Persistent Microglial Activation Research Articles

Expression of ChAT, Iba-1, and nNOS in the Central Nervous System following Facial Nerve Injury.

Facial nerve injury can cause significant functional impairment, impacting both the peripheral and central nervous systems. The present study evaluated changes in facial motor function, numbers of cholinergic neurons and microglia, and nNOS levels in the facial nucleus of the central nervous system (CNS) following peripheral facial nerve injury. Facial nerve function, as determined by eyeblink and whisker-movement reflexes, was evaluated at baseline and 1, 2, 3, 4, 8, and 12 weeks after inducing facial nerve injury through compression or axotomy. The expression of choline acetyltransferase (ChAT), ionized calcium-binding adaptor molecule 1 (Iba-1), and neuronal nitric oxide synthase (nNOS) in the facial nucleus of the CNS was analyzed 2, 4, and 12 weeks after peripheral facial nerve injury. Compression-induced facial nerve injury was found to lead to temporary facial motor impairment, whereas axotomy resulted in persistent impairment. Moreover, both compression and axotomy reduced ChAT expression and increased Iba-1 and nNOS expression in the facial nucleus, indicating upregulation of an inflammatory response and neurodegeneration. These results indicate that, compared with compression-induced injury, axotomy-induced facial nerve injury results in greater facial motor dysfunction and more persistent microglial and nitric oxide activation in the facial nucleus of the CNS.

Mechanisms of sepsis‐induced inflammation in the aging brain

AbstractBackgroundSepsis affects millions worldwide with adults over the age of 65 at particularly high risk. Up to 70% of sepsis patients develop delirium and the severity and length of delirium are strong predictors of acquisition of Alzheimer’s Disease and Related Dementias (AD, ADRD). We hypothesize that older adults and those at risk for AD have augmented neuroinflammation during sepsis that correlates development of delirium and persistence of long‐term cognitive impairment.MethodMale and female wild‐type (young‐3 months and aged‐18 months) and 4 month old Tau P301S transgenic AD mice were used to test the effects of polymicrobial sepsis on markers of neuroimflammation and behavior. Sepsis was induced by intraperitoneal injection of a slurry of cecal contents. Plasma and brain were collected from 8 hours to 7 days post infection for measurement of pro‐inflammatory cytokines (IL‐6, CXCL‐1, TNF‐a) and markers of neuroinflammation (GFAP, IBA1 and s100B) using a combination of protein quantification, multiplex and immunohistochemical approaches. Behavior, including measures of locomotor activity, motor learning (rotarod) and cognition (Y‐maze, nest building) were tested up to 7 days post infection.ResultSepsis increases plasma markers of acute neuroinflammation between 8 and 48 hours that resolves by 7 days. Despite this, mice who have recovered from sepsis have persistent microglial activation in the hippocampus at least 7 days later suggesting prolonged damage. Some recovered mice also have persistent deficits in behavioral tasks including activity and nest building despite appearing healthy and fully recovered. Older WT mice are more susceptible to sepsis as measured by severity of illness (Figure) and mortality and mice genetically predisposed to AD have greater deficits in behavioral measures compared to their WT counterparts following infection.ConclusionSepsis is accompanied acutely by marked neuroinflammation and chronically by microglial activation and behavior deficits. Older age and presence of even early stages of AD‐relevant pathology augment these deficits. Persistent microglial activation may underlie this pathology and prime the aged brain to develop an ADRD‐like syndrome.

