Figure 1.
Reduced synaptic density in human postmortem pneumococcal meningitis neocortical brain tissue samples.
A. Schematic representation of the analyzed neocortical regions. B, C. Decreased synapsin I (B) and the PSD95 (C) staining densities in layers I–II of the frontal neocortex of human post-mortem samples from S. pneumoniae meningitis cases (S. pneumoniae) vs. post mortem samples of cases who experienced rapid non-neurological death (Non-meningitis). D. Representative tissue samples (layer II) with anti-synapsin I immunohistochemistry. Scale bar: 10 µm. E. 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.
Table 1.
Clinical histories of the individual patients from the non-meningitis histology group.
Table 2.
Clinical histories of the individual patients from the Streptococcus pneumoniae meningitis histology group.
Figure 2.
Reduced synaptic density in mouse pneumococcal meningitis neocortical brain tissue samples.
A. Reduced synapsin staining in layers I–II of the neocortex in animals with meningitis by PLY-producing bacteria vs. all other groups 36 h after injection. * p<0.05. (D39) indicates the group of mice injected intracerebrally with the pneumolysin (PLY)-producing D39 S. pneumoniae strain; (PLY-) mice indicates those infected with the PLY-deficient D39 strain. B. Reduced staining was observed for PSD95 in layers I–II of the frontal neocortex of mice injected with the PLY-producing strain vs. 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. C. 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. D. 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.
Figure 3.
Dendritic and synaptic changes caused by pneumolysin in acute brain slices.
A. Equivalent cell lysis (LDH release) between slices that were mock treated or treated with 0.2 µg/ml PLY for 8 h. B. 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. C. Magnified dendritic fragments, demonstrating the dendrite configuration and the morphology of the dendritic spines. Scale bars: 10 µm. D. Increased number of dendritic swellings after exposure to 0.2 µg/ml PLY for 5 h. *** p<0.001. E. Decreased number of dendritic spines following 5 h of exposure to 0.2 µg/ml PLY. ** p<0.01. F. 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. G. Unchanged number of synapsin I-positive fluorescent puncta in the neocortices of slices treated with 0.2 µg/ml PLY for 5 h. H. 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. Unchanged protein expression levels of synapsin I and PSD95 in acute mouse brain slices (normalized to the corresponding levels of actin). J. 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.
Figure 4.
Kinetics of toxin tissue binding.
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.
Figure 5.
NMDA dependence of the dendritic changes caused by pneumolysin.
A. 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. B. 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. C. Reversal of the PSD95 density loss by PLY when incubated with 10 µM MK801. D. 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.
Figure 6.
Increased glutamate release and calcium changes caused by pneumolysin.
A. 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. B. 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) vs. 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. C. 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. D. Unchanged cytosolic calcium concentration following an identical incubation as in C., but under calcium-free extracellular buffer conditions. E. Preserved glutamate uptake in brain slices following 0.2 µg/ml PLY challenge for 4 h; n = 3 experiments.