Neurodegeneration is defined as the loss of the structure and function of neurons . Neurodegeneration due to microglial activation and inflammation is seen in many Central Nervous System (CNS) pathologies, especially neurodegenerative diseases (ND) [2, 3]. NDs that involve neurodegeneration include Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), and Parkinson’s disease (PD) [1, 4]. Although these diseases are characterized by neurodegeneration, they differ in the area of the brain that is affected, leading to the different pathologies that exist for each type of ND . In Alzheimer’s disease, chronic inflammation causes neuronal cell death in the areas of the hippocampus and frontal cortex . Inflammation destroys motor neurons in the spinal cord, brain stem, and cortex in Amyotrophic Lateral Sclerosis [4, 5]. In Parkinson’s disease, chronic inflammation causes the loss of dopaminergic receptors in the substantia nigra . Lastly, Multiple Sclerosis is an autoimmune disorder where inflammatory cells attack the myelin sheath that surrounds the axons of neurons .
The brain is separated from the periphery by the blood brain barrier, which allows the brain to be immuno-priviledged . Inflammation is an activated immune state and is considered a normal self-defense mechanism that is implemented by the body in order to fight pathogens . Inflammation in the body recruits immune cells to the area that is under attack, which will then clear the system of the antigen . When inflammation occurs in the CNS, microglia are recruited to the affected area . Microglia are considered the “resident macrophage” of the brain [2, 4]. When in their resting state, microglia perform routine maintenance and immune surveillance [4, 5]. Once activated, either by injury or an immune stimulus, microglia secrete a variety of pro-inflammatory molecules, such as Nitric Oxide (NO), superoxide, inflammatory cytokines, reactive oxygen species (ROS), and glutamate [2–6]. Activated microglia also express inducible Nitric Oxide synthase (iNOS), as well as cyclo-oxygenase-2 (COX-2), which cause the production and subsequent release of NO and pro-inflammatory cytokines . Neuronal cell death will not occur if the inflammatory response is transient . However, if a prolonged inflammatory response occurs, chronic inflammation, neurodegeneration and neuronal cell death will also occur [2, 4]. Neuronal cell death leads to reactive microgliosis, the activation of microglia as a result of neuronal death . Reactive microgliosis is toxic to surrounding neurons and results in continued microglial activation, inflammation, and neuronal death .
Microglia can be activated in a number of ways, including injury or immunological stimuli [3, 4]. Microglial activation, due to immunological stimuli, occurs through a Toll-Like Receptor (TLR) pathways . TLR pathways are considered the first line of defense against viral and bacterial pathogens . Toll-like receptors are a family of nine receptors (TLR1-9) that are found on the cell’s plasma membrane and on the surface of endosomal vesicles, which specifically recognize conserved pathogen-associated molecular patterns (PAMPs) that recognize a variety of pathogens (bacteria, viruses, parasites, yeast and fungi) . Microglial expression of TLRs is undetectable when in their resting state . However, once activated, microglia rapidly express a variety of toll-like receptors (TLR1-9) at differing intesities . It should be noted that over stimulation of TLRs can result in chronic inflammation, leading to many inflammatory diseases .
Microglial TLRs can be activated by exogenous and endogenous TLR ligands, including Lipopolysaccharide (LPS) and Polyinosinic-Polycytidylic acid (Poly I:C) . LPS, an endotoxin found in gram-negative bacteria activates TLR-4 receptors expressed on microglia [2, 8]. Poly I:C activates TLR-3 receptors on microglia by mimicking the viral double stranded RNA observed during viral replication . When microglial TLRs are stimulated by LPS or Poly I:C, signaling occurs and causing the production and subsequent secretion of inflammatory molecules, reactive oxygen species, and glutamate . These molecules are neurotoxic and cause neurodegeneration . The extent of neurodegeneration that occurs depends on the intensity and length of microglial activation .