Microglia-derived CCL20 deteriorates neurogenesis following intraventricular hemorrhage

Intraventricular hemorrhage (IVH) commonly occurs as an extension of intracerebral hemorrhage (ICH) into the brain ventricular system, leading to worse outcomes without effective management. Using a mouse model of IVH, we found that impaired neurogenesis is evident in the subventricular zone (SVZ), along with persistent microglia activation, leukocyte infiltration and cell death. Pharmacological depletion of microglia using PLX3397, an inhibitor of colony stimulating factor 1 receptor (CSF1R), promotes neurogenesis, and alleviated delayed functional impairments in IVH mice. Meanwhile, an elevated level of microglia-derived CC chemokine ligand 20 (CCL20) is observed in the SVZ following IVH, which can induce the upregulation of pro-inflammatory factors in microglia and impair the proliferation and survival of neural stem cells (NSCs) in vitro. Blocking CCL20 in microglia leads to downregulation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/the nuclear factor-κB (NF-κB) signaling pathway, which may contribute to CCL20-dependent pro-inflammatory responses and neural injury. These findings demonstrate a detrimental role of microglia in the neurogenesis and neurorepair after IVH in which CCL20 likely plays a role.

Persistent inflammation and neuronal loss in the mouse brain induced by a modified form of attenuated herpes simplex virus type I

Herpes simplex virus-1 (HSV-1) is a widespread neurotropic virus that can reach the brain and cause a rare but acute herpes simplex encephalitis (HSE) with a high mortality rate. Most patients present with changes in neurological and behavioral status, and survivors suffer long-term neurological sequelae. To date, the pathogenesis leading to brain damage is still not well understood. HSV-1 induced encephalitis in the central nervous system (CNS) in animals are usually very diffuse and progressing rapidly, and mostly fatal, making the analysis difficult. Here, we established a mouse model of HSE via intracerebral inoculation of modified version of neural-attenuated strains of HSV-1 (deletion of ICP34.5 and inserting a strong promoter into the latency-associated transcript region), in which the LMR-αΔpA strain initiated moderate productive infection, leading to strong host immune and inflammatory response characterized by persistent microglia activation. This viral replication activity and prolonged inflammatory response activated signaling pathways in neuronal damage, amyloidosis, Alzheimer's disease, and neurodegeneration, eventually leading to neuronal loss and behavioral changes characterized by hypokinesia. Our study reveals detailed pathogenic processes and persistent inflammatory responses in the CNS and provides a controlled, mild and non-lethal HSE model for studying long-term neuronal injury and increased risk of neurodegenerative diseases due to HSV-1 infection.

Prenatal alcohol exposure causes persistent microglial activation and age- and sex- specific effects on cognition and metabolic outcomes in an Alzheimer’s Disease mouse model

ABSTRACT Background: Prenatal alcohol exposure (PAE) causes behavioral deficits and increases risk of metabolic diseases. Alzheimer’s Disease (AD) is a neurodegenerative disease that has a higher risk in adults with metabolic diseases. Both present with persistent neuroinflammation. Objectives: We tested whether PAE exacerbates AD-related cognitive decline in a mouse model (3xTg-AD; presenilin/amyloid precursor protein/tau), and assessed associations among cognition, metabolic impairment, and microglial reactivity. Methods: Alcohol-exposed (ALC) pregnant 3xTg-AD mice received 3 g/kg alcohol from embryonic day 8.5–17.5. We evaluated recognition memory and associative memory (fear conditioning) in 8–10 males and females per group at 3 months of age (3mo), 7mo, and 11mo, then assessed glucose tolerance, body composition, and hippocampal microglial activation at 12mo. Results: ALC females had higher body weights than controls from 5mo (p < .0001). Controls showed improved recognition memory at 11mo compared with 3mo (p = .007); this was not seen in ALC mice. Older animals froze more during fear conditioning than younger, and ALC mice were hyper-responsive to the fear-related cue (p = .017). Fasting blood glucose was lower in ALC males and higher in ALC females than controls. Positive associations occurred between glucose and fear-related context (p = .04) and adiposity and fear-related cue (p = .0002) in ALC animals. Hippocampal microglial activation was higher in ALC than controls (p < .0001); this trended to correlate with recognition memory. Conclusions: ALC animals showed age-related cognitive impairments that did not interact with AD risk but did correlate with metabolic dysfunction and somewhat with microglial activation. Thus, metabolic disorders may be a therapeutic target for people with FASDs.

