Apoptosis of cells in the hippocampal dentate gyrus is a characteristic form of brain damage in BM [3, 21]. In experimental models an association of injury to the dentate gyrus with learning and memory deficits has been shown . The present study demonstrates that treatment with vitamin B6 reduces the number of apoptotic cells in the hippocampal dentate gyrus. We investigated the mechanisms underlying this neuroprotective effect by studying the influence of vitamin B6 on the transcriptome and on cellular energy stores.
In the model used, hippocampal apoptosis starts to occur during the acute phase of BM (18-24 h) with a peak in the sub-acute phase at 36 h, and reaches control levels in the late phase of the disease (46-72 h). Recent studies in our lab showed that in the acute phase of BM, genes that are significantly expressed in the hippocampus and cortex are mainly involved in processes of the immune and inflammatory response . The innate immune system has a predominant role in immune defense in the otherwise immune privileged brain tissue. However, neurological complications secondary to BM  suggest that the host defense mechanisms are inefficient in eliminating the pathogen and furthermore, that the host inflammatory reactions contribute considerably to the brain damage observed in the disease. As shown previously, the major events on transcriptional level concerning the regulation of the host immune response occur within the first 3 days after infection .
A promising candidate for pre-treatment of BM is vitamin B6 administered during the acute phase of the disease. A prerequisite for the application of a therapeutic target is the understanding of the processes leading to the desired effects, in the case of vitamin B6 to a reduction of hippocampal apoptosis. Thus, in the present study, we focused on the mechanisms that take place as a consequence of vitamin B6 treatment in the acute phase of BM. The time point for the second application of vitamin B6 (18 h post-infection) and the endpoint at 24 h post-infection were chosen during the acute phase of BM according to recent findings which define a time window for therapeutic interventions during this phase of the disease.
In order to handle the huge data mass resulting from a microarray study, we clustered the significantly regulated transcripts according to their involvement in given biological processes (Figure 4).
The first step in immune activation is the recognition of bacterial products by pattern recognition receptors, such as Toll-like receptors (TLRs), expressed on resident cells of the central nervous system (CNS) . Experimental studies showed that primary mouse microglial cells, the resident macrophages of the CNS, are activated upon stimulation with agonists of several TLRs. Activated microglia release inflammatory mediators such as nitric oxide (NO) or activin A. Activin A is a member of the transforming growth factor-β (TGF-β) family which plays a central role in different aspects of cell growth and differentiation . Previous studies showed that activin A is increased in the CSF of patients suffering from BM. Furthermore, activin A may decrease the secretion of NO and pro-inflammatory mediators by activated microglia, and enhances microglial proliferation, suggesting that activin A may counteract the activation of microglial cells in BM . The biological functions of members of the TGF-β family are mediated by 2 types of transmembrane serine/threonine kinase receptors (type I and II) that are expressed on activated microglial cells. Therefore, microglia is not only the source but also the target cells of activin A in the CNS . The down-regulation of the activin A receptor type II-like 1 (ID 382555, Figure 4A) upon vitamin B6 treatment may indicate a reduced activation of microglia and thus, a decreased release of tissue-destructive mediators.
G protein coupled receptors (GPCRs) change their conformation upon stimulation by an activating signal. This activated state is controlled by regulators of G protein signaling (RGS) [26, 27]. RGS2 (ID 377853) as well as GPCR3 (ID 435019) are up-regulated in vitamin B6-treated rats compared to saline-treated animals, suggesting an involvement of G protein signaling in cellular processes leading to a reduction of hippocampal apoptosis (Figure 4A). Recent studies documented that among the RGS family, RGS2 plays a prominent role in regulating synaptic transmission and plasticity in hippocampal neurons . It is therefore likely that increased RGS2 levels modulate synaptic output, probably leading to an elevated survival of neurons upon vitamin B6 treatment.
The GPCR3 endothelinB (ETB) has been found to be important for the physiological reduction of neuronal apoptosis in the dentate gyrus during postnatal development. Furthermore, ETB receptors are involved in pathological apoptosis in the dentate gyrus, especially in a rabbit model of pneumococcal meningitis . These data suggest a role for the ETB receptor as an anti-apoptotic factor in the dentate gyrus, consistent with the up-regulation of ET
receptor gene as a result of vitamin B6 treatment.
