Purpose
Infection with Plasmodium falciparum, leading to cerebral malaria (CM), remains a major health threat in sub-Saharan African children. Malaria retinopathy (MR) often occurs concomitantly with CM. The brain and retina show similar neurovascular pathology including breakdown of the blood barriers and localised inflammation. This commonality offers a feasible avenue for early detection and prediction of risk and severe disease progression in the cerebral cortex. Animal models of CM have been widely used although the pathological events occurring in the retina have not been well characterised. Moreover, assessing key molecular events occurring in the retina and cerebral cortex during malaria could provide important insights into precisely how the parasite drives localised inflammation and neurovascular pathology. This project has been examining the common pathology between CM and MR in brain and retina in a murine model. In parallel in vitro experiments, we have also been assessing the impact of P. falciparum infected red blood cell (iRBC) lysates on retinal and brain endothelial barrier integrity.
Methods
C57BL6 mice were infected with Plasmodium berghei ANKA and monitored for up to 10 days post infection. Levels of parasitaemia and CM-related criteria such as abnormal gait, poor reflexes and slow movement were measured daily. In mice with CM and non-affected controls, fundus imaging, optical coherence tomography (OCT) and fluorescein angiography (FA) were performed. Following sacrifice, retinal flatmounts were stained with TER119 (n=4-6), a RBC marker, to assess haemocoagulation in the retinal vasculature. Also in both mouse groups, microglia were labelled using IBA1 and assessed for ramified (resting) or amoeboid (activated) morphology. To measure barrier integrity in vitro, brain and retinal endothelial cells, transendothelial electrical resistance was measured using the xCelligence system following addition of Plasmodium falciparum lysates.
Results
When compared to controls, the retinal fundus of mice that had CM (n=8) showed a distinct whitening in the vasculature which appeared to be linked to sequestration of red blood cells in localised regions. No gross changes were observed with OCT in either group . Post-mortem assessments of retinal flat mounts revealed areas of haemocoagulation and vessel occlusion, especially in the superficial plexi in the retinas of mice infected with CM. In many cases, this resulted in clear breakdown of the blood retinal barrier, including appearance of RBCs in the neuropile. Parallel in vitro studies investigating the effect of Plasmodium falciparum lysates on brain and retinal endothelial cells demonstrated a loss of barrier integrity as assessed by transendothelial electrical resistance (p< 0.0001).
Conclusion
Plasmodium berghei infected mice are a useful model of MR. In the current study we have characterised some aspects of pathophysiology in the retina of mice with CM. We can draw distinct parallels between the neurovascular changes occurring in the brain during CM and MR in the murine model. We are currently harnessing the power of spatial transcriptomics to further analyse key molecular mechanisms that drive neurovascular pathology in the retinal and brain. .