Gut Microbiota Dysbiosis after Traumatic Brain Injury Contributes to Persistent Microglial Activation Associated with Upregulated Lyz2 and Shifted Tryptophan Metabolic Phenotype.

Traumatic brain injury (TBI) is a common cause of disability and mortality, affecting millions of people every year. The neuroinflammation and immune response post-TBI initially have neuroprotective and reparative effects, but prolonged neuroinflammation leads to secondary injury and increases the risk of chronic neurodegenerative diseases. Persistent microglial activation plays a critical role in chronic neuroinflammation post-TBI. Given the bidirectional communication along the brain–gut axis, it is plausible to suppose that gut microbiota dysbiosis post-TBI influences microglial activation. In the present study, hippocampal microglial activation was observed at 7 days and 28 days post-TBI. However, in TBI mice with a depletion of gut microbiota, microglia were activated at 7 days post-TBI, but not at 28 days post-TBI, indicating that gut microbiota contributes to the long-term activation of microglia post-TBI. In addition, in conventional mice colonized by the gut microbiota of TBI mice using fecal microbiota transplant (FMT), microglial activation was observed at 28 days post-TBI, but not at 7 days post-TBI, supporting the role of gut microbiota dysbiosis in persistent microglial activation post-TBI. The RNA sequencing of the hippocampus identified a microglial activation gene, Lyz2, which kept upregulation post-TBI. This persistent upregulation was inhibited by oral antibiotics and partly induced by FMT. 16s rRNA gene sequencing showed that the composition and function of gut microbiota shifted over time post-TBI with progressive dysbiosis, and untargeted metabolomics profiling revealed that the tryptophan metabolic phenotype was differently reshaped at 7 days and 28 days post-TBI, which may play a role in the persistent upregulation of Lyz2 and the activation of microglia. This study implicates that gut microbiota and Lyz2 are potential targets for the development of novel strategies to address persistent microglial activation and chronic neuroinflammation post-TBI, and further investigations are warranted to elucidate the specific mechanism.

Flavonoids Ameliorate Stress-Induced Depression by Preventing NLRP3 Inflammasome Priming

ObjectivesChronic stress activates danger-associated molecular patterns (DAMPs), stimulating the NLRP3 inflammasome. NLRP3 activation triggers the release of pro-inflammatory cytokine IL‐1β. The activity of the NLRP3 inflammasome propagates pro-inflammatory signaling cascades implicated in the onset of depression. Our previous studies show that polyphenolic compounds were found to ameliorate stress induced depression in mouse models. However, the relevant mechanism has not been identified. This study examined the effect of administering polyphenols on DAMP signaling in enriched mice microglia.MethodsThis study examined the effect of administering polyphenols on DAMP signaling in mice microglia. To recapitulate stress-induced depression, mice underwent chronic unpredictable stress (CUS). Microglia were isolated at various time points throughout the CUS protocol. We also assessed long-term persistent changes after CUS and susceptibility to subthreshold unpredictable stress (US) re-exposure.ResultsInterestingly, the development of US-induced depression and anxiety depended upon a previous exposure to CUS. We found that CUS caused robust upregulation of IL-1β mRNA in enriched microglia, an effect that persists for up to 4 weeks following CUS exposure. Following the subthreshold US re-exposure, we observed the upregulation of pro- IL-1β as well as pro-receptor for advanced glycation end products (RAGE). Toll-like receptor 4 (TLR-4) was not. We also observed an increase in RAGE mRNA expression when mice were exposed to US prior to the start of the CUS paradigm. Importantly, a primary exposure to US, was sufficient to increase RAGE mRNA expression. We found that polyphenol administration significantly improved CUS-induced depressive-like phenotypes and also reversed neuroinflammation in mice. Treatment with dietary flavonoids prevented upregulation of IL-1β, RAGE mRNA, which reflects the ability of polyphenols that may have begun following the primary exposure to US.ConclusionsTaken all together, the results provide evidence of the role of dietary polyphenols in preventing persistent microglial activation, which has been shown to result in reduced long term vulnerability to depressive-like behaviors following expose to chronic stress.Funding SourcesThis study was supported by a P50 CARBON Center grant from the NCCIH/ODS.

