Characterization of a pneumococcal meningitis mouse model

Abstract

Background: S. pneumoniae is the most common causative agent of meningitis, and is associated with high morbidity and mortality. We aimed to develop an integrated and representative pneumococcal meningitis mouse model resembling the human situation.

Methods: Adult mice (C57BL/6) were inoculated in the cisterna magna with increasing doses of S. pneumoniae serotype 3 colony forming units (CFU; n = 24, 104, 105, 106 and 107 CFU) and survival studies were performed. Cerebrospinal fluid (CSF), brain, blood, spleen, and lungs were collected. Subsequently, mice were inoculated with 104 CFU S. pneumoniae serotype 3 and sacrificed at 6 (n = 6) and 30 hours (n = 6). Outcome parameters were bacterial outgrowth, clinical score, and cytokine and chemokine levels (using Luminex®) in CSF, blood and brain. Meningeal inflammation, neutrophil infiltration, parenchymal and subarachnoidal hemorrhages, microglial activation and hippocampal apoptosis were assessed in histopathological studies.

Results: Lower doses of bacteria delayed onset of illness and time of death (median survival CFU 104, 56 hrs; 105, 38 hrs, 106, 28 hrs. 107, 24 hrs). Bacterial titers in brain and CSF were similar in all mice at the end-stage of disease independent of inoculation dose, though bacterial outgrowth in the systemic compartment was less at lower inoculation doses. At 30 hours after inoculation with 104 CFU of S. pneumoniae, blood levels of KC, IL6, MIP-2 and IFN- γ were elevated, as were brain homogenate levels of KC, MIP-2, IL-6, IL-1β and RANTES. Brain histology uniformly showed meningeal inflammation at 6 hours, and, neutrophil infiltration, microglial activation, and hippocampal apoptosis at 30 hours. Parenchymal and subarachnoidal and cortical hemorrhages were seen in 5 of 6 and 3 of 6 mice at 6 and 30 hours, respectively.

Conclusion: We have developed and validated a murine model of pneumococcal meningitis.

Figure 1

Kaplan-Meier survival curve. Four groups of 6 mice inoculated via direct intracisternal injection with 10⁴, 10⁵, 10⁶ and 10⁷ CFU of S. pneumoniae/mouse respectively.

Figure 2

Bacterial outgrowth. Bacterial titers in CSF, brain (central nervous system compartment), and blood, spleen, and lung (systemic compartment) at the end-stage of disease after inoculation with 10⁴, 10⁵, 10⁶ and 10⁷ CFU S. pneumoniae per mouse. Titers are expressed mean CFU/ml +/- S.E.M.

Figure 3

Cytokine levels in plasma, brain homogenates and CSF. Median concentrations (expressed in pg/ml) of cytokines in mice inoculated with either NaCl or 10⁴ CFU/mouse of S. pneumoniae and sacrificed after 30 hours, or 6 and 30 hours respectively. Comparisons of cytokine levels between groups were calculated using the Mann-Whitney U test. (*P < 0.05; **P < 0.01).

Figure 4

Brain pathology of mice with pneumococcal meningitis. Nissl staining of the cortex of mice infected with S. pneumoniae, showing extensive leptomeningeal inflammatory infiltrate (A; 100× magnification), perivascular lymphocytic cuffing (B; 100× magnification), perivascular lymphocytic infiltration combined with bacterial overgrowth (C; 200× magnification) and further bacterial overgrowth combined with perivascular necrosis (D; 100× magnification). Iba-1 immunohistochemistry revealed microglial activation (E; 100× magnification). Hematoxylin and eosin staining showed subarachnoidal and cortical hemorrhages (F; 100× magnification).

Figure 5

Neuronal apoptosis. Caspase-3 immunohistochemistry of 10 μm sections of the middle portion of the hippocampus of the right cerebral hemisphere of mice inoculated with 10⁴ CFU S. pneumoniae at 30 hrs post infection (panel A; 200× magnification). Mice inoculated with S. pneumoniae showed significantly more hippocampal apoptosis at 30 hrs post infection than NaCl infected control mice (panel B, expressed in mean number of Caspase-3 positive cells/section +/- S.E.M.; groups were compared using a Student's t-test; *P < 0.001).