Coma in fatal adult human malaria is not caused by cerebral oedema

Abstract

Background: The role of brain oedema in the pathophysiology of cerebral malaria is controversial. Coma associated with severe Plasmodium falciparum malaria is multifactorial, but associated with histological evidence of parasitized erythrocyte sequestration and resultant microvascular congestion in cerebral vessels. To determine whether these changes cause breakdown of the blood-brain barrier and resultant perivascular or parenchymal cerebral oedema, histology, immunohistochemistry and image analysis were used to define the prevalence of histological patterns of oedema and the expression of specific molecular pathways involved in water balance in the brain in adults with fatal falciparum malaria.

Methods: The brains of 20 adult Vietnamese patients who died of severe malaria were examined for evidence of disrupted vascular integrity. Immunohistochemistry and image analysis was performed on brainstem sections for activation of the vascular endothelial growth factor (VEGF) receptor 2 and expression of the aquaporin 4 (AQP4) water channel protein. Fibrinogen immunostaining was assessed as evidence of blood-brain barrier leakage and perivascular oedema formation. Correlations were performed with clinical, biochemical and neuropathological parameters of severe malaria infection.

Results: The presence of oedema, plasma protein leakage and evidence of VEGF signalling were heterogeneous in fatal falciparum malaria and did not correlate with pre-mortem coma. Differences in vascular integrity were observed between brain regions with the greatest prevalence of disruption in the brainstem, compared to the cortex or midbrain. There was a statistically non-significant trend towards higher AQP4 staining in the brainstem of cases that presented with coma (P = .02).

Conclusions: Histological evidence of cerebral oedema or immunohistochemical evidence of localised loss of vascular integrity did not correlate with the occurrence of pre-mortem coma in adults with fatal falciparum malaria. Enhanced expression of AQP4 water channels in the brainstem may, therefore, reflect a mix of both neuropathological or attempted neuroprotective responses to oedema formation.

Figure 1

Histological types of oedema in severe malaria. Left column: Examples of the different histological types of oedema observed in the brain of fatal severe malaria in adults visualised with haematoxylin and eosin (A, C, E, G) or Luxol fast blue cresyl violet (LBCV, I). Right column: graphs indicating the incidence (% of cases) of the different histological types of oedema in cortex (yellow), diencephalon (orange) and brainstem (brown) of cerebral malaria (CM) and non-CM cases. No error bars are given as the bar chart represents the percentage of cases with or without a categorical histological feature. A. Rarefaction of the perivascular space characterized by separation of the compact parenchyma by fluid filled spaces (indicated by dashed line). B. There was no significant difference in the prevalence of perivascular rarefaction between CM and non-CM cases or between different brain regions. C-D. Perivascular pools of proteinaceous material indicated with a star (C). There was a greater prevalence in the brainstem compared with the diencephalon (P = .006) of CM cases (D). E-F. Vacuolar parenchymal oedema characterized by small isolated spaces (star) compare with compact parenchyma (diamond); (E). There was no difference between CM and non-CM cases or between different brain regions (F). G-H. Oedema between fibres of white matter tracts (star) compare with compact fibre tract (diamond); (G). There was a hierarchy of prevalence: brainstem > diencephalon > cortex. However, there was no difference between CM and non-CM cases (H). I-J. Decreased staining intensity of LBCV as a result of increased fluid-filled spaces between myelin fibres. Note myelin pallor in area indicated by dashed line radiating from the vessel in the bottom right corner (I). There was no significant difference between CM and non-CM cases or between different brain regions (J). Scale bar in A = 50 μm (for images A-G); scale bar in I = 100 μm.

Figure 2

Assessment of frank vascular damage in the form of haemorrhages. A-B. Haemorrhages associated with small (A) and large (B) vessels visualized with haematoxylin and eosin stain. Scale bar = 50 μm. C-D. Quantitation of haemorrhages in severe malaria expressed as either the incidence of any haemorrhages in a case (% cases; C) or number of haemorrhages per mm² (error bars indicate SEM; D) in cortex (yellow), diencephalon (orange) and brainstem (brown). There was no significant difference in the prevalence or number of haemorrhages between CM and non-CM cases or between different brain regions.

Figure 3

Extravasation of fibrinogen reflecting vasogenic oedema. Left column: Different fibrinogen staining patterns in the brain parenchyma: diffuse (A), glial- (C) or neuronal-associated (E). Combinations of these patterns could be observed on the same section. Right column: The frequency of the different fibrinogen staining patterns were evaluated using the semi-quantitative scoring system: no staining (0), < 1% cells or vessels staining/grade 1, 1 - 10% cells or vessels staining/grade 2, > 10% cells or vessels staining/grade 3 in addition to measurement of total fibrinogen load (see Figures 4 & 5). The diffuse pattern of staining may reflect a more recent leak compared to cellular uptake of fibrinogen that may indicate an older lesion or cell injury. There was no significant difference in the frequency of fibrinogen extravasation between CM and non-CM cases (B, D, F) but there were differences between different brain regions. Greater numbers of vessels showed diffuse fibrinogen leakage in the brainstem compared with the cortex of severe malaria cases (B). Neuronal uptake of fibrinogen was more frequently observed in the brainstem compared with diencephalon or cortex (F).

Figure 4

Quantitation of immunolabelling for pKDR, fibrinogen, AQP4 and GFAP and patterns of AQP4 immunolabelling in post-mortem brainstem sections. A-B, F-G. Quantitation of low, medium and high levels of immunolabelling for pKDR (A), Fibrinogen (B), AQP4 (F), and GFAP (G). Comparisons are made between severe malaria patients with cerebral malaria (CM) and those that died from other severe complications of malarial disease (non-CM). Data are presented as box and whisker plots showing median, lower quartile, the upper quartile and outliers. C. Enhanced perivascular labelling for AQP4 around small vessels containing parasites. D. Perineuronal AQP4 labelling in a CM patient (black arrows) in an area without histological evidence of oedema. E. AQP4 labelled astrocyte (empty arrow) in an area showing vacuolar oedema (white arrows). Scale bar = 50 μm (C-E).

Figure 5

Quantitation of AQP4, GFAP and fibrinogen using digital image analysis. Brain maps showing low (blue), medium (green) and high (red) levels of immunolabelling for Aquaporin 4 (top row), Glial fibrillary acidic protein (GFAP; middle row) and Fibrinogen (bottom row). Maps of pKDR are not shown since all images appear almost entirely blue at this magnification. Each image represents half a transverse section of brainstem separated from the other half for presentation purposes at the 4^th ventricle (4V) to the anterior median fissure (AMF). For orientation, other histological features have been labelled on the middle image, first row: medial lemniscus (ML), inferior olivary nucleus (IOL) and pyramidal tracts (PT). Each column shows serial sections from the same patient staining for the different markers. Tissue sections were digitized, regions of low, medium and high expression were selected by density thresholding and areas were calculated and summated using SigmaScan Pro5.