No description available.

Several studies have shown that dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) are beneficial in neurodegenerative diseases, although the mechanism of action is not agreed upon. Because most neurodegenerative diseases have an inflammatory component, it is possible that docosahexaenoic acid (DHA) is anti-inflammatory in the brain as it is known to be in several non-neural tissues. Specialized pro-resolving mediators (SPM) are metabolized from DHA and are leading candidates to explain the anti-inflammatory effects of DHA. The goal of this work was to investigate the role and potential mechanisms of action of DHA in neuroinflammation. In our first approach, fat-1 transgenic mice had higher phospholipid and unesterified DHA levels in their hippocampi, and attenuated lipopolysaccharide (LPS)-induced neuroinflammation, compared to wildtype littermates. Feeding wildtype littermates n-3 PUFA mimicked hippocampal DHA levels and LPS-induced neuroinflammatory responses of fat-1 mice, indicating DHA is anti-neuroinflammatory whether derived from the diet or the activity of the fat-1 protein. In an attempt to further augment hippocampal DHA levels, feeding n-3 PUFA adequate mice an n-3 PUFA diet increased phospholipid but not unesterified DHA levels, and did not attenuate LPS-induced neuroinflammation, highlighting the potential importance of unesterified DHA. Directly infusing unesterified DHA into a cerebral ventricle throughout LPS-induced neuroinflammation mimicked several aspects of the attenuated neuroinflammatory response seen with our chronic dietary and transgenic models, as did infusing its 17S-hydroperoxy-DHA (17S-HpDHA) derivative, a precursor to SPM. The metabolism of DHA to SPM in the brain was found to be distinct from non-neural tissues, characterized by the presence of protectin D1 and maresin 1, and the absence of resolvin D1 or D2. Further, infusing 17S-HpDHA increased protectin D1 concurrent to attenuating neuroinflammation, suggesting protectin D1 is responsible for some of the anti-neuroinflammatory effects of DHA. In conclusion, DHA is anti-neuroinflammatory in a mouse model of neuroinflammation, in part, via its metabolism to SPM.

Microglia, the resident immune cells of the central nervous system (CNS), become activated in response to danger signals given out by other cells when homeostasis has been disturbed. Microglial activation is a multifaceted phenomenon that includes numerous distinct phenotypes. The type of activation often influences the survival of surrounding CNS tissue, and thus gaining a better understanding of how microglial activation is regulated has important therapeutic implications. Currently, it is known that the phenotype of activated microglia depends on both the type of CNS insult and the specific activating agent. The aim of this thesis was to investigate the potential involvement of other determining factors. Extrinsic regulators of microglial activation, including the severity of CNS insult and the stimulation strength of activating agents, were examined. Intrinsic differences among different microglial populations, namely differences in region of origin and age of origin, were also investigated. To study microglial behavior without interference from other cells, rat primary cultures were used as the system of study. With regard to extrinsic factors, it was found that different severities of hypoxic neuronal injury induced distinct microglial phenotypes. Among the activating agents released by injured neurons, adenosine 5-triphosphate (ATP) was studied in isolation and was found to induce trophic and toxic effectors in microglia depending on the strength of ATP stimulation. In regards to intrinsic factors, it was found that microglia derived from different regions of the brain had distinct responses to activators, with cortical and hippocampal microglia generating more toxic responses than brainstem, striatal, and thalamic microglia. Microglia derived from various ages of origin also responded differentially to activators, with neonatal and aged microglia being more reactive than microglia derived from other age groups. Together, the results here present several novel concepts, that the phenotype of activated microglia are dependent not only on the type of activating stimulus, but the strength of that stimulus, and that in addition to stimuli from other cells, the regional and age differences among microglia themselves are also crucial in determining their activation phenotype.

