Pneumococcal cell wall-induced meningitis impairs adult hippocampal neurogenesis

Abstract

Bacterial meningitis is a major infectious cause of neuronal degeneration in the hippocampus. Neurogenesis, a continuous process in the adult hippocampus, could ameliorate such loss. Yet the high rate of sequelae from meningitis suggests that this repair mechanism is inefficient. Here we used a mouse model of nonreplicative bacterial meningitis to determine the impact of transient intracranial inflammation on adult neurogenesis. Experimental meningitis resulted in a net loss of neurons, diminished volume, and impaired neurogenesis in the dentate gyrus for weeks following recovery from the insult. Inducible nitric oxide synthase (iNOS) immunoreactivity was prominent in microglia in nonproliferating areas of the dentate gyrus and hilus region after meningitis induction. Treatment with the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine restored neurogenesis in experimental meningitis. These data suggest that local central nervous system inflammation in and of itself suppresses adult neurogenesis by affecting both proliferation and neuronal differentiation. Repair of cognitive dysfunction following meningitis could be improved by intervention to interrupt these actively suppressive effects.

FIG. 1.

Rate of proliferation of new neurons following acute meningeal inflammation. (A) Nuclei in both DG that accumulated BrdU over 3 days were enumerated. (B) The percentage of BrdU-positive cells expressing the early neuronal differentiation marker Hu-D was determined at 3 days and 3 weeks after meningeal challenge with cell wall. P values were calculated by one-way ANOVA followed by Student-Newman-Keuls post hoc testing.

FIG. 2.

Effects of meningitis on hippocampal growth. (A and B) The absolute number of granule cells (A) and volume (B) of the DG bilaterally were compared over a 5-week observation period in mice with and without challenge with PCW. (C) To follow the long-term fate of new neurons appearing after cell wall challenge, all animals received BrdU on days 21 to 23 after surgery. Although meningitis animals (solid bars) showed an increase of BrdU-positive cells in the DG on day 24 compared to control animals (hatched bars), equal numbers of newly formed neurons were present in both groups 4 weeks later. P values were calculated by one-way ANOVA followed by Student-Newman-Keuls post hoc testing.

FIG. 3.

Immunohistology of the DG. (A and B) Bright-field microscopic BrdU immunostaining was used to quantify newly formed cells in the DG (A) and SVZ (B). (C) Double staining for BrdU (red) and Hu-D (green) immediately after BrdU exposure was used for the early quantification of newly formed cells adopting a neuronal phenotype. Inset, confocal demonstration of colocalization. (D) Double staining for BrdU (red) and NeuN (green) 4 weeks after BrdU exposure was used to quantify surviving new neurons. (E) Apoptosis of NPCs was examined 3 days after meningitis induction by costaining for activated caspase 3 (red), nestin (green), and chromatin (blue). (F) Triple staining for active caspase 3 (green), NeuN (blue), and BrdU (red) 3 days after meningitis was used to indicate apoptosis of mature neurons. (G and H) Double staining for BrdU (blue) and CD11b (red) revealed more CD11b-positive microglia 3 days after meningitis (G) compared to the control (H) but no colocalization of proliferating cells with these activated microglia. (I and J) Activated microglia (CD11b, green) expresses iNOS protein (red) in meningitis mice (I) but not in controls (J). (K through M) Double staining for BrdU (red) and nitrotyrosine (green), a footprint of peroxynitrite, reveals nitrotyrosine formation in untreated meningitis (K) but not in controls (L) or in meningitis mice treated with the iNOS inhibitor l-NIL (M).

FIG. 4.

Restoration of neurogenesis in the acute phase of meningitis by iNOS inhibition. (A) In nonmeningitic animals, the number of newly formed, BrdU-incorporating cells in the SGZ is not significantly changed by the specific iNOS inhibitor l-NIL. Control animals (open bars) and animals challenged with cell wall (solid bars) were treated with the iNOS inhibitor l-NIL as indicated. Neurogenesis was measured by BrdU-positive cells in both DG. (B) Early neuronal differentiation of BrdU-positive cells was demonstrated by Hu-D costaining. P values were calculated by Student-Newman-Keuls tests after one-way ANOVA. (C) Behavioral effect of l-NIL in the inhibitory avoidance paradigm 4 weeks after induction of experimental meningitis. Compared to PBS-treated controls, the proportion of animals avoiding the dark compartment of the test apparatus for more than 180 s (i.e., successful learners) has a tendency to be lower after challenge with PCW (P = 0.26), and this effect is abolished by chronic treatment with l-NIL (P = 0.23 versus untreated meningitis and P = 1.0 versus PBS-treated controls by Fisher's exact test).