Inhibition of the kynurenine-NAD+ pathway leads to energy failure and exacerbates apoptosis in pneumococcal meningitis

Pneumococcal meningitis causes neurological sequelae, including learning and memory deficits in up to half of the survivors. In both humans and in animal models of the disease, there is apoptotic cell death in the hippocampus, a brain region involved in learning and memory function. We previously demonstrated that in an infant rat model of pneumococcal meningitis, there is activation of the kynurenine (KYN) pathway in the hippocampus, and that there was a positive correlation between the concentration of 3-hydroxykynurenine and the extent of hippocampal apoptosis. To clarify the role of the KYN pathway in the pathogenesis of hippocampal apoptosis in pneumococcal meningitis, we specifically inhibited 2 key enzymes of the KYN pathway and assessed hippocampal apoptosis, KYN pathway metabolites, and nicotinamide adenine dinucleotide (NAD) concentrations by high-performance liquid chromatography. Pharmacological inhibition of kynurenine 3-hydroxylase and kynureninase led to decreased cellular NAD levels and increased apoptosis in the hippocampus. The cerebrospinal fluid levels of tumor necrosis factor and interleukin-1? and -? were not affected. Our data suggest that activation of the KYN pathway in pneumococcal meningitis is neuroprotective by compensating for an increased NAD demand caused by infection and inflammation;this mechanism may prevent energy failure and apoptosis in the hippocampus.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

Tracking the transcriptional host response from the acute to the regenerative phase of experimental pneumococcal meningitis

Despite the availability of effective antibiotic therapies, pneumococcal meningitis (PM) has a case fatality rate of up to 30% and causes neurological sequelae in up to half of the surviving patients. The underlying brain damage includes apoptosis of neurons in the hippocampus and necrosis in the cortex. Therapeutic options to reduce acute injury and to improve outcome from PM are severely limited.With the aim to develop new therapies a number of pharmacologic interventions have been evaluated. However, the often unpredictable outcome of interventional studies suggests that the current concept of the pathophysiologic events during bacterial meningitis is fragmentary. The aim of this work is to describe the transcriptomic changes underlying the complex mechanisms of the host response to pneumococcal meningitis in a temporal and spatial context using a well characterized infant rat model.

Adjuvant glycerol is not beneficial in experimental pneumococcal meningitis

Bacterial meningitis in children causes high rates of mortality and morbidity. In a recent clinical trial, oral glycerol significantly reduced severe neurological sequelae in paediatric meningitis caused by Haemophilus influenzae type b, and a tendency towards a benefit of adjunctive glycerol was seen in pneumococcal meningitis.

Attenuation of cerebrospinal fluid inflammation by the nonbacteriolytic antibiotic daptomycin versus that by ceftriaxone in experimental pneumococcal meningitis

Antibiotic-induced bacteriolysis exacerbates inflammation and brain damage in bacterial meningitis. Here the quality and temporal kinetics of cerebrospinal fluid (CSF) inflammation were assessed in an infant rat pneumococcal meningitis model for the nonbacteriolytic antibiotic daptomycin versus ceftriaxone. Daptomycin led to lower CSF concentrations of interleukin 1beta (IL-1beta), IL-10, IL-18, monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 alpha (MIP-1alpha) (P < 0.05). In experimental pneumococcal meningitis, daptomycin treatment resulted in more rapid bacterial killing, lower CSF inflammation, and less brain damage than ceftriaxone treatment.

Bacteremia causes hippocampal apoptosis in experimental pneumococcal meningitis

Bacteremia and systemic complications both play important roles in brain pathophysiological alterations and the outcome of pneumococcal meningitis. Their individual contributions to the development of brain damage, however, still remain to be defined.

Adjunctive dexamethasone affects the expression of genes related to inflammation, neurogenesis and apoptosis in infant rat pneumococcal meningitis

Streptococcus pneumoniae is the most common pathogen causing non-epidemic bacterial meningitis worldwide. The immune response and inflammatory processes contribute to the pathophysiology. Hence, the anti-inflammatory dexamethasone is advocated as adjuvant treatment although its clinical efficacy remains a question at issue. In experimental models of pneumococcal meningitis, dexamethasone increased neuronal damage in the dentate gyrus. Here, we investigated expressional changes in the hippocampus and cortex at 72 h after infection when dexamethasone was given to infant rats with pneumococcal meningitis. Nursing Wistar rats were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis or were sham-infected with pyrogen-free saline. Besides antibiotics, animals were either treated with dexamethasone or saline. Expressional changes were assessed by the use of GeneChip® Rat Exon 1.0 ST Arrays and quantitative real-time PCR. Protein levels of brain-derived neurotrophic factor, cytokines and chemokines were evaluated in immunoassays using Luminex xMAP® technology. In infected animals, 213 and 264 genes were significantly regulated by dexamethasone in the hippocampus and cortex respectively. Separately for the cortex and the hippocampus, Gene Ontology analysis identified clusters of biological processes which were assigned to the predefined categories "inflammation", "growth", "apoptosis" and others. Dexamethasone affected the expression of genes and protein levels of chemokines reflecting diminished activation of microglia. Dexamethasone-induced changes of genes related to apoptosis suggest the downregulation of the Akt-survival pathway and the induction of caspase-independent apoptosis. Signalling of pro-neurogenic pathways such as transforming growth factor pathway was reduced by dexamethasone resulting in a lack of pro-survival triggers. The anti-inflammatory properties of dexamethasone were observed on gene and protein level in experimental pneumococcal meningitis. Further dexamethasone-induced expressional changes reflect an increase of pro-apoptotic signals and a decrease of pro-neurogenic processes. The findings may help to identify potential mechanisms leading to apoptosis by dexamethasone in experimental pneumococcal meningitis.