Another important receptor also belonging to the GPCR superfamily is the neuromedin B receptor (NMBR, ID 438637, Figure 4A). NMBR is localized in brain microvascular endothelial cells that form the blood–brain barrier. It is coupled to the phospholipase C transducing system controlling the K+ efflux out of the brain . A down-regulation of NMBR in vitamin B6-treated rats may be indicative for blood–brain barrier disruption induced by BM.
The pro-inflammatory mediator IL-1 consists of 4 molecular species (IL-1α, IL-1β, IL-1γ and IL-1δ) that exert similar biological activity through IL-1 receptor (IL-1R) type I. In vivo studies showed that IL-1R−/− mice, lacking an intact IL-1 signal, are more susceptible to develop pneumococcal meningitis, and that the disease is associated with higher mortality in IL-1R−/− mice compared to wild-type animals. These results suggest a protective role of locally produced IL-1 in the first-line defense against pathogens during pneumococcal meningitis . However, in the present study, IL-1R type I (ID 381937, Figure 4A) was down-regulated, indicating that upon vitamin B6 treatment the host inflammatory reaction is down-modulated.
Highly raised concentrations of CXC-chemokines (e.g. IL-8) that attract neutrophils, and of CC-chemokines (e.g. monocyte chemoattractant protein MCP-1, macrophage inflammatory proteins MIP-1α and MIP-1β) that attract monocytes and lymphocytes, were observed in the CSF during BM [31, 32]. Chemokine receptors are also GPCRs. In this study, CCR2 (ID 2818300), the receptor of MCP-1, and CCR5 (ID 382626), the receptor of MIP-1α and MIP-1β, are down-regulated (Figure 4A). Both receptors are expressed on glial and neuronal cells in the adult brain as well as on neural progenitor cells isolated from the subventricular zone where neurogenesis occurs. The localization of chemokine receptors in these regions suggests an involvement of CCR2 and CCR5 in the regulation of adult neural progenitor cells in physiological or pathological conditions . Other studies showed that CCR2 is one of the most prominent chemokine receptor associated with neuro-inflammatory diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis . However, the down-regulation of CCR2 and CCR5 following vitamin B6 treatment may result in a reduced production of neuro-inflammatory mediators by glial or neuronal cells. Furthermore, recruitment of monocytes and lymphocytes to the CSF may also be reduced. Finally, it could also influence the neurogenetic processes observed in the hippocampal dentate gyrus (see below).
Following inflammation, microglial cells become activated and produce inflammatory mediators causing brain damage in a variety of neurodegenerative disorders. Since inflammation may exacerbate brain damage, the control and reduction of brain inflammation is pathophysiologically important. IL-13 is an anti-inflammatory cytokine which minimizes the production of inflammatory mediators from activated microglia (e.g. TNFα and MIP-1α) . Moreover, experimental studies showed that exogenous IL-13 selectively induces apoptotic death of activated microglia . Another study demonstrated that neurons and microglia cooperatively down-regulate brain inflammation by inducing endogenous IL-13 expression in microglia, resulting in microglial death and elevation of neuronal survival . Suggesting a reduced inflammatory reaction as assessed by a down-regulation of pro-inflammatory cytokines (IL-1R type I) and chemokines (CCR2 and CCR5) in vitamin B6-treated rats, the requirement for anti-inflammatory cytokines such as IL-13 is reduced. This suggestion is consistent with the down-modulation of the IL-13 receptor alpha 1 (ID 432716, Figure 4A) gene upon vitamin B6 treatment.
In summary, vitamin B6 down-modulates the inflammatory response as evidenced by reduced RNA levels encoding for pro-inflammatory cytokines and chemokines, and by transcriptional indication for diminished activation of microglia. Because the brain damage observed in BM, including hippocampal apoptosis, is mainly due to the host inflammatory reaction , a down-modulated immune reaction may decisively contribute to diminished hippocampal apoptosis observed in vitamin B6-treated rats. Evidence for strong anti-inflammatory effects of vitamin B6 in patients with systemic inflammatory symptoms has also been provided by others [38–40].
