Dendritic and synaptic changes caused by pneumolysin in acute brain slices.

<p><i>A.</i> Equivalent cell lysis (LDH release) between slices that were mock treated or treated with 0.2 µg/ml PLY for 8 h. <i>B.</i> A DiI-stained pyramidal neuron in the neocortex of an acute mouse slice demonstrated a normal spine and dendrite morphology (mock) in contrast to a PLY-treated slice (0.2 µg/ml for 5 h), which showed a reduction in spine number and multiple dendritic enlargements (swellings). Scale bars: 10 µm. <i>C.</i> Magnified dendritic fragments, demonstrating the dendrite configuration and the morphology of the dendritic spines. Scale bars: 10 µm. <i>D.</i> Increased number of dendritic swellings after exposure to 0.2 µg/ml PLY for 5 h. *** p<0.001. <i>E.</i> Decreased number of dendritic spines following 5 h of exposure to 0.2 µg/ml PLY. ** p<0.01. <i>F.</i> Reduced number of PSD95-positive fluorescent puncta in the neocortices of slices treated with 0.2 µg/ml PLY for 5 h. * p<0.05. <i>G.</i> Unchanged number of synapsin I-positive fluorescent puncta in the neocortices of slices treated with 0.2 µg/ml PLY for 5 h. <i>H.</i> Western blot analysis of the protein levels of synapsin I, PSD95 and actin in acute mouse brain slices treated with 0.2 µg/ml PLY for 5 h or in mock-treated slices. <i>I.</i> Unchanged protein expression levels of synapsin I and PSD95 in acute mouse brain slices (normalized to the corresponding levels of actin). <i>J.</i> The delta6 non-pore forming mutant of PLY did not produce varicosity increase and dendritic spine loss. All values are presented as the mean ± SEM; n = 6 slices from at least 3 independent experiments.</p

NMDA dependence of the dendritic changes caused by pneumolysin.

<p><i>A.</i> Inhibition of the formation of dendritic swellings caused by treatment with 0.2 µg/ml PLY for 5 h by the application of 10 µM of the non-competitive NMDA-receptor inhibitor MK801. *** p<0.001 vs. all. <i>B.</i> Preserved dendritic spine number following treatment with 10 µM MK801 together with 0.2 µg/ml PLY for 5 h. * p<0.05, ** p<0.01. <i>C.</i> Reversal of the PSD95 density loss by PLY when incubated with 10 µM MK801. <i>D.</i> Complete inhibition of dendritic swelling formation caused by treatment with 0.2 µg/ml PLY for 5 h using a 50 µM of the competitive NMDA-receptor antagonist D-AP5. *** p<0.001. All values represent the mean ± SEM; n = 5–6 slices from at least 3 independent experiments.</p

Increased glutamate release and calcium changes caused by pneumolysin.

<p><i>A.</i> Representative sample of three experiments demonstrating increased neocortical glutamate content (via electrochemical detection in an acute slice; a diagram of the electrode is presented) following 0.2 µg/ml PLY exposure. <i>B.</i> Elevation of glutamate release on the surface of a monolayer of mouse astrocytes by a treatment with 0.1 µg/ml PLY in buffer containing 2 mM extracellular calcium (Ca-rich) <i>vs.</i> unchanged glutamate levels in calcium-free buffer (Ca-free). A permeabilization diagram (propidium iodide-positive cells) is presented above the glutamate release diagram. The values are presented as the means ± SEM; n = 3–5 experiments. <i>C.</i> Increase in cytosolic calcium (Fura-2-loaded mouse astrocytes) following treatment with 0.1 µg/ml PLY and a 10 µM ionomycin control at 800 s in 2 mM calcium-containing buffer (representative experiment). The experiments were repeated 5 times with identical results. <i>D.</i> Unchanged cytosolic calcium concentration following an identical incubation as in <i>C.</i>, but under calcium-free extracellular buffer conditions. <i>E.</i> Preserved glutamate uptake in brain slices following 0.2 µg/ml PLY challenge for 4 h; n = 3 experiments.</p

Reduced synaptic density in human postmortem pneumococcal meningitis neocortical brain tissue samples.

<p><i>A.</i> Schematic representation of the analyzed neocortical regions. <i>B</i>, <i>C.</i> Decreased synapsin I (<i>B</i>) and the PSD95 (<i>C</i>) staining densities in layers I–II of the frontal neocortex of human post-mortem samples from <i>S. pneumoniae</i> meningitis cases (S. pneumoniae) <i>vs.</i> post mortem samples of cases who experienced rapid non-neurological death (Non-meningitis). <i>D.</i> Representative tissue samples (layer II) with anti-synapsin I immunohistochemistry. Scale bar: 10 µm. <i>E.</i> There was no difference in the number of TUNEL-positive nuclei in neocortical layers I–II between non-meningitis and meningitis samples. All values represent the mean ± SEM, and samples from 5 to 6 cases per group were analyzed.</p

Clinical histories of the individual patients from the <i>Streptococcus pneumoniae</i> meningitis histology group.

<p>Note: the synaptic/cell death analysis in our tissue samples involved non-necrotic tissue areas.</p

Kinetics of toxin tissue binding.

<p>Measurement of the fluorescence intensity of GFP-tagged PLY (PLY) in the medium following incubation of brain slices (6 slices per well) challenged with either 0.5 or 2 µg/ml PLY-GFP. The initial toxin concentration in the medium was high but rapidly (within minutes) decreased due to tissue binding. In the enlarged diagram (upper right), a rescaled y-axis fragment of the 0.5 µg/ml PLY experiment is presented.</p

Reduced synaptic density in mouse pneumococcal meningitis neocortical brain tissue samples.

<p><i>A.</i> Reduced synapsin staining in layers I–II of the neocortex in animals with meningitis by PLY-producing bacteria <i>vs.</i> all other groups 36 h after injection. * p<0.05. (D39) indicates the group of mice injected intracerebrally with the pneumolysin (PLY)-producing D39 <i>S. pneumoniae</i> strain; (PLY-) mice indicates those infected with the PLY-deficient D39 strain. <i>B.</i> Reduced staining was observed for PSD95 in layers I–II of the frontal neocortex of mice injected with the PLY-producing strain <i>vs.</i> the PLY-deficient D39 strain animals after 36 h. All values are presented as the mean ± SEM. There were 5 animals in the mock group and 10–13 in the meningitis group. <i>C.</i> Representative tissue sample images with anti-synapsin I immunohistochemistry of layers I–III with magnification of equivalent areas of interest in layer I. Scale bar: 15 µm. <i>D.</i> Representative images of the TUNEL-FITC staining of equivalent areas in layers I/II of the neocortex of mice infected with D39 and PLY-deficient pneumococcal strain, where no TUNEL-positive cells are present. All nuclei were counterstained with propidium iodide (PI). TUNEL-negative control (enzyme missing) and TUNEL-positive control (pretreatment with DNAseI) are presented for staining validation. Scale bar: 20 µm.</p