Prenatal disruption of blood–brain barrier formation via cyclooxygenase activation leads to lifelong brain inflammation

Gestational maternal immune activation (MIA) in mice induces persistent brain microglial activation and a range of neuropathologies in the adult offspring. Although long-term phenotypes are well documented, how MIA in utero leads to persistent brain inflammation is not well understood. Here, we found that offspring of mothers treated with polyriboinosinic–polyribocytidylic acid [poly(I:C)] to induce MIA at gestational day 13 exhibit blood–brain barrier (BBB) dysfunction throughout life. Live MRI in utero revealed fetal BBB hyperpermeability 2 d after MIA. Decreased pericyte–endothelium coupling in cerebral blood vessels and increased microglial activation were found in fetal and 1- and 6-mo-old offspring brains. The long-lasting disruptions result from abnormal prenatal BBB formation, driven by increased proliferation of cyclooxygenase-2 (COX2; Ptgs2)-expressing microglia in fetal brain parenchyma and perivascular spaces. Targeted deletion of the Ptgs2 gene in fetal myeloid cells or treatment with the inhibitor celecoxib 24 h after immune activation prevented microglial proliferation and disruption of BBB formation and function, showing that prenatal COX2 activation is a causal pathway of MIA effects. Thus, gestational MIA disrupts fetal BBB formation, inducing persistent BBB dysfunction, which promotes microglial overactivation and behavioral alterations across the offspring life span. Taken together, the data suggest that gestational MIA disruption of BBB formation could be an etiological contributor to neuropsychiatric disorders.

Low-grade peripheral inflammation affects brain pathology in the AppNL-G-Fmouse model of Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by the accumulation of amyloid β (Aβ) and neurofibrillary tangles. The last decade, it became increasingly clear that neuroinflammation plays a key role in both the initiation and progression of AD. Moreover, also the presence of peripheral inflammation has been extensively documented. However, it is still ambiguous whether this observed inflammation is cause or consequence of AD pathogenesis. Recently, this has been studied using amyloid precursor protein (APP) overexpression mouse models of AD. However, the findings might be confounded by APP-overexpression artifacts. Here, we investigated the effect of low-grade peripheral inflammation in the APP knock-in (AppNL-G-F) mouse model. This revealed that low-grade peripheral inflammation affects (1) microglia characteristics, (2) blood-cerebrospinal fluid barrier integrity, (3) peripheral immune cell infiltration and (4) Aβ deposition in the brain. Next, we identified mechanisms that might cause this effect on AD pathology, more precisely Aβ efflux, persistent microglial activation and insufficient Aβ clearance, neuronal dysfunction and promotion of Aβ aggregation. Our results further strengthen the believe that even low-grade peripheral inflammation has detrimental effects on AD progression and may further reinforce the idea to modulate peripheral inflammation as a therapeutic strategy for AD.

Sirtuins as Potential Therapeutic Targets for Mitigating Neuroinflammation Associated With Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, which is associated with memory deficit and global cognitive decline. Age is the greatest risk factor for AD and, in recent years, it is becoming increasingly appreciated that aging-related neuroinflammation plays a key role in the pathogenesis of AD. The presence of β-amyloid plaques and neurofibrillary tangles are the primary pathological hallmarks of AD; defects which can then activate a cascade of molecular inflammatory pathways in glial cells. Microglia, the resident macrophages in the central nervous system (CNS), are the major triggers of inflammation; a response which is typically intended to prevent further damage to the CNS. However, persistent microglial activation (i.e., neuroinflammation) is toxic to both neurons and glia, which then leads to neurodegeneration. Growing evidence supports a central role for sirtuins in the regulation of neuroinflammation. Sirtuins are NAD+-dependent protein deacetylases that modulate a number of cellular processes associated with inflammation. This review examines the latest findings regarding AD-associated neuroinflammation, mainly focusing on the connections among the microglial molecular pathways of inflammation. Furthermore, we highlight the biology of sirtuins, and their role in neuroinflammation. Suppression of microglial activity through modulation of the sirtuin activity has now become a key area of research, where progress in therapeutic interventions may slow the progression of Alzheimer’s disease.