The circadian rhythm is generated by a set of interacting genes and proteins (see Additional file 3: Figure S2). For example in mammals, the protein products of the clock and Bmal1 genes act together to induce the expression of other clock genes including period (PER) . The up-regulation of period homolog transcripts (PER1, ID 395923, and PER2, ID 392428) in vitamin B6- compared to placebo-treated rats suggests an involvement of the circadian rhythm in the regulation of apoptotic processes (Figure 4B).
Recent studies demonstrated a circadian periodicity of the TRP metabolism via the KYN pathway . However, TRP metabolism in the brain mainly occurs via 2 different pathways, the methoxyindole and the KYN pathway. In experimental models as well as in humans, melatonin, the main metabolite of the methoxyindole pathway, acts as neuroprotective agent. It inhibits the NMDA receptor and thus, protects the neurons from excitotoxic damage. The same effect is mediated by KYNA, a neuroprotective metabolite of the KYN pathway. The inhibition of the NMDA receptor activity partially depends on the reduction of the NO synthase activity, therefore decreasing the amount of NO produced as a result of NMDA activation. Melatonin also follows a circadian rhythmic pattern, mainly determined by the pineal gland that increases the production of melatonin upon physiological stimuli such as darkness. Activation of either the methoxyindole or the KYN pathway reaches an equilibrium in normal conditions by an increase in the TRP degradation via the KYN pathway during the day and via the methoxyindole pathway during the night . This equilibrium is lost under conditions of stress including febrile and epileptic seizures and probably also in other pathological situations . BM displaying a stress situation could influence the equilibrium between the methoxyindole and the KYN pathway. Because vitamin B6 acts as a cofactor for 2 key enzymes of the KYN pathway and also positively affects the pineal production of melatonin , administration of vitamin B6 could restore this equilibrium. Therefore, melatonin as a immunomodulatory agent could play an important role in neuroinflammation and subsequent brain injury .
The elevation of cellular NAD+ levels through the vitamin B6 induced activation of the KYN pathway observed in this study, may also have an influence on factors involved in the circadian rhythm described above. NAD+ has been shown to act as a central circadian regulator. Concerning the role of NAD+ in cellular energy stores, a molecular coupling between the circadian rhythm and energy metabolism has been proposed [46–48]. Moreover, a link between disruption of circadian rhythm and hippocampal learning and memory has been reported in rats using the water maze task. Chronic stress, sleep deprivation and decreases in melatonin secretion are some of the many side effects of circadian disruption. By its anti-oxidant and neuroprotective role in the brain, melatonin deprivation may contribute to brain damage in individuals suffering from chronic circadian disruption. In transgenic mouse models of Alzheimer’s disease, melatonin treatment may reduce the deposition of β-amyloid and protects against oxidative stress. A possible speculation is that with decreasing levels of melatonin, individuals suffering from chronic circadian disruption become more vulnerable to brain damage associated with learning and memory impairment . Another study showed that the clock gene could have an important role on spatial learning in mice, as assessed by water maze . Furthermore, experimental mouse models suggest that cell cycle and apoptotic processes may be regulated by circadian clock genes in bone marrow .
Neurogenesis, the continuous production of new neurons from a population of dividing neural progenitor cells, occurs in the hippocampal dentate gyrus. It is influenced by pathological situations such as ischemia or inflammation. BM may affect the production of neuronal survival factors such as brain-derived neurotrophic factor (BDNF, ID 382982, Figure 4C) gene, thereby promoting the survival of neuronal cells and thus, having an impact on neurogenetic processes .
Recent studies demonstrated that the expression of BNDF and its receptor TrkB is increased in mature neurons during the acute phase of pneumococcal meningitis . BDNF protein co-localizes with cells expressing TrkB in the hippocampal CA3/4 region and the hilus adjacent to the subgranular zone of the dentate gyrus where the proliferation of progenitor cells is increased. These findings indicate an involvement of endogenous BDNF and TrkB signaling in neurogenesis after BM . However, the persistence of neurological sequelae in up to 50% of survivors from BM [1, 3] suggests that endogenous mechanisms responsible for neuroregeneration are inefficient.
