatment inhibited the up-regulation of alpha-D-galactose expression and reactive morphological changes in microglia during FMCM. These results suggest that disruption of the CNS milieu by entry of plasma constituents, or circulating malaria parasites in the absence of an immune response, by themselves are insufficient to induce the reactive microglial changes that are characteristic of FMCM. In addition, dexamethasone-sensitive event(s), presumably associated with immune system activation, occurring within the first few days of malaria infection are essential for the development of reactive microglia and subsequent fatal neurological complications.

Figure 1.

Micrographs show the amelioration of morphological changes of microglia in dexamethasone-treated FMCM mice at day 7 p.i. A−C: Microglia visualized with the GS lectin (A) and NDPase histochemistry (B), and the density of GS lectin-labeled microglia (C) at the terminal stage of FMCM. D−F: FMCM mice treated with dexamethasone on days 3 and 4 p.i. There was a marked reduction in the average density of microglia visualized using the GS lectin (compare C with F). This reduction in density was concomitant with a reduction in the extent of microglial morphological changes visualized with the GS lectin (D) and NDPase (E) histochemistry. Intensely stained GS lectin- and NDPase-labeled microglia with retracted processes and enlarged somas were still evident. However, these changes were mild compared with those in FMCM mice not treated with dexamethasone. G−I: FMCM mice treated with dexamethasone at days 0 and 1 p.i. The average density of GS lectin-labeled microglia (I) was similar to uninfected mice (21 versus 17 cells/mm²). Similarly, microglia labeled with the GS lectin had a morphology typical of resting microglia (G). NDPase-labeled microglia also showed a typical morphology of resting microglia (H). However, the number of microglia visualized was greater than the number of GS lectin-labeled microglia (compare arrows in G and H), because NDPase labels the total microglial population. C, F, and I: Retinal maps showing the density of GS lectin-labeled microglia from FMCM mice treated with dexamethasone. The number of labeled microglia was determined within a 1-mm region, and the cells/mm were converted to a gray scale. Each map is of a single retina representative of the changes observed. Each micrograph is representative of 6 mice.

Figure 2.

Microglial response to an infarct (A), a single injection of saline containing 1.6 mol/L arabinose, 2% (w/v) Evans blue, and 1 μg/ml polymyxin B (D, F, and H) and saline control containing 2% (w/v) Evans blue and 1 μg/ml polymyxin B (C, E, and G). Retinae were prepared for nucleoside diphosphatase (NDPase) histochemistry at 12 (C and D), 24 (E and F), or 36 (G and H) hours p.i. A: NDPase-labeled retinal microglia devoid of processes in mice with an experimentally-induced infarct (arrow). B: NDPase-labeled microglia in the contralateral retina at 36 hours post-i.c. injection of arabinose (arrow). As with contralateral retinae at 12 and 24 hours, there were no major changes in microglia morphology or distribution. C, E, and G: NDPase labeling of microglia in the retina, ipsilateral to the injection site of vehicle (arrow). No major morphological or spatial distributional changes in microglia were seen in mice injected with vehicle. D, F, and H: An equivalent region in the ipsilateral retina from mice treated with the arabinose mixture. D: At 12 hours post-i.c. injection of arabinose, microglia displayed tortuous processes with prominent distensions and a hypertrophied cell body (arrow). F and H: From 24 hours post-i.c. injection of arabinose, microglia displayed shorter processes and further enlargement of the cell body (arrow). Each micrograph is representative of 6 mice.

Figure 3.

Maps showing representative densities of NDPase-labeled microglia from mice given an intracarotid injection of 1.6 mol/L arabinose + 2% (w/v) Evans blue and polymyxin B. The number of labeled microglia was determined within a 1-mm region, and the cells/mm were converted to a gray scale. Retinal maps show that there was a change in microglial density in response to osmotic barrier opening at 12 (A), 24 (B), and 36 (C) hours after i.c. arabinose injection. D−F: Despite the progressive morphological changes of microglia from 12 to 24 hours after arabinose administration, there was no increase in the total number of microglia visualized using NDPase histochemistry. The black dots over the cell bodies of microglia in D, E, and F illustrate that although the microglia with their long and tortuous processes cover larger areas of the retina at 12 (D) and 24 (E) hours after arabinose compared with those at 36 hours (F), the total number of microglia does not change dramatically. Each map is a single mouse retina. Each micrograph is representative of 6 mice.

Figure 4.

Tracings of photographic montages showing microglial distributional changes in response to intracarotid injection of 1.6 mol/L arabinose, 2% (w/v) Evans blue, and 1 μg/ml polymyxin B or vehicle alone. A: In the ipsilateral retina from mice given an intracarotid injection of vehicle, microglia were regularly distributed like those found in untreated animals. B: At 36 hours after injection of arabinose there was a marked redistribution of microglia toward the arterial side of the vascular tree. The arteries are the narrower caliber vessels with frequent stippling; the veins are the wider caliber vessels with sparse stippling.