Cytokines are secreted by immune cells under a variety of conditions . Cytokines regulate inflammatory processes and are also key regulators of ND pathologies . As the brain ages, the blood–brain barrier becomes compromised, leading to an increase in the synthesis of pro-inflammatory cytokines such as IL-6, TNF-α and IL-1β . This causes continued microglial activation and neuro-inflammation . IL-6, TNF-α and IL-1β induce expression of the cyclo-oxygenase 2 (COX-2) enzyme. Over expression of COX-2 has been shown to be involved in neuronal apoptosis . TNF-α and IL-1β have also been shown to influence synaptic transmission in vitro. TNF-α is also thought to cause neurodegeneration by silencing cell survival signals and activating caspase-dependent pathways . TNF-α causes glutamate release by activated microglia, leading to excitoneurotoxicity and neuronal damage . Excessive IL-6 and IL-1β have been identified as being neurotoxic . However, it is not known if IL-6 and IL-1β are directly causing neurotoxicity, or it is mediated by other molecules such as ROS or glutamate . In regards to specific neurodegenerative diseases, brains affected by Alzheimer’s disease have increased levels of pro-inflammatory cytokines . Pro-inflammatory cytokines have also been suspected of being able to determine the extent of neurodegeneration that is seen in Multiple Sclerosis .
Excessive NO production is associated with both acute and chronic inflammation . Neurons are very susceptible to Nitric Oxide-induced death and very low concentrations can cause extensive neuronal damage and death [3, 13]. Recent studies suggest that activated microglia kill co-cultured neurons through a NO and ROS-mediated mechanism . Glutamate-mediated excitotoxicity has also been implicated in causing neuronal injury and death . The following mechanism for Nitric Oxide and glutamate-mediated neuronal cell death has been proposed: NO is released by activated microglia and inhibits neuronal respiration, causing glutamate to be released by neurons . Glutamate then binds to NMDA receptors present on neuronal cells, causing an extreme calcium influx, and ultimately neuronal cell death .
ROS and oxidative stress have been implicated in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis . In Alzheimer’s disease, plaque formation and accumulation lead to an inflammatory response that causes the production of ROS, and cause oxidative damage to surrounding neurons . Plaque accumulation also causes oxidative damage, which leads to mitochondrial dysfunction . Together, mitochondrial dysfunction and oxidative damage can also cause amyloid aggregation and tau polymerization . In Parkinson’s disease, increases in the generation of reactive oxygen species, as well as lipid peroxidation, are seen in the substantia nigra [4, 14]. Lastly, the pathogenesis of familial and sporadic Amyotrophic Lateral Sclerosis has been shown to involve ROS and oxidative stress .
Recent studies suggest that Docosahexaenoic Acid (DHA), a 22-carbon, long-chain polyunsaturated fatty acid (LC-PUFA), could have beneficial effects in brain diseases . The combination of aspirin and DHA has been shown to generate a number of anti-inflammatory species . DHA is a major fatty acid that makes up about 12-16% of total fatty acids in the grey matter of the brain . DHA is important in proper brain development, and plays a role in maintaining a homeostatic environment in the CNS [15, 18, 19]. DHA has been shown to modulate important neurochemical processes, synaptic plasticity, memory formation, neuroprotection, gene expression, and intracellular calcium concentrations [15, 19]. DHA promotes neural stem cell differentiation into neurons, as well as neurogenesis . DHA and its derivatives, whether made by peroxidation or enzymatic processing, all have potent anti-inflammatory properties in both acute and chronic ND . DHA has been shown to reduce the number of activated microglia and reduce pro-inflammatory molecule production .
DHA is known for its anti-inflammatory properties and has been shown to reduce pro-inflammatory cytokine production in microglia [7, 15]. The NF-κB signaling pathway is important for mediating the expression of genes such as COX-2 and iNOS, which encode pro-inflammatory molecules, such as Nitric Oxide and prostaglandins [7, 12, 20]. DHA inhibits pro-inflammatory cytokine production by preventing NK-κB translocation to the nucleus . This causes a decrease in the transcription of pro-inflammatory genes (COX-2 and iNOS) . Ultimately, pro-inflammatory cytokines and Nitric Oxide production is reduced .
