A survey of neuroimmune changes in pregnant and postpartum female rats.

During pregnancy and the postpartum period, the adult female brain is remarkably plastic exhibiting modifications of neurons, astrocytes and oligodendrocytes. However, little is known about how microglia, the brain's innate immune cells, are altered during this time. In the current studies, microglial density, number and morphological phenotype were analyzed within multiple regions of the maternal brain that are known to show neural plasticity during the peripartum period and/or regulate peripartum behavioral changes. Our results show a significant reduction in microglial density during late pregnancy and the early-mid postpartum period in the basolateral amygdala, medial prefrontal cortex, nucleus accumbens shell and dorsal hippocampus. In addition, microglia numbers were reduced postpartum in all four brain regions, and these reductions occurred primarily in microglia with a thin, ramified morphology. Across the various measures, microglia in the motor cortex were unaffected by reproductive status. The peripartum decrease in microglia may be a consequence of reduced proliferation as there were fewer numbers of proliferating microglia, and no changes in apoptotic microglia, in the postpartum hippocampus. Finally, hippocampal concentrations of the cytokines interleukin (IL)-6 and IL-10 were increased postpartum. Together, these data point to a shift in the maternal neuroimmune environment during the peripartum period that could contribute to neural and behavioral plasticity occurring during the transition to motherhood.

Determining the role of IL-4 induced neuroinflammation in microglial activity and amyloid-ß using BV2 microglial cells and APP/PS1 transgenic mice.

BACKGROUND: Microglia are considered the resident immune cells of the central nervous system (CNS). In response to harmful stimuli, an inflammatory reaction ensues in which microglia are activated in a sequenced spectrum of pro- and antiinflammatory phenotypes that are akin to the well-characterized polarization states of peripheral macrophages. A "classically" activated M1 phenotype is known to eradicate toxicity. The transition to an "alternatively" activated M2 phenotype encompasses neuroprotection and repair. In recent years, inflammation has been considered an accompanying pathology in response to the accumulation of extracellular amyloid-ß (Aß) in Alzheimer's disease (AD). This study aimed to drive an M2a-biased immune phenotype with IL-4 in vitro and in vivo and to determine the subsequent effects on microglial activation and Aß pathology. METHODS: In vitro, exogenous IL-4 was applied to BV2 microglial cell cultures to evaluate the temporal progression of microglial responses. In vivo, intracranial injections of an adeno-associate-virus (AAV) viral vector were performed to assess long-term expression of IL-4 in the frontal cortex and hippocampus of Aß-depositing, APP/PS1 transgenic mice. Quantitative real-time PCR was used to assess the fold change in expression of biomarkers representing each of the microglial phenotypes in both the animal tissue and the BV2 cells. ELISAs quantified IL-4 expression and Aß levels. Histological staining permitted quantification of microglial and astrocytic activity. RESULTS: Both in vitro and in vivo models showed an enhanced M2a phenotype, and the in vivo model revealed a trend toward a decreased trend in Aß deposition. CONCLUSIONS: In summary, this study offers insight into the therapeutic potential of microglial immune response in AD.

Transition from an M1 to a mixed neuroinflammatory phenotype increases amyloid deposition in APP/PS1 transgenic mice.

BACKGROUND: The polarization to different neuroinflammatory phenotypes has been described in early Alzheimer's disease, yet the impact of these phenotypes on amyloid-beta (Aß) pathology remains unknown. Short-term studies show that induction of an M1 neuroinflammatory phenotype reduces Aß, but long-term studies have not been performed that track the neuroinflammatory phenotype. METHODS: Wild-type and APP/PS1 transgenic mice aged 3 to 4 months received a bilateral intracranial injection of adeno-associated viral (AAV) vectors expressing IFNγ or green fluorescent protein in the frontal cortex and hippocampus. Mice were sacrificed 4 or 6 months post-injection. ELISA measurements were used for IFNγ protein levels and biochemical levels of Aß. The neuroinflammatory phenotype was determined through quantitative PCR. Microglia, astrocytes, and Aß levels were assessed with immunohistochemistry. RESULTS: AAV expressing IFNγ induced an M1 neuroinflammatory phenotype at 4 months and a mixed phenotype along with an increase in Aß at 6 months. Microglial staining was increased at 6 months and astrocyte staining was decreased at 4 and 6 months in mice receiving AAV expressing IFNγ. CONCLUSIONS: Expression of IFNγ through AAV successfully induced an M1 phenotype at 4 months that transitioned to a mixed phenotype by 6 months. This transition also appeared with an increase in amyloid burden suggesting that a mixed phenotype, or enhanced expression of M2a and M2c markers, could contribute to increasing amyloid burden and disease progression.