Necroptosis increases with age in the brain and contributes to age-related neuroinflammation

Chronic inflammation of the central nervous system (CNS), termed neuroinflammation, is a hallmark of aging and a proposed mediator of cognitive decline associated with aging. Neuroinflammation is characterized by the persistent activation of microglia, the innate immune cells of the CNS, with damage-associated molecular patterns (DAMPs) being one of the well-known activators of microglia. Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we hypothesized that an age-associated increase in necroptosis contributes to increased neuroinflammation with age. The marker of necroptosis, phosphorylated form of MLKL (P-MLKL), and kinases in the necroptosis pathway (RIPK1, RIPK3, and MLKL) showed a region-specific increase in the brain with age, specifically in the cortex layer V and the CA3 region of the hippocampus of mice. Similarly, MLKL-oligomers, which cause membrane binding and permeabilization, were significantly increased in the cortex and hippocampus of old mice relative to young mice. Nearly 70 to 80% of P-MLKL immunoreactivity was localized to neurons and less than 10% was localized to microglia, whereas no P-MLKL was detected in astrocytes. P-MLKL expression in neurons was detected in the soma, not in the processes. Blocking necroptosis using Mlkl−/− mice reduced markers of neuroinflammation (Iba-1 and GFAP) in the brains of old mice, and short-term treatment with the necroptosis inhibitor, necrostatin-1s, reduced expression of proinflammatory cytokines, IL-6 and IL-1β, in the hippocampus of old mice. Thus, our data demonstrate for the first time that brain necroptosis increases with age and contributes to age-related neuroinflammation in mice.

Astrocyte inflammatory signaling mediates α-synuclein aggregation and dopaminergic neuronal loss following viral encephalitis

Viral infection of the central nervous system (CNS) can cause lasting neurological decline in surviving patients and can present with symptoms resembling Parkinson's disease (PD). The mechanisms underlying postencephalitic parkinsonism remain unclear but are thought to involve increased innate inflammatory signaling in glial cells, resulting in persistent neuroinflammation. We therefore studied the role of glial cells in regulating neuropathology in postencephalitic parkinsonism by studying the involvement of astrocytes in loss of dopaminergic neurons and aggregation of α–synuclein protein following infection with western equine encephalitis virus (WEEV). Infections were conducted in both wildtype mice and in transgenic mice lacking NFκB inflammatory signaling in astrocytes. For 2 months following WEEV infection, we analyzed glial activation, neuronal loss and protein aggregation across multiple brain regions, including the substantia nigra pars compacta (SNpc). These data revealed that WEEV induces loss of SNpc dopaminergic neurons, persistent activation of microglia and astrocytes that precipitates widespread aggregation of α-synuclein in the brain of C57BL/6 mice. Microgliosis and macrophage infiltration occurred prior to activation of astrocytes and was followed by opsonization of ⍺-synuclein protein aggregates in the cortex, hippocampus and midbrain by the complement protein, C3. Astrocyte-specific NFκB knockout mice had reduced gliosis, α-synuclein aggregate formation and neuronal loss. These data suggest that astrocytes play a critical role in initiating PD-like pathology following encephalitic infection with WEEV through innate immune inflammatory pathways that damage dopaminergic neurons, possibly by hindering clearance of ⍺-synuclein aggregates. Inhibiting glial inflammatory responses could therefore represent a potential therapy strategy for viral parkinsonism.

Sex-specific acute and chronic neurotoxicity of acute diisopropylfluorophosphate (DFP)-intoxication in juvenile Sprague-Dawley rats

Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of cognitive deficits in juvenile rats acutely intoxicated with OPs.

Gliosis Precedes Amyloid-β Deposition and Pathological Tau Accumulation in the Neuronal Cell Cycle Re-Entry Mouse Model of Alzheimer's Disease.

