Early microvascular changes in murine cerebral malaria detected in retinal wholemounts

Abstract

Changes in the cerebral microvasculature such as breakdown of the blood-brain barrier, petechial hemorrhages, congestion, and edema are observed in the later stages of murine cerebral malaria. These changes have been described from histologic sections of brain, but the need to section the material makes direct observation of the microvasculature in situ difficult. The retinal vasculature, in contrast, offers a unique opportunity to study rheologic, barrier, and functional properties of the microvasculature as a wholemount preparation with normal spatial relationship with other tissues and as an intact vascular plexus. A combination of techniques, including intravascular perfusion of Evan's Blue, Bisbenzimide and Monastral Blue, and fluorescence and transmitted light observation of retinal wholemounts, were developed to examine the progressive microvascular changes in murine cerebral malaria. These techniques allowed detection of phenomena such as monocyte adherence to endothelial cells, congestion, small hemorrhages, and breakdown of the blood-retinal barrier, with details of the location of this leakage, earlier than was possible by studying brain sections. Because the retina is intact, the phenomena were seen in greater detail and some, such as occlusion of vessel segments, were detectable only in retinal wholemounts. In addition, the covisualization of the blood elements, barrier properties, and vascular endothelial integrity that are possible with retinal wholemounts allowed detailed analysis of the interaction of different cellular elements in the pathogenesis of cerebral malaria. Except for detection of edema, the retinal wholemount technique offers a more powerful and less time-consuming technique for detecting early microvascular changes in murine cerebral malaria. This technique could find wider application in the study of other diseases that affect the microvasculature of the central nervous system, such as experimental allergic encephalitis and meningitis.