Cardiopulmonary arrest and resuscitation disrupts cholinergic anti-inflammatory processes: a role for cholinergic α7 nicotinic receptors.

Cardiac arrest is a leading cause of death worldwide. While survival rates following sudden cardiac arrest remain relatively low, recent advancements in patient care have begun to increase the proportion of individuals who survive cardiac arrest. However, many of these individuals subsequently develop physiological and psychiatric conditions that likely result from ongoing neuroinflammation and neuronal death. The present study was conducted to better understand the pathophysiological effects of cardiac arrest on neuronal cell death and inflammation, and their modulation by the cholinergic system. Using a well validated model of cardiac arrest, here we show that global cerebral ischemia increases microglial activation, proinflammatory cytokine mRNA expression (interleukin-1ß, interleukin-6, tumor necrosis factor-α), and neuronal damage. Cardiac arrest also induces alterations in numerous cellular components of central cholinergic signaling, including a reduction in choline acetyltransferase enzymatic activity and the number of choline acetyltransferase-positive neurons, as well as, reduced acetylcholinesterase and vesicular acetylcholine transporter mRNA. However, treatment with a selective agonist of the α7 nicotinic acetylcholine receptor, the primary receptor mediating the cholinergic anti-inflammatory pathway, significantly decreases the neuroinflammation and neuronal damage resulting from cardiac arrest. These data suggest that global cerebral ischemia results in significant declines in central cholinergic signaling, which may in turn diminish the capacity of the cholinergic anti-inflammatory pathway to control inflammation. Furthermore, we provide evidence that pharmacological activation of α7 nicotinic acetylcholine receptors provide significant protection against ischemia-related cell death and inflammation within a clinically relevant time frame.

Cannabinoids attenuate the effects of aging upon neuroinflammation and neurogenesis.

WIN-55,212-2 (WIN-2) can elicit anti-inflammatory and cognitive-enhancing effect in aged rats. The current study was designed to determine the differential role of the endocannabinoid receptor sub-types 1 (CB1) and 2 (CB2) and transient receptor potential vanilloid 1 receptor (TRPV1) in the reduction of age-associated brain inflammation and their effects on neurogenesis in the dentate gyrus of aged rats. Our results demonstrate that 1) the antagonist actions of WIN-2 at the TRPV1 receptor are responsible for the reduction in microglial activation and 2) the agonist actions of WIN-2 at CB1/2 receptors can trigger neurogenesis in the hippocampus of aged rats. Chronic treatment with WIN-2 established an anti-inflammatory cytokine profile within the hippocampus. Our results provide insight into the role of the endocannabinoid and vanilloid systems upon two different and detrimental aspects of normal and pathological aging, chronic neuroinflammation and decline in neurogenesis.

Inflammation and aging: can endocannabinoids help?

Aging often leads to cognitive decline due to neurodegenerative process in the brain. As people live longer, there exists a growing concern linked to long-term, slowly debilitating diseases, such as Alzheimer's disease for which a cure has not yet been found. Recently, the role of neuroinflammation has attracted attention due to its slow onset, chronic nature and its possible role in the development of many different neurodegenerative diseases. In the future, treatment of chronic neuroinflammation may help counteract aspects of neurodegenerative disease. Our recent studies have focused upon the endocannabinoid system for its unique effects on the expression of neuroinflammation. The basis for the manipulation of the endocannabinoid system in the brain in combination with existing treatments for Alzheimer's disease will be discussed in this review.