Since treatment with exogenous BDNF results in the reduction of various forms of cell death in experimental pneumococcal meningitis , one can speculate that the up-regulated expression level of BDNF in vitamin B6-treated animals plays an important role in diminishing hippocampal apoptosis.
BDNF induces the expression of many genes in hippocampal cells in culture, including activity regulated cytoskeletal-associated protein (ARC, ID 382171, Figure 4C) gene. ARC itself is involved in memory consolidation and long-term potentiation . Because injury to the hippocampal dentate gyrus is associated with learning and memory deficits , the up-regulation of ARC RNA in our study provides further evidence for a role of BDNF in the reduction of hippocampal apoptosis.
Another gene involved in neuronal signaling processes is early growth response 2 (EGR2, ID 438557, Figure 4C). EGR2 is an important mediator of the growth-suppressive signal of phosphatase and tensin homolog (PTEN) and plays a key role in the PTEN-induced apoptotic pathway. It alters the permeability of mitochondrial membranes, resulting in the release of cytochrome c which in turn activates caspase-3, -8 and −9. As an alternative route, EGR2 may directly induce the expression of pro-apoptotic factors of the Bcl-2 family . In the present study, EGR2 is up-regulated by vitamin B6 treatment. This result is not consistent with a reduction of apoptotic cell death by vitamin B6. This discrepancy between an induction of apoptosis by EGR2 and an up-regulation of EGR2 under circumstances that have been proven to diminish apoptosis may be due to different experimental conditions. In both studies, the molecular mechanisms of the apoptotic pathway were analyzed by microarrays, but we used an in vivo model system of BM, whereas cancer-derived cells served as in vitro culture system for the study performed by Unoki and Nakamura . Furthermore, posttranslational mechanisms such as phosphorylation, important for the biological activity of PTEN, are not considered in microarray experiments.
Members of the nuclear receptor subfamily 4 group A (NR4A) are classified as early response genes expressed in a wide variety of metabolically demanding and energy dependent tissues such as the brain. They are induced by a broad range of signals, including stress, growth factors, inflammatory cytokines, hormones, calcium, neurotransmitters and physical stimuli. Consistent with the pleiotropic physiological stimuli inducing the NR4A members, these receptors have been implicated in cell cycle regulation, apoptosis, neurological disease, inflammation, carcinogenesis and atherogenesis . Since BM is an inflammatory disease associated with brain damage due to hippocampal apoptosis and often leads to neurological deficits, the NR4A subfamily may play an essential role in this disease. In the present study, both member 1 and 2 of the NR4A family (NR4A1, ID 382110, and NR4A2, ID 438446) are up-regulated, suggesting an involvement in apoptotic processes (Figure 4C). Recent studies showed that the role of the Nr4A members in cancer is largely defined by the implication of the subfamily in the regulation of apoptosis . Furthermore, experimental studies with macrophages demonstrated an involvement of NR4A1 in modulating apoptosis in the inflammatory response. Recent work also suggested that in certain cell lines NR4A1 translocates to the mitochondria to release cytochrome c .
Platelet activating factor (PAF) is an extremely potent activator of inflammatory cells owing to the expression of its receptor by numerous cells of the innate immune system. Accordingly, hydrolysis of PAF by extracellular or intracellular PAF acetylhydrolases is predicted to inhibit inflammatory signaling. Indeed, expression of plasma PAF acetylhydrolase is increased by stimulation with inflammatory agonists such as LPS, and decreased by anti-inflammatory drugs . Given the possible anti-inflammatory effect of vitamin B6 as suggested by reduced levels of pro-inflammatory mediators (e.g. cytokines and chemokines) and diminished activation of inflammatory cells (e.g. microglia), vitamin B6 may down-regulate the expression of PAF hydrolase. This hypothesis was tested by the vitamin B6 induced attenuation of PAF acetylhydrolase 2 (PAFAH2, ID 549642, Figure 4D) levels in our study.