Background: Cerebral ischemia is a leading cause of death and disability with limited treatment options. Although inflammatory and immune responses participate in ischemic brain injury, the molecular regulators of neuroinflammation after ischemia remain to be defined. Translocator protein 18 kDa (TSPO) mainly localized to the mitochondrial outer membrane is predominantly expressed in glia within the central nervous system during inflammatory conditions. This study investigated the effect of a TSPO agonist, etifoxine, on neuroinflammation and brain injury after ischemia/reperfusion. Methods: We used a mouse model of middle cerebral artery occlusion (MCAO) to examine the therapeutic potential and mechanisms of neuroprotection by etifoxine. Results: TSPO was upregulated in Iba1(+) or CD11b(+) CD45(int) cells from mice subjected to MCAO and reperfusion. Etifoxine significantly attenuated neurodeficits and infarct volume after MCAO and reperfusion. The attenuation was pronounced in mice subjected to 30, 60, or 90 min MCAO. Etifoxine reduced production of pro-inflammatory factors in the ischemic brain. In addition, etifoxine treatment led to decreased expression of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and inducible nitric oxide synthase by microglia. Notably, the benefit of etifoxine against brain infarction was ablated in mice depleted of microglia using a colony-stimulating factor 1 receptor inhibitor. Conclusions: These findings indicate that the TSPO agonist, etifoxine, reduces neuroinflammation and brain injury after ischemia/reperfusion. The therapeutic potential of targeting TSPO requires further investigations in ischemic stroke.

No description available.

Neuroinflammation has been linked to the pathogenesis of many diseases, including chronic traumatic encephalopathy (CTE). CTE is a progressive neurodegenerative disease caused by exposure to repeated head impacts (RHI) from a variety of sources, including contact sports and military injury. CTE is characterized neuropathologically by the deposition of hyperphosphorylated tau (p-tau) in neurons as neurofibrillary tangles (NFT) and neurites at the depths of the cortical sulci in an irregular pattern. In addition to p-tau accumulation, there is also an accumulation of pigment-containing macrophages around small blood vessels in the white matter and widespread microglial inflammation in CTE. Macrophage and microglial inflammation can be beneficial to tissue repair, but if persistent, can precipitate neurodegeneration. This study quantified the density of perivascular CD68+ macrophages in the dorsolateral prefrontal (DLF) white matter, a brain region known to be affected early in CTE in post-mortem brain tissue from 46 individuals, 7 controls (mean age: 46.14, SD: 11.39, range: 22-55), 20 individuals exposed to RHI without CTE (mean age: 22.75, SD: 3.65, range: 17- 29), and 19 individuals, all of them American football players, with pathologically verified CTE (mean age: 25.11, SD: 2.92, range: 18-29). Brain tissue was provided by the Injury and Traumatic Encephalopathy (UNITE) brain bank and the post-traumatic stress disorder (PTSD) brain bank. Comparisons were made between controls, individuals exposed to RHI without CTE, and individuals with CTE. Fixed tissue samples of the DLF cortex and white matter were cut at 10μm and stained with CD68 to mark perivascular macrophages. Slides were imaged with a brightfield microscope at 40x magnification and analyzed using the HALO image software analysis platform. In the total population, a one-way test of variance (ANOVA) revealed a statistically significant increase in perivascular macrophages, indicated by CD68 positive pixels, in Stage III CTE compared to controls (p<0.05), a significant increase in Stage II compared to Stage I CTE (p<0.05), and a statistically significant increase in Stage III compared to Stage I CTE (p<0.01). The analysis also revealed a trend toward more CD68 pixels in Stage II CTE compared to controls (p=0.0883) and a trend toward more pixels in Stage III CTE compared to RHI no CTE (p=0.0705). Among the American football players, analyses revealed that Stage II CTE had significantly more perivascular macrophages than Stage I CTE (p<0.01), Stage II CTE had significantly more than controls (p<0.01), Stage III CTE had significantly more than Stage I CTE (p<0.05), and Stage III CTE had significantly more than controls (p<0.05). In summary, this study demonstrates that there is an increase in perivascular CD68 positive macrophages in individuals exposed to RHI with and without CTE. Perivascular macrophages and other neuroinflammatory molecules may play a critical role in the pathogenesis of CTE.