Adjunctive daptomycin attenuates brain damage and hearing loss more efficiently than rifampin in infant rat pneumococcal meningitis

Exacerbation of cerebrospinal fluid (CSF) inflammation in response to bacteriolysis by beta-lactam antibiotics contributes to brain damage and neurological sequelae in bacterial meningitis. Daptomycin, a nonlytic antibiotic acting on Gram-positive bacteria, lessens inflammation and brain injury compared to ceftriaxone. With a view to a clinical application for pediatric bacterial meningitis, we investigated the effect of combining daptomycin or rifampin with ceftriaxone in an infant rat pneumococcal meningitis model. Eleven-day-old Wistar rats with pneumococcal meningitis were randomized to treatment starting at 18 h after infection with (i) ceftriaxone (100 mg/kg of body weight, subcutaneously [s.c.], twice a day [b.i.d.]), (ii) daptomycin (10 mg/kg, s.c., daily) followed 15 min later by ceftriaxone, or (iii) rifampin (20 mg/kg, intraperitoneally [i.p.], b.i.d.) followed 15 min later by ceftriaxone. CSF was sampled at 6 and 22 h after the initiation of therapy and was assessed for concentrations of defined chemokines and cytokines. Brain damage was quantified by histomorphometry at 40 h after infection and hearing loss was assessed at 3 weeks after infection. Daptomycin plus ceftriaxone versus ceftriaxone significantly (P < 0.04) lowered CSF concentrations of monocyte chemoattractant protein 1 (MCP-1), MIP-1α, and interleukin 6 (IL-6) at 6 h and MIP-1α, IL-6, and IL-10 at 22 h after initiation of therapy, led to significantly (P < 0.01) less apoptosis, and significantly (P < 0.01) improved hearing capacity. While rifampin plus ceftriaxone versus ceftriaxone also led to lower CSF inflammation (P < 0.02 for IL-6 at 6 h), it had no significant effect on apoptosis and hearing capacity. Adjuvant daptomycin could therefore offer added benefits for the treatment of pediatric pneumococcal meningitis.

Grafted neuronal precursor cells differentiate and integrate in injured hippocampus in experimental pneumococcal meningitis

Bacterial meningitis (BM) frequently causes persisting neurofunctional sequelae. Autopsy studies in patients dying from BM show characteristic apoptotic brain injury to the stem cell niche in the subgranular zone of the hippocampal dentate gyrus (DG), and this form of brain damage is associated with learning and memory deficits in experimental BM. With an eye to potential regenerative therapies, the survival, migration, and differentiation of neuronal precursor cells (NPCs) were evaluated after engraftment into the injured hippocampus in vitro and in vivo in an infant rat model of pneumococcal meningitis. Green fluorescent protein (GFP)-expressing NPCs were grafted into the DG of organotypic hippocampal slice cultures injured by challenge with live Streptococcus pneumoniae. Seven days after engraftment, NPCs had migrated from the site of injection into the injured granular layer of the DG and electro-functionally integrated into the hippocampal network. In vivo, GFP-expressing NPCs migrated within 1 week from the injection site in the hilus region to the injured granular layer of the hippocampal DG and showed neuronal differentiation at 2 and 4 weeks after transplantation. Hippocampal injury induced by BM guides grafted NPCs to the area of brain damage and provides a microenvironment for neuronal differentiation and functional integration.

Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.