PAF induces apoptosis independent of its receptor, but the mechanism underlying this ability is not fully understood . However, PAFAH2 hydrolyzes not only PAF but also short-chain phospholipids . These substrates are pro-apoptotic, pointing to an essential role of PAFAH2 as anti-apoptotic agent . Recent studies reported that a transfection of the plasma PAFAH2 gene reduces glutamate-induced apoptosis in cultured rat cortical neurons. Moreover, studies using a mouse model of focal cerebral ischemia showed that PAFAH2 exerts strong neuroprotective effects against ischemic injury in the CNS by protecting neurons against oxidative stress . In this context, it seems that down-regulated PAFAH2 does not contribute to the processes leading to the reduced hippocampal apoptosis in vitamin B6-treated rats.
Beside the role of matrix metalloproteinases (MMPs) in blood–brain barrier disruption and extravasation of inflammatory cells into the CNS [3, 59], recent studies suggested an involvement of MMPs in glial and neuronal cell death. Furthermore, an excessive increase of MMP-9 (ID 382260, Figure 4D) in BM has been identified as a risk factor for the development of neurological sequelae . Therefore, the down-regulation of MMP-9 upon vitamin B6 treatment indicates a long-term effect of vitamin B6 in terms of reduced learning and memory impairments.
MMPs are also increased by antimicrobial peptides. Antimicrobial peptides are effector molecules of the innate immune system with antibiotic function. Aside from their antibiotic functions, they may be involved in immune responses and inflammatory disease. For example, they may amplify inflammation by activation of cytokine and chemokine expression in immune cells . Lysozyme (Lyz) is an antimicrobial protein belonging to the defensin family of host-defense proteins which are distributed widely in biological fluids and tissues. Experimental studies with transgenic mice showed that Lyz raises the levels of antioxidant reserves that are required to manage non-pathological amounts of reactive oxygen species. These antioxidant properties are partly mediated via negative regulation of stress response genes (e.g. c-Jun) and also involve the blockade of cellular apoptosis in vitro. However, Brandenburg et al. reported that there is no increase of Lyz in the CSF and serum samples from patients with meningitis . In the present study, we found a down-regulation of Lyz 2 (ID 378051, Figure 4D) in vitamin B6-treated rats when compared to saline-treated animals. This down-regulation could be a further indication of a reduced inflammation and in this context, would explain the reduced levels of pro-inflammatory cytokines and chemokines.
Recent studies showed that adjuvant BDNF protects the brain from caspase 3-dependent hippocampal apoptosis in experimental BM . In the present study, up-regulated endogenous BDNF is also involved in apoptotic processes as indicated by the apoptotic cell death cluster (Figure 4D). This result provides further evidence for a crucial role of BDNF in reducing hippocampal apoptosis upon vitamin B6 treatment.
But how does vitamin B6 induce BDNF expression? Several studies showed that BDNF expression in neuronal cells is induced by activation of calcium channels and recruitment of calcium-sensitive transcription factors . The excitatory amino acid glutamate which is increased in interstitial brain fluid in BM  induces a calcium influx by binding to the NMDA receptor and thus, may stimulate the production of BDNF . On the contrary, KYNA, the neuroprotective intermediate of the KYN pathway, is an antagonist of the NMDA receptor and therefore, inhibits calcium influx. Moreover, in vitro studies using rat cerebral cortex nerve terminals showed that vitamin B6 inhibits glutamate release through the suppression of calcium influx .
However, other studies reported that high levels of IL-1β decrease BDNF mRNA expression in the rat hippocampus . Thus, the increased amount of BDNF transcripts in vitamin B6-treated rats may result from decreased levels of IL-1β. This suggestion is also supported by the down-regulation of the IL-1R type I gene as discussed previously.
A related phenomenon could be observed in the brains of rats administered an antibiotic plus dexamethasone. Given the up-regulation of BDNF RNA and protein in this study, Li et al. hypothesize that the adjuvant therapy with dexamethasone might have a beneficial effect on BM via up-regulation of neuroprotective BDNF. In addition, this study demonstrated a dose-dependent down-regulation of BDNF RNA and protein in rats treated with antibiotics alone. A possible reason for this finding is the lysis of bacteria caused by the antibiotic treatment, resulting in the release of bacterial components that stimulate the expression of pro-inflammatory mediators such as IL-1β .