Eclampsia is defined as de novo seizure in a woman with the hypertensive complication of pregnancy known as preeclampsia (PE), and is a leading cause of maternal and fetal morbidity and mortality worldwide. The pathogenesis of eclamptic seizure remains unknown, but is considered a form of hypertensive encephalopathy where an acute rise in blood pressure causes loss of cerebral blood flow (CBF) autoregulation and hyperperfusion of the brain that results in vasogenic edema formation and subsequent seizure. However, eclamptic seizure can occur during seemingly uncomplicated pregnancies, in the absence of hypertension and PE, suggesting that normal pregnancy may predispose the brain to hypertensive encephalopathy or seizure, independently of PE. The overall goal of this dissertation was to investigate the effect of pregnancy and PE on the cerebrovasculature and neurophysiological properties that may promote brain injury and eclamptic seizure. For this dissertation project, a rat model of PE was established that combined placental ischemia, induced by restricting blood flow to the uteroplacental unit, and maternal endothelial dysfunction that was induced by a prolonged high cholesterol diet. Rats with PE developed several PE-like symptoms, including elevated blood pressure, fetal growth restriction, placental dysfunction, and were in a state of oxidative stress and endothelial dysfunction. We found that pregnancy had an overall protective effect on the maintenance of CBF that was potentially due to a nitric-oxide dependent enhancement of the vasodilation of cerebral arteries to decreased intravascular pressure. Further, maintenance of CBF during acute hypertension was similar in pregnancy and PE. Thus, it does not appear that pregnancy and PE are states during which CBF autoregulation is compromised in a manner that would promote the development of hypertensive encephalopathy. However, the brain was found to be in a hyperexcitable state during normal pregnancy that was augmented in PE, and could contribute to onset of eclamptic seizure. Under chloral hydrate anesthesia, generalized seizure was induced by timed infusion of the convulsant pentylenetetrazole (PTZ), with simultaneous electroencephalography that was stopped at the first onset of spikewave discharge indicative of electrical seizure. Seizure threshold was determined as the amount of PTZ required to elicit seizure. Compared to the nonpregnant state, seizure threshold was ~44% lower in pregnant rats and ~80% lower in rats with PE. Further, pregnant rats were more susceptible to seizure-induced vasogenic edema formation than the nonpregnant state. Mechanisms by which pregnancy and PE lowered seizure threshold appeared to be through pregnancy-associated decreases in cortical gamma-aminobutyric acid type A receptor (GABAAR) subunits and PE-induced disruption of the blood-brain barrier (BBB) and microglial activation, indicative of neuroinflammation. Magnesium sulfate (MgSO4), the leading treatment for seizure prophylaxis in women with PE, restored seizure threshold to control levels by reversing neuroinflammation in PE rats, without affecting BBB permeability. Overall, this dissertation provides evidence that pregnancy increases susceptibility of the brain to seizure and vasogenic edema formation that likely contribute to the onset of eclampsia during seemingly uncomplicated pregnancies. Further, the pathogenesis of eclampsia during PE likely involves breakdown of the BBB and subsequent neuroinflammation, resulting in a state of greater seizure susceptibility that is ameliorated by MgSO4 treatment.

Neuroinflammation is considered to be an important underlying process in the pathology of major depressive disorder (MDD) within a subpopulation of patients. MDD is associated with increased levels of proinflammatory cytokines in the blood, and cytokine-based treatments can induce depression. In mice, the induction of systemic inflammation with lipopolysaccharide (LPS) can induce depressive-like behaviours that are associated with symptoms of MDD. Microglia mediate the neuroinflammatory response within the brain and have a critical role in inflammation-induced depressive- like behaviours. Microglia within the brain exist in low O2 conditions (~5 %), though experimentation in vitro is typically carried out in high O2 conditions (20 %). The NLRP3 inflammasome is a molecular complex central to the production of the proinflammatory cytokine IL-1β and the propagation of the inflammatory response. NLRP3 inflammasome activity has been implicated in chronic stress and inflammation-based models of depressive-like behaviours in mice. The aims of this thesis were to study LPS-induced depressive-like behaviour in C57BL/6J mice, the role of NLRP3 in the behavioural output and the influence of oxygen (O2) availability on NLRP3 inflammasome activity in microglia cell cultures. Acute LPS induced depressive-like behaviours were observed in hedonia-based tasks but not in the forced swim test (FST). However, acute LPS induces a brief period of inflammation that does not address the sustained nature of depression. A FST depressive-like behaviour was observed in a novel 3-day increasing dose LPS model of sustained inflammation, whilst circumventing the development of LPS tolerance. The LPS-induced sickness was partially dependent upon NLRP3, though the resulting depressive-like behaviour was not. NLRP3 inflammasome signalling in microglia was studied in 5 % O2 conditions to replicate the hypoxic environment within the brain. Primary microglia isolated from mixed glial cultures by mild trypsinisation exhibited functional NLRP3 inflammasome expression and activity. When exposed to 5 % O2 (24 hours), NLRP3 inflammasome activity and adenosine triphosphate (ATP)-induced cell death was attenuated, whilst the production of other proinflammatory cytokines were unaffected. These data demonstrate the O2 sensitivity of NLRP3 inflammasome signalling in microglia. This thesis demonstrates a novel model of sustained inflammation and that inhibiting NLRP3 signalling may provide a target for attenuating neuroinflammation and the resulting behavioural changes. The importance of understanding the influence of O2 in microglia function and neuroinflammation was highlighted by the sensitivity of NLRP3 inflammasome activity to low O2.