Background:The presence of cell cycle markers in postmortem Alzheimer’s disease (AD) brains suggest a potential role of cell cycle activation in AD. It was shown that cell cycle activation in postmitotic neurons in mice produces Aβ and tau pathologies from endogenous mouse proteins in the absence of AβPP or tau mutations.Objective:In this study, we examined the microglial and astrocytic responses in these mice since neuroinflammation is another key pathological feature in AD.Methods:Our neuronal cell cycle re-entry (NCCR) mouse model are bitransgenic mice heterozygous for both Camk2a-tTA and TRE-SV40T. Using this tet-off system, we triggered NCCR in our animals via neuronal expression of SV40T starting at 1 month of age. TRE-SV40T Tg mice were used as SV40T transgene controls. The animals were examined at following time points: 2, 3, 4, 6, and 12 months of age. The microglia and astrocyte responses in our mice were determined by image analysis and stereology on brain sections immunofluorescently labeled using the following antibodies: Iba1, CD45, CD68, MHCII, and GFAP. Cellular senescent marker p16 was also used in this study.Results:Our NCCR mice demonstrate early and persistent activation of microglia and astrocytes. Additionally, proinflammatory and senescent microglia phenotype and brain leukocyte infiltration is present at 12 months of age.Conclusion:In the absence of FAD gene mutations, our NCCR mice simultaneously display many of the pathological changes associated with AD, such as ectopic neuronal cell cycle re-entry, Aβ and tau pathologies, neuroinflammation, and neurodegeneration. These animals represent a promising alternative AD mouse model.

Safe Focused Ultrasound-Mediated Blood-Brain Barrier Opening and Repair is Not Mediated by Tight Junction Degradation

Focused ultrasound (FUS) with microbubbles opens the blood-brain barrier (BBB) to allow targeted drug delivery into the brain; however, the mechanisms of BBB opening and the response of the neurovascular unit at either low acoustic pressures, known to open the BBB transiently, or high pressures that cause brain damage, remain unclear. Employing a transgenic mouse strain where tight junctions (TJs) are labelled with eGFP, we examined TJ strand morphology at 1 and 72 hours post-FUS at both low and high pressures. We find that transient BBB opening due to FUS is not caused by TJ strand damage, unless FUS is used at high pressures, and this is localized primarily in arterioles and capillaries. At high pressures, TJ strands are obliterated and remain unrepaired even at 72 hours, allowing for fibrinogen passage, and persistent microglial activation. Our findings suggest that at low safe pressures, upregulation of transcytosis may likely increase transiently BBB permeability to deliver therapeutics to the brain.

Flavonoids Ameliorate Stress-Induced Depression by Preventing NLRP3 Inflammasome Priming

Abstract Objectives Chronic stress activates danger-associated molecular patterns (DAMPs), stimulating the NLRP3 inflammasome. NLRP3 activation triggers the release of pro-inflammatory cytokine IL-1β. The activity of the NLRP3 inflammasome propagates pro-inflammatory signaling cascades implicated in the onset of depression. Our previous studies show that polyphenolic compounds were found to ameliorate stress induced depression in mouse models. However, the relevant mechanism has not been identified. This study examined the effect of administering polyphenols on DAMP signaling in enriched mice microglia. Methods This study examined the effect of administering polyphenols on DAMP signaling in mice microglia. To recapitulate stress-induced depression, mice underwent chronic unpredictable stress (CUS). Microglia were isolated at various time points throughout the CUS protocol. We also assessed long-term persistent changes after CUS and susceptibility to subthreshold unpredictable stress (US) re-exposure. Results Interestingly, the development of US – induced depression and anxiety depended upon a previous exposure to CUS. We found that CUS caused robust upregulation of IL-1β mRNA in enriched microglia, an effect that persists for up to 4 weeks following CUS exposure. Following the subthreshold US re-exposure, we observed the upregulation of pro- IL-1β as well as pro-receptor for advanced glycation end products (RAGE). Toll-like receptor 4 (TLR-4) was not. We also observed an increase in RAGE mRNA expression when mice were exposed to US prior to the start of the CUS paradigm. Importantly, a primary exposure to US, was sufficient to increase RAGE mRNA expression. We found that polyphenol administration significantly improved CUS-induced depressive-like phenotypes and also reversed neuroinflammation in mice. Treatment with dietary flavonoids prevented upregulation of IL-1β, RAGE mRNA, which reflects the ability of polyphenols that may have begun following the primary exposure to US. Conclusions Taken all together, the results provide evidence of the role of dietary polyphenols in preventing persistent microglial activation, which has been shown to result in reduced long term vulnerability to depressive-like behaviors following expose to chronic stress. Funding Sources This study was supported by a P50 CARBON Center grant from the NCCIH/ODS.