Alzheimer's Amyloid-ß is an Antimicrobial Peptide: A Review of the Evidence.

The amyloid-ß (Aß) peptide has long been considered to be the driving force behind Alzheimer's disease (AD). However, clinical trials that have successfully reduced Aß burden in the brain have not slowed the cognitive decline, and in some instances, have resulted in adverse outcomes. While these results can be interpreted in different ways, a more nuanced picture of Aß is emerging that takes into account the facts that the peptide is evolutionarily conserved and is present throughout life in cognitively normal individuals. Recent evidence indicates a role for Aß as an antimicrobial peptide (AMP), a class of innate immune defense molecule that utilizes fibrillation to protect the host from a wide range of infectious agents. In humans and in animal models, infection of the brain frequently leads to increased amyloidogenic processing of the amyloid-ß protein precursor (AßPP) and resultant fibrillary aggregates of Aß. Evidence from in vitro and in vivo studies demonstrates that Aß oligomers have potent, broad-spectrum antimicrobial properties by forming fibrils that entrap pathogens and disrupt cell membranes. Importantly, overexpression of Aß confers increased resistance to infection from both bacteria and viruses. The antimicrobial role of Aß may explain why increased rates of infection have been observed in some of the AD clinical trials that depleted Aß. Perhaps progress toward a cure for AD will accelerate once treatment strategies begin to take into account the likely physiological functions of this enigmatic peptide.

The Physiological Roles of Amyloid-ß Peptide Hint at New Ways to Treat Alzheimer's Disease.

Amyloid-ß (Aß) is best known as the misfolded peptide that is involved in the pathogenesis of Alzheimer's disease (AD), and it is currently the primary therapeutic target in attempts to arrest the course of this disease. This notoriety has overshadowed evidence that Aß serves several important physiological functions. Aß is present throughout the lifespan, it has been found in all vertebrates examined thus far, and its molecular sequence shows a high degree of conservation. These features are typical of a factor that contributes significantly to biological fitness, and this suggestion has been supported by evidence of functions that are beneficial for the brain. The putative roles of Aß include protecting the body from infections, repairing leaks in the blood-brain barrier, promoting recovery from injury, and regulating synaptic function. Evidence for these beneficial roles comes from in vitro and in vivo studies, which have shown that the cellular production of Aß rapidly increases in response to a physiological challenge and often diminishes upon recovery. These roles are further supported by the adverse outcomes of clinical trials that have attempted to deplete Aß in order to treat AD. We suggest that anti-Aß therapies will produce fewer adverse effects if the known triggers of Aß deposition (e.g., pathogens, hypertension, and diabetes) are addressed first.

Safety and Efficacy of Anti-Amyloid-ß Immunotherapy in Alzheimer's Disease: A Systematic Review and Meta-Analysis.

Immunotherapeutics targeting amyloid-ß (Aß) have had mixed results in clinical trials. The present study aims to evaluate the safety and clinical efficacy of immunotherapeutic agents targeting Aß in Alzheimer's disease. Randomised controlled trials of at least two weeks duration were included in the review. Fourteen randomised controlled trials (n = 5554) were identified in a systematic search of eight electronic databases. Upon pooling of data, there was no increased risk of any adverse event, serious adverse events, or death with the exception of a near fivefold increase in amyloid-related imaging abnormalities (ARIA; OR 4.79, 95% CI 1.24-18.55; p = 0.02). Of the cognitive indicators, the Mini-Mental State Examination (MMSE) showed a small statistically significant improvement (diff in means =0.44; p = 0.02), while the others (ADAS-cog, ADCS-ADL, and CDR-sb) showed no change. Therefore, immunotherapeutic agents have been relatively well tolerated, with some promise for cognitive improvements if the occurrence of ARIA can be mitigated.