In neurodegenerative diseases, the CNS becomes inflamed through activation of pathways, including the NF-B pathway. Some of the therapies for those diseases target neuroinflammatory pathways. Here, we explore the mechanisms for the upregulation of a subset of genes following a restimulation of the NF-B pathway. We discover that this upregulation occurs independent of IRF1 expression and type 1 interferon signaling. A knockdown of IRF1 using siRNA and an inhibition of JAK proteins using inhibitor AG490 both had no effect on priming. A secreted factor was found to upregulate the expression of both this subset of genes and genes encoding pro-inflammatory cytokines induced by NF-B activation. We also explored the role of IRF1 in a mouse model of multiple sclerosis. We found that the deletion of IRF1 from oligodendrocytes diminished EAE severity. A deletion of IRF1 from myeloid cells within mice did not diminish EAE severity, however showed a promising decrease in the expression of certain inflammatory genes. Thus, IRF1 plays a critical role in fine-tuning inflammatory responses in the brain.

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder and the most common form of dementia. The hallmark pathologies of AD are extracellular aggregates of amyloid-beta, intracellular aggregates of microtubule associated protein tau and increased neuroinflammation. Current therapeutics offer only symptomatic relief and clinical trials investigating therapeutic benefits of non-steroidal anti-inflammatory drugs have yielded no positive results. Therefore, recent work has focused on immunomodulators, such as CD200 and fractalkine, as potential therapeutic targets for AD. Fractalkine (CX3CL1; FKN) is expressed as a transmembrane protein with an N-terminal chemokine domain followed by a long, mucin-like stalk. FKN can signal as a membrane-bound protein or, upon cleavage, as a soluble ligand (sFKN). Upon binding its receptor, FKN reduces expression of pro-inflammatory genes in activated microglia. Disrupting FKN signaling has been shown to exacerbate neurodegeneration in a number or neurodegenerative diseases. Relevant to this study, there have been conflicting reports on how FKN signaling affects AD pathology and whether a soluble FKN is beneficial or not. Here, we examine the ability of soluble FKN over expression to impact tauopathy and the resulting cognitive deficits in the rTg4510 mouse model of tauopathy, focusing on cognitive improvement after the onset of tau deposition. Furthermore, we explore the functional activity of proteolytic fragments of FKN on activated microglia in vitro to rectify the contradictory findings in the literature. We observed that sFKN over expression can significantly reduce both soluble and insoluble phospho-tau in both a preventative and an early interventional study design. However, in animals with significant pathology and neurodegeneration we did not observe an impact of sFKN over expression on tau pathology. Interestingly, in these late stage animals we did observe an improvement in spatial learning and memory as well as a reduction in hyperactivity. This suggests that earlier intervention would likely be most beneficial in reducing tau pathology but in late stage AD FKN signaling can still have benefits on cognition, likely due to reductions in the inflammatory milieu. Current publications suggest that different proteolytic fragments of FKN may have different functional signaling. Here we demonstrate that the this may be due to differences in receptor binding. sFKN (which includes the mucin-like stalk) exhibited a lower EC50 than the ckFKN (soluble chemokine domain), which leads to reduced functional efficacy of ckFKN at low concentrations. More interestingly, we also observed that high concentrations of FKN, regardless of cleavage variant, is ineffective at reducing pro-inflammatory activation of microglial and may in fact elicit a proinflammatory response. We hypothesize that FKN may signal through an alternative receptor at high concentrations, suggesting an as yet unidentified signaling pathway for FKN. Furthermore, we show that the ckFKN does not rescue pathology in the rTg4510 mouse, as sFKN does. These data may clarify conflicts in the literature and demonstrate that care must be taken with respect to in vitro and in vivo studies using FKN.