Murine cerebral malaria is associated with a vasospasm-like microcirculatory dysfunction, and survival upon rescue treatment is markedly increased by nimodipine

Abstract

Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.

Figure 1

Cumulative survival (A), course of parasitemia (B), and rectal temperature (C) of PbA-infected mice that did (n = 19) or did not (n = 4) develop CM and in uninfected control mice (n = 10). Rectal temperature was lower in CM mice than in uninfected controls (P = 0.0004). Data in (B) and (C) are the mean ± SEM.

Figure 2

PbA infection leads to decreased blood flow in pial vessels. Arteriolar (A) and venular (B) blood flow in PbA-infected mice with or without CM and in uninfected control mice. Results are expressed as the percentage change in relation to baseline measurements performed before infection. Flow was significantly decreased on day 6 in mice that developed CM (arteriolar: P = 0.0003; venular: P = 0.0003). Data are the mean ± SEM. C: Arteriolar blood flow in mice with CM, at the time of presentation of clinical signs (irrespective of the day of infection). Flow was significantly decreased in mice with CM (P < 0.0001). Values shown for uninfected controls mice, and infected mice without CM are the lowest recorded for each mouse on days 5 to 8. Bars indicate the mean value.

Figure 3

Changes in diameters and in RBC velocities in arterioles during PbA infection. A: Mice with CM showed a significant decrease in arteriolar diameters compared with uninfected controls (P = 0.0151). B: Mice with CM also showed significant decreases in RBC velocities in relation to uninfected controls (P < 0.0001). Bars indicate the mean value.

Figure 4

CM is associated with impaired perfusion in pial vessels. A–D: Four consecutive sections of the same venule showing how adherent leukocytes can cause marked reduction in luminal diameters and impair perfusion (a more detailed dynamic view is available at the supplemental video S3, at http://ajp.amjpathol.org). Asterisks show adherent leukocytes. The luminal diameter in specific sections is shown (black bars with the respective diameter value); note the large variations in diameter mostly attributable to adherent leukocytes, and a site of major constriction in B with apparent damage of the vessel structure. Bright spots in A and D are flowing fluorescent PbA-GFP pRBCs. Adherent leukocytes can completely block blood flow in small venules. E and F: Two consecutive frames showing vessels stained with albumin-FITC, one of them is nonperfused (arrow) blocked by an adherent leukocyte (asterisk; E) and the same area showing the leukocyte stained with anti-CD45-TxR antibodies (F; see dynamic view in supplemental video S5, at http://ajp.amjpathol.org). G and H: Collapse of nonperfused small vessels: disappearance of small vessels with no flow (white arrows), and associated nonflowing vessels (black arrows).

Figure 5

PbA pRBCs do not directly adhere to pial endothelial cells but may be found trapped by adherent leukocytes. A: A fluorescent PbA-GFP pRBC attached to a leukocyte (arrow). Other visible adherent leukocytes are indicated by asterisks. B: The same vessel section evidencing the adherent leukocytes, highlighted after changing the filter to detect TxR fluorescence. The arrow points to the leukocyte with the attached pRBC. See also supplemental video S7 at http://ajp.amjpathol.org.

Figure 6

CM is associated with vascular collapse. A–C: Collapse of a major pial vessel and branches (arrows) on day 6 of infection. D–F: Collapse of two branches (arrows) of a vessel and microhemorrhages (encircled); note that vessel collapse occurred between two observations in the same day (6), in between which the mouse developed clinical CM. G–I: Collapse of virtually the entire pial vascular network under the cranial window on day 6 of infection. J–L: Collapse of the vascular network under the window on day 6 after a major hemorrhage (arrow) on days 4 and 5.

Figure 7

Nimodipine improves the death-rescuing capacity of artemether on mice with established CM. A: Cumulative survival of PbA-infected mice presenting clinical CM and treated with artemether-nimodipine (1.3 mg/kg, n = 12; 4 mg/kg, n = 20; and 12 mg/kg, n = 15) or artemether-vehicle (n = 31). A total of nine experiments were conducted. Survival was significantly increased in mice treated with 4 mg/kg (P = 0.0217) and 12 mg/kg (P = 0.0474), but not with 1.3 mg/kg. B: Survivor mice treated with artemether plus nimodipine (n = 20) presented faster clinical recovery than survivor mice treated with artemether-vehicle (n = 9) with significantly higher scores at 48 (P = 0.0402) and 72 (P = 0.0238) hours. C: Efficacy of artemether treatment: parasitemia decreased fast after artemether administration in mice treated with nimodipine or vehicle. Data are the mean ± SEM.

Figure 8

Nimodipine increases blood flow in CM mice through vasorelaxation. A: Administration of nimodipine at 4 mg/kg IP caused an increase in arteriolar blood flow in mice with clinical CM (n = 3) and in uninfected control mice (n = 3). B: Increase in arteriolar blood flow was attributable to a sustained increase in vascular diameter. Data are the mean ± SEM.