Pharmacological Agents Targeting γ-Secretase Increase Risk of Cancer and Cognitive Decline in Alzheimer's Disease Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Drugs targeting γ-secretase in Alzheimer's disease (AD) have failed to demonstrate efficacy in clinical trials. OBJECTIVE: To perform a meta-analysis of randomized controlled trials (RCTs) to evaluate the efficacy and safety of drugs targeting γ-secretase in AD. METHODS: Ten trials were identified involving 5,227 patients using electronic databases and manual review of reference lists. RCTs of at least two weeks duration involving a drug targeting γ-secretase were eligible. The main outcomes examined were adverse events and cognitive measures (ADAS-cog, MMSE, ADCS-ADL, and CDR-sb). A sub-group analysis was performed, excluding the γ-secretase modulator tarenflurbil, to evaluate the safety and efficacy of γ-secretase inhibitors only. RESULTS: There was an increased risk of adverse events (Odds Ratio (OR) 1.38, 95% CI 1.09-1.73; p = 0.01), serious adverse events (OR 1.50, 95% CI 1.22-1.84; p < 0.001), and skin cancers (OR 4.77, 95% CI 2.83-8.06; p < 0.001). There was significantly increased risk of infections (OR 1.36, 95% CI 1.13-1.63; p < 0.001) in the subgroup analysis excluding tarenflurbil. Pooled results also revealed a worsening in ADAS-cog (difference in means 1.33, 95% CI 0.58-2.08; p < 0.001) and MMSE (difference in means -0.66, 95% CI -0.96 to 0.35; p < 0.001), but not ADCS-ADL or CDR-sb. CONCLUSION: The use of γ-secretase inhibitors is associated with significantly increased risk of serious adverse events including skin cancers, and worsening in cognitive indicators. This evidence indicates that γ-secretase may not be an appropriate target for clinical treatment of AD.

Age and duration of inflammatory environment differentially affect the neuroimmune response and catecholaminergic neurons in the midbrain and brainstem.

Neuroinflammation and degeneration of ascending catecholaminergic systems occur early in the neurodegenerative process. Age and the duration of a pro-inflammatory environment induced by continuous intraventricular lipopolysaccharide (LPS) differentially affect the expression profile of pro- and anti-inflammatory genes and proteins as well as the number of activated microglia (express major histocompatibility complex II; MHC II) and the integrity and density of ascending catecholaminergic neural systems originating from the locus coeruleus (LC) and substantia nigra pars compacta (SNpc) in rats. LPS infusion increased gene expression and/or protein levels for both pro- and anti-inflammatory biomarkers. Although LPS infusion stimulated a robust increase in IL-1ß gene and protein expression, this increase was blunted with age. LPS infusion also increased the density of activated microglia cells throughout the midbrain and brainstem. Corresponding to the development of a pro-inflammatory environment, LC and SNpc neurons immunopositive for tyrosine-hydroxylase (the rate-limiting synthetic enzyme for dopamine and norepinephrine) decreased in number, along with a decrease in tyrosine-hydroxylase gene expression in the midbrain and/or brainstem region. Our data support the concept that continuous exposure to a pro-inflammatory environment drives exaggerated changes in the production and release of inflammatory mediators that interact with age to impair functional capacity of the SNpc and LC.

ß-amyloid deposition is shifted to the vasculature and memory impairment is exacerbated when hyperhomocysteinemia is induced in APP/PS1 transgenic mice.

INTRODUCTION: Vascular dementia is the second most common cause of dementia after Alzheimer's disease (AD). In addition, it is estimated that almost half of all AD patients have significant cerebrovascular disease comorbid with their AD pathology. We hypothesized that cerebrovascular disease significantly impacts AD pathological progression. METHODS: We used a dietary model of cerebrovascular disease that relies on the induction of hyperhomocysteinemia (HHcy). HHcy is a significant clinical risk factor for stroke, cardiovascular disease and type 2 diabetes. In the present study, we induced HHcy in APP/PS1 transgenic mice. RESULTS: While total ß-amyloid (Aß) load is unchanged across groups, Congophilic amyloid deposition was decreased in the parenchyma and significantly increased in the vasculature as cerebral amyloid angiopathy (CAA; vascular amyloid deposition) in HHcy APP/PS1 mice. We also found that HHcy induced more microhemorrhages in the APP/PS1 mice than in the wild-type mice and that it switched the neuroinflammatory phenotype from an M2a biased state to an M1 biased state. Associated with these changes was an induction of the matrix metalloproteinase protein 2 (MMP2) and MMP9 systems. Interestingly, after 6 months of HHcy, the APP/PS1 mice were cognitively worse than wild-type HHcy mice or APP/PS1 mice, indicative of an additive effect of the cerebrovascular pathology and amyloid deposition. CONCLUSIONS: These data show that cerebrovascular disease can significantly impact Aß distribution in the brain, favoring vascular deposition. We predict that the presence of cerebrovascular disease with AD will have a significant impact on AD progression and the efficacy of therapeutics.

