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Apoptosis of neurogenic cells in an invitro model of bacterial meningitis

Abstract number: O85

Hofer S., Grandgirard D., Trachsel T., Leib S.L.

Background: Bacterial meningitis (BM) leads to learning deficits in up to 50% of survivors. The histomorphologic correlates of the deficits include apoptosis of neurogenic cells in the hippocampus, a brain structure involved in learning.

Objective: To use an in vitro system of hippocampal neuronal differentiation to (i) identify triggers and molecular mechanism of apoptosis and, (ii) to investigate whether the differentiation stage during neurogenesis determines the vulnerability for apoptosis.

Methods: Stem- and neuronal progenitor-cells were isolated from postnatal (P4–6) rat hippocampus. Dissociated cells were driven into neuronal differentiation by the addition of BDNF for 21d. Cells were challenged with different stimuli characteristic for BM: Growth factor deprivation (GFD) by omission of BDNF, EGF and FGF for 24 h, application of TNF-a (20ng/ml) for 24 h and exposure to live bacteria (Streptococcus pneumoniae), together with penicillin and streptomycin (10ng/ml) to cause bacteriolysis, for 2 h. Differentiation stage was documented by immunoassaying for nestin (stem cells), doublecortin (immature neurons) and MAP2 (neurons). Neuronal differentiation in vitro was documented by a shift in the predominant staining pattern from nestin at 1d, doublecortin at 7–14d and MAP2 at 21d (Figure 1A). Caspase dependent and independent apoptosis was immunoassayed by active caspase-3 and annexin-V, respectively.

Figure 1.

Results: Caspase-3 dependent apoptosis was observed after GFD (27±7% of cells) and TNF-a (29±3%) but less after bacteria (5±3%) in mixed cell population at 7d. In contrast, apoptosis identified by annexin-V staining was induced by bacteria (25±6%) and to a lesser extent by GFD (16±7%) and TNF-a (17±3%). Vulnerability to apoptosis peaked at 7d when stem cells and immature neurons were specifically susceptible for caspase-3 dependent apoptosis induced by GFD 10±11% and 17±12% and by TNF-a 13±17% and 28±12% respectively (Figure 1B).

Conclusions: In a novel in vitro system of hippocampal neuronal differentiation, GFD and TNF-a induced caspase dependent apoptosis while bacteria induced caspase independent apoptosis. Stem cells and immature neurons are specifically vulnerable to undergo apoptosis in paradigms of BM. These results suggest that hippocampal injury in BM includes apoptosis of stem cells. The compromised regenerative potential of the hippocampus may contribute to the persistence of neurological deficits after BM.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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