Pneumococcal meningitis causes accumulation of neurotoxic kynurenine metabolites in brain regions prone to injury

Pneumococcal meningitis (PM) is characterized by an intense inflammatory host reaction that contributes to the development of cortical necrosis and hippocampal apoptosis. Inflammatory conditions in the brain are known to induce tryptophan degradation along the kynurenine pathway, resulting in accumulation of neurotoxic metabolites. In the present study, we investigated the contribution of the kynurenine pathway to brain injury in experimental PM by measuring the concentration of its metabolites and the enzymatic activities and mRNA levels of its major enzymes in the vulnerable brain regions. In the late phase of acute PM, we found a significant transcriptional upregulation of kynurenine-3-hydroxylase and an accumulation of the neurotoxic metabolites 3-hydroxykynurenine (3-HKYN) and 3-hydroxyanthranilic acid in cortex and hippocampus. The positive correlation between the concentration of 3-HKYN and the extent of hippocampal apoptosis adds support to the concept that 3-HKYN contributes to brain injury in PM.

Doxycycline reduces mortality and injury to the brain and cochlea in experimental pneumococcal meningitis

Bacterial meningitis is characterized by an inflammatory reaction to the invading pathogens that can ultimately lead to sensorineural hearing loss, permanent brain injury, or death. The matrix metalloproteinases (MMPs) and tumor necrosis factor alpha-converting enzyme (TACE) are key mediators that promote inflammation, blood-brain barrier disruption, and brain injury in bacterial meningitis. Doxycycline is a clinically used antibiotic with anti-inflammatory effects that lead to reduced cytokine release and the inhibition of MMPs. Here, doxycycline inhibited TACE with a 50% inhibitory dose of 74 microM in vitro and reduced the amount of tumor necrosis factor alpha released into the cerebrospinal fluid by 90% in vivo. In an infant rat model of pneumococcal meningitis, a single dose of doxycycline (30 mg/kg) given as adjuvant therapy in addition to ceftriaxone 18 h after infection significantly reduced the mortality, the blood-brain barrier disruption, and the extent of cortical brain injury. Adjuvant doxycycline (30 mg/kg given subcutaneously once daily for 4 days) also attenuated hearing loss, as assessed by auditory brainstem response audiometry, and neuronal death in the cochlear spiral ganglion at 3 weeks after infection. Thus, doxycycline, probably as a result of its anti-inflammatory properties, had broad beneficial effects in the brain and the cochlea and improved survival in this model of pneumococcal meningitis in infant rats.

Gene expression in cortex and hippocampus during acute pneumococcal meningitis

BACKGROUND: Pneumococcal meningitis is associated with high mortality (approximately 30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.We used an infant rat model of pneumococcal meningitis to assess gene expression profiles in cortex and hippocampus at 22 and 44 hours after infection and in controls at 22 h after mock-infection with saline. To analyze the biological significance of the data generated by Affymetrix DNA microarrays, a bioinformatics pipeline was used combining (i) a literature-profiling algorithm to cluster genes based on the vocabulary of abstracts indexed in MEDLINE (NCBI) and (ii) the self-organizing map (SOM), a clustering technique based on covariance in gene expression kinetics. RESULTS: Among 598 genes differentially regulated (change factor > or = 1.5; p < or = 0.05), 77% were automatically assigned to one of 11 functional groups with 94% accuracy. SOM disclosed six patterns of expression kinetics. Genes associated with growth control/neuroplasticity, signal transduction, cell death/survival, cytoskeleton, and immunity were generally upregulated. In contrast, genes related to neurotransmission and lipid metabolism were transiently downregulated on the whole. The majority of the genes associated with ionic homeostasis, neurotransmission, signal transduction and lipid metabolism were differentially regulated specifically in the hippocampus. Of the cell death/survival genes found to be continuously upregulated only in hippocampus, the majority are pro-apoptotic, while those continuously upregulated only in cortex are anti-apoptotic. CONCLUSION: Temporal and spatial analysis of gene expression in experimental pneumococcal meningitis identified potential targets for therapy.

Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis.

Endothelin inhibition improves cerebral blood flow and is neuroprotective in pneumococcal meningitis

By using an infant rat model of pneumococcal meningitis, we determined whether endothelins contribute to neuronal damage in this disease. Cerebrospinal fluid analysis demonstrated a significant increase of endothelin-1 in infected animals compared with uninfected controls. Histopathological examination 24 hours after infection showed brain damage in animals treated with ceftriaxone alone (median, 9.2% of cortex; range, 0-49.1%). In infected animals treated intraperitoneally with the endothelin antagonist bosentan (30 mg/kg, every 12 hours) also, injury was reduced to 0.5% (range, 0-8.6%) of cortex. Cerebral blood flow was reduced in infected animals (6.5 +/- 4.0 ml/min/100 g of brain vs 14.9 +/- 9.1 ml/min/100 g in controls. Treatment with bosentan restored cerebral blood flow to levels similar to controls (12.8 +/- 5.3 ml/min/100 g). Improved blood flow was not mediated by nitric oxide production, because bosentan had no effect on cerebrospinal fluid or plasma nitrite/nitrate concentrations at 6, 12, or 18 hours. These data indicate that endothelins contribute to neuronal injury in this model of pneumococcal meningitis by causing cerebral ischemia.