Age-associated alterations in the time-dependent profile of pro- and anti-inflammatory proteins within the hippocampus in response to acute exposure to interleukin-1ß.

The pro-inflammatory cytokine IL-1ß is known to play a role in several models of aging, neuroinflammation, and neurodegenerative diseases. Here, we document a detailed time- and age-dependent pattern of pro- and anti-inflammatory biomarkers following bilateral intrahippocampal injection of interleukin-1ß. During the first 12h several pro- and anti-inflammatory cytokines increased in the aged (24 mo old) rats, some of which returned to baseline levels by 24h post-injection while others remained elevated for 72 h post-injection. In contrast, no such increases were observed in the young (3 mo old) rats. Interestingly, young rats up-regulated mRNA of two pro-inflammatory cytokines, interleukin-1ß and tumor necrosis factor-α, but did not translate these transcripts into functional proteins, which may be related to expression of suppressor of cytokine signaling type-2. These results contribute to our understanding of how neuroinflammation may contribute to the pathogenesis of age-related neurodegenerative disorders due to an age-related bias towards a hyper-reactive immune response that is not selective for a pro- or anti-inflammatory phenotype following an inflammatory stimulus.

Are inflammatory profiles the key to personalized Alzheimer's treatment?

There is currently no disease-modifying treatment for Alzheimer's disease (AD) and the need is great as the number of people diagnosed with AD is predicted to steadily increase. Inflammation is associated with AD, and is predictive of more advanced disease pathology and cognitive impairment. Moreover, preventing inflammation reduces the risk of developing AD. However, clinical trials with anti-inflammatory treatment have not been successful. One reason may be that there is diversity in the immune response and reducing immune activity with anti-inflammatories is not appropriate in all conditions. Recently, we have begun to apply categorizations, used to characterize the peripheral immune response, to the immune processes of the brain. When we do this, we are able to describe an individual's inflammatory profile within this spectrum. We have observed that patients with early AD are distributed across two broad categories of immune activation. If we recognize the diversity within this cohort of individuals with early AD and use information about immune phenotypes to guide the choice of treatment, then we may expect better clinical outcomes.

Differential effects of duration and age on the consequences of neuroinflammation in the hippocampus.

The current study investigated the hypothesis that the duration of the proinflammatory environment plays a critical role in the brain's response that results in negative consequences on cognition, biochemistry, and pathology. Lipopolysaccharide or artificial cerebrospinal fluid was slowly (250 ηg/h) infused into the fourth ventricle of young (3-month-old), adult (9-month-old), or aged (23-month-old) male F-344 rats for 21 or 56 days. The rats were then tested in the water pool task and endogenous hippocampal levels of pro- and anti-inflammatory proteins and genes and indicators of glutamatergic function were determined. The duration of the lipopolysaccharide infusion, compared with the age of the rat, had the greatest effect on (1) spatial working memory; (2) the density and distribution of activated microglia within the hippocampus; and (3) the cytokine protein and gene expression profiles within the hippocampus. The duration- and age-dependent consequences of neuroinflammation might explain why human adults respond positively to anti-inflammatory therapies and aged humans do not.

Riluzole partially rescues age-associated, but not LPS-induced, loss of glutamate transporters and spatial memory.