DHA also causes an increased level of intracellular Glutathione (GSH), a potent antioxidant molecule that is found in the brain at high concentrations . High levels of GSH have been shown to be important for suppressing NF-κB activation . Glutathione is also a cofactor for Glutathione Peroxidase (GPx), an enzyme that converts hydrogen peroxide to water . High expression of GPx has been shown to inhibit the degredation of IκB, as well as inhibit the activation of NF-κB . Increased levels of GSH, caused by DHA, enhance the activity of GPx, which further inhibits the transcription and translation of pro-inflammatory molecules through NF-κB signaling . DHA also increases the activity of Glutathione Reductase (GR), an enzyme that is important in maintaining the anti-oxidative capacity of cells by a GSH-based mechanism [12, 17]. In turn, the occurrence of up-regulated GPx and GR in the brain causes an increase in GSH, and an enhancement of the anti-oxidative defense mechanism employed by the brain .
Nitric Oxide mediates inflammatory processes . Recently, DHA has been shown to reduce iNOS expression and NO production in microglia . DHA has also been shown to inhibit iNOS expression and NO production in murine macrophages . DHA also down-regulates the expression of genes involved in ROS production . It is thought that this reduction occurs through up-regulation of the anti-oxidative capacity of the macrophages by enhancing a Glutathione-mediated anti-oxidative mechanism .
Dietary intake is the main source of DHA in humans and when adequate, it offers visual, neurological and cardiovascular health benefits [12–15, 17, 18]. Decreased DHA intake can lead to oxidative damage, and has been shown to cause cognitive insufficiencies and impaired vision [4, 18]. Reduced DHA intake in adults has been shown to contribute to age-related cognitive deficiencies, as well as neuronal dysfunction . Low DHA in the blood is hypothesized to be an important risk factor in developing Alzheimer’s disease [17, 23].
Increased dietary intake has been shown to significantly alter DHA levels in the brain . This suggests that DHA supplementation could be used to directly influence brain function . Clinical studies suggest dietary DHA supplementation can alter the risk of developing Alzheimer’s disease . Participants with the highest level of DHA in their blood also had a decreased risk of developing dementia . Moderate increases of DHA in the daily diet have been shown to reduce the risk of developing Alzheimer’s disease by 60% . It should be noted that elderly people who eat fish and seafood enriched with omega-3 PUFAs (i.e.: DHA) at least once a week have a decreased risk of developing dementia and Alzheimer’s disease . The aforementioned data suggests that DHA could be an effective therapy for preventing Alzheimer’s disease, as well as other neurodegenerative diseases .
Non-steroidal anti-inflammatory drugs (NSAIDs) have been proposed as a possible preventative treatment for NDs . NSAIDs, as their name describes, have anti-inflammatory properties and can be selective for cyclo-oxygenase (COX) -1, COX-2, or both . NSAIDs inhibit the production of Nitric Oxide, as well as pro-inflammatory cytokine production  NSAIDs that specifically target COX-2 have been shown to reduce microglial activation, block the production of pro-inflammatory cytokines, and reduce the risk of Alzheimer’s disease . NSAIDs have also been shown to block the production and accumulation of degenerative proteins, thereby reducing the risk of Alzheimer’s disease . NSAIDs that target COX-2 have been shown to improve cognitive and motor functions in mice [4, 23]. Epidemiological studies have shown an inverse relationship between NSAID intake and the development of Alzheimer’s and Parkinson’s diseases [23, 27]. NSAIDs are also proposed to have an impact on the inflammatory component of Multiple Sclerosis and Amyotrophic Lateral Sclerosis . Ibuprofen, a NSAID that is non-selective in terms of COX-1 and COX2, has been proposed through epidemiological studies as a possible preventative treatment for Alzheimer’s disease . However, Ibuprofen has concerning side effects that have prevented it from being used in clinical trials for Alzheimer’s prevention .
COX-2 in neurons, neurodegeneration caused by excitotoxicity, as well as neurodegernation caused by microglia, are proposed as the main targets of NSAIDs . With this information, classical NSAIDs are logically an attractive option for delaying the onset and slowing the progression of neurodegenerative diseases . Combination therapies of different types of anti-inflammatory agents are also proposed as a preventative therapy for neurodegenerative diseases because they can work through different mechanisms .
This research project sought to elucidate whether microglial activation can be modulated by combining Aspirin, a classical NSAID, with Docosahexaenoic Acid, a naturally occurring anti-inflammatory agent. The combined ability of Aspirin and DHA to modulate activated microglia was determined in the context of pro-inflammatory cytokines, Nitric Oxide levels, Reactive Oxygen Species, as well as total Glutathione levels.