Nogo-A Expression in the Brain of Mice with Cerebral Malaria

Cerebral malaria (CM) is associated with a high rate of transient or persistent neurological sequelae. Nogo-A, a protein that is highly expressed in the endoplasmic reticulum (ER) of the mammalian central nervous system (CNS), is involved in neuronal regeneration and synaptic plasticity in the injured CNS. The current study investigates the role of Nogo-A in the course of experimental CM. C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. Brain homogenates of mice with different clinical severity levels of CM, infected animals without CM and control animals were analyzed for Nogo-A up-regulation by Western blotting and immunohistochemistry. Brain regions with Nogo-A upregulation were evaluated by transmission electron microscopy. Densitometric analysis of Western blots yielded a statistically significant upregulation of Nogo-A in mice showing moderate to severe CM. The number of neurons and oligodendrocytes positive for Nogo-A did not differ significantly between the studied groups. However, mice with severe CM showed a significantly higher number of cells with intense Nogo-A staining in the brain stem. In this region ultrastructural alterations of the ER were regularly observed. Nogo-A is upregulated during the early course of experimental CM. In the brain stem of severely affected animals increased Nogo-A expression and ultrastructural changes of the ER were observed. These data indicate a role of Nogo-A in neuronal stress response during experimental CM

Representative micrographs of an animal with severe CM (A, C, E) and a non infected control animal (B, D, F). A, B, E, F: bregma -6 (cerebellum and brainstem); C, D: bregma -2 (cortex, hippocampus and thalamic nuclei).

<p>In CNT animals, brain sections showed mild labeling for Nogo-A (B, D, F). In CM animals more intense neuronal and oligodendroglial Nogo-A labeling was observed (A, C, E) especially in the brainstem (E). Magnifications: A–D: 1x, E-F: 20x.</p

Transmission electron microscopic micrographs of animals with CM (A-C) and an uninfected control animal (D).

<p>In animals with CM, ultrastructural changes indicating alterations of the endoplasmic reticulum (ER) were observed. In most neurons ribosomes and / or polyribosomes appeared conspicuously clustered (A-B, arrows). Some neurons even showed dilated ER (C, arrow-heads) and Golgi apparatus (A,C: g). Mitochondria (m) frequently showed altered cristae (B: asterisks). Control specimens from the same brain regions did not show signs of neuronal or oligodendroglial degeneration (D). Golgi apparatus are marked by g; mitochondria by m, nuclei by n. Bars in A, B, C, D correspond to 1 µm. Framed areas in A, B, D are shown at higher magnification in the respective inserts, where the bars correspond to 0.5 µm.</p

Representative Western blot (A) for Nogo-A (200 kDa) and alpha-tubulin (50 kDa) and densitometric analysis of western blot experiments (B) in mice with different stages of cerebral malaria (mild, CM1 n = 4; moderate, CM2 n = 9; severe CM3 n = 6), infected mice without neurological involvement (NCM n = 6) and uninfected control animals (CNT n = 5).

<p>A significant increase of densitometric measures for Nogo-A was observed in animals with CM compared to NCM and non-infected control animals. Comparing subgroups of animals with different clinical levels of severity CM2 and CM3 mice showed significantly higher Nogo-A levels compared to CNT mice. ***, p>0.001; **, p>0.01; *, p<0.05.</p

Representative micrographs of a non infected control animal.

<p>A: Corpus callosum (bregma -2); B: Cerebellum (bregma -6). Positive immunolabeling for Nogo-A was observed in cells showing morphological characteristics of oligodendrocytes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025728#pone-0025728-g002" target="_blank">figure 2A</a>) and neurons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025728#pone-0025728-g002" target="_blank">figure 2B</a>). Framed areas in the respective inserts delineate regions of interest presented in the micrographs. Magnifications: A: 20x, left inlay 100x; B: 20x.</p

Stereological analysis of the number of Nogo-A positive cells per mm² (A) and the ratio of Nogo-A positive cells per mm² with intense labeling in relation to mildly labeled cells (B) in defined regions of the brain (bregma +2, 0, -2, -4, -6 mm).

<p>The total amount of parenchymal cells positive for Nogo-A did not differ significantly between the studied groups (A). In the brain stem animals with severe CM showed a significantly higher number of neurons and oligodendrocytes with intense Nogo-A labeling (B). **, p<0.001 for the comparison CM3 vs. CM2, CM1, CNT and p<0.01 for the comparison CM3 vs. NCM; *, p<0.01 for the comparison CM3 vs. NCM and p<0.05 for the comparison CM3 vs. CM1, CNT. Mean and SEM are shown.</p