Impaired memory may result from synaptic glutamatergic dysregulation related to chronic neuroinflammation. GLT1 is the primary excitatory amino acid transporter responsible for regulating extracellular glutamate levels in the hippocampus. We tested the hypothesis that if impaired spatial memory results from increased extracellular glutamate due to age or experimentally induced chronic neuroinflammation in the hippocampus, then pharmacological augmentation of the glutamate transporter GLT1 will attenuate deficits in a hippocampal-dependent spatial memory task. The profile of inflammation-related genes and proteins associated with normal aging, or chronic neuroinflammation experimentally-induced via a four-week LPS infusion into the IV(th) ventricle, were correlated with performance in the Morris water maze following treatment with Riluzole, a drug that can enhance glutamate clearance by increasing GLT1 expression. Age-associated inflammation was qualitatively different from LPS-induced neuro-inflammation in young rats. LPS produced a pro-inflammatory phenotype characterized by increased IL-1ß expression in the hippocampus, whereas aging was not associated with a strong central pro-inflammatory response but with a mixed peripheral immune phenotype. Riluzole attenuated the spatial memory impairment, the elevation of serum cytokines and the decrease in GLT1 gene expression in Aged rats, but had no effect on young rats infused with LPS. Our findings highlight the therapeutic potential of reducing glutamatergic function upon memory impairment in neurodegenerative diseases associated with aging.

Time-Dependent Compensatory Responses to Chronic Neuroinflammation in Hippocampus and Brainstem: The Potential Role of Glutamate Neurotransmission.

Chronic neuroinflammation is characteristic of neurodegenerative diseases and is present during very early stages, yet significant pathology and behavioral deficits do not manifest until advanced age. We investigated the consequences of experimentally-induced chronic neuroinflammation within the hippocampus and brainstem of young (4 mo) F-344 rats. Lipopolysaccharide (LPS) was infused continuously into the IVth ventricle for 2, 4 or 8 weeks. The number of MHC II immunoreactive microglia in the brain continued to increase throughout the infusion period. In contrast, performance in the Morris water maze was impaired after 4 weeks but recovered by 8 weeks. Likewise, a transient loss of tyrosine hydroxylase immunoreactivity in the substantia nigra and locus coeruleus was observed after 2 weeks, but returned to control levels by 4 weeks of continuous LPS infusion. These data suggest that direct activation of microglia is sufficient to drive, but not sustain, spatial memory impairment and a decrease in tyrosine hydroxylase production in young rats. Our previous studies suggest that chronic neuroinflammation elevates extracellular glutamate and that this elevation underlies the spatial memory impairment. In the current study, increased levels of GLT1 and SNAP25 in the hippocampus corresponded with the resolution of performance deficit. Increased expression of SNAP25 is consistent with reduced glutamate release from axonal terminals while increased GLT1 is consistent with enhanced clearance of extracellular glutamate. These data demonstrate the capacity of the brain to compensate for the presence of chronic neuroinflammation, despite continued activation of microglia, through changes in the regulation of the glutamatergic system.

The neuron-astrocyte-microglia triad in normal brain ageing and in a model of neuroinflammation in the rat hippocampus.

Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.

Caffeine attenuates lipopolysaccharide-induced neuroinflammation.

Caffeine is an antagonist at A1 and A2A adenosine receptors and epidemiological evidence suggests that caffeine consumption reduces the risk of Alzheimer's and Parkinson's diseases. Neuroinflammation plays a role in the etiology of these diseases and caffeine may provide protection through the modulation of inflammation. Adenosine has a known role in the propagation of inflammation and caffeine may reduce microglia activation directly by blocking adenosine receptors on microglia. Chronic neuroinflammation is associated with an increase in extracellular levels of glutamate and drugs that limit the effects of glutamate at neuronal receptors have been shown to indirectly reduce the neuroinflammatory response of microglia cells. A1 and A2A receptors have been shown to regulate the pre-synaptic release of glutamate, therefore, caffeine may also reduce neuroinflammation via its ability to regulate glutamate release. Caffeine was administered at various doses to young rats with experimentally induced neuroinflammation by chronic infusion of lipopolysaccharide (LPS) over two or four weeks into the 4th ventricle and to aged rats with naturally elevated levels of microglia activation. Caffeine attenuated the number of activated microglia within the hippocampus of animals with LPS-induced and age-related inflammation.