Innate sensing of mechanical properties of brain tissue by microglia.

External organic or inorganic objects (foreign bodies) that are inadvertently or purposefully placed in the human or animal tissues can trigger local tissue responses that aim at the elimination and/or segregation of foreign bodies from the tissue. The foreign body response (FBR) may have major implications for neurodegeneration associated with the formation of aberrant protein-based aggregates or plaques. The distinct physical features of the plaques, including high rigidity and varying surface properties, may trigger microglial mechanosensing of the plaque as a foreign body. The microglial FBR may have a dual function by promoting and/or suppressing the plaque driven neurodegeneration. Microglial contact with the plaque may trigger inflammatory activation of microglia and support microglia-driven neuronal damage. Conversely, persistent microglial activation may trigger the formation of a microglia-supported cell barrier that segregates and compacts the plaques thus preventing further plaque-induced damage to healthy neurons.

Persistent Hydrocephalus and Activated Microglia Drive the Spatial Memory Decline After Intraventricular Hemorrhage in Rodents

Abstract INTRODUCTION Intraventricular hemorrhage (IVH) can result in significant cognitive deficits and spatial memory impairments that can prevent over half of IVH patients from returning to work after recovery. The mechanism of this memory decline, however, is incompletely understood. METHODS To understand the role of mechanical trauma caused by rapid ventricular expansion vs the presence of blood components, such as thrombin, in IVH deficits, we developed a rodent model of IVH by injecting 200 μL of autologous arterial blood into the animals' ventricles. The IVH animals’ memories were then assessed along with sham (no injection), vehicle control (200 μL aCSF), and intraventricular thrombin (IVT) (20 U thrombin in 5 μL aCSF) animals using Morris water maze. RESULTS The IVH group performed worse on this spatial memory task compared to all other groups, indicating that ventricular expansion and presence of blood components both contribute to memory deficits. Based on magnetic resonance imaging (MRI) results, the IVH group was also the only group that developed persistent hydrocephalus. Contrary to our hypothesis that the cognitive deficits of IVH are due to decreased hippocampal neurogenesis, we did not find any statistical difference in the numbers of dentate gyrus progenitor cells (labeled with BrdU and DCX) or neurons (labeled with NeuN) between the different groups. The number of microglia (labeled with Iba1) was also compared among the groups and found to be not statistically different. The microglia in IVH and vehicle control animals, however, were found to have significantly lower fractal dimension (P &lt; .001) and higher lacunarity (P &lt; .001) numbers compared to those of sham animals, indicating a more activated state, likely caused by the mechanical trauma. CONCLUSION Based on our results, persistent hydrocephalus microglial activation seem to be the drivers of spatial memory deficit after IVH.

P2‐091: SV40T‐MEDIATED NEURONAL CELL CYCLE DYSREGULATION IN MICE INDUCES Aβ AND TAU PATHOLOGIES AND NEUROINFLAMMATION

The majority of the Alzheimer's disease (AD) cases are sporadic and generate Aβ plaques and neurofibrillary tangles in the absence of mutations in the APP or tau proteins. The presence of cell cycle markers in postmortem AD brains has led a number of researchers to suggest a potential role of cell cycle activation in AD. My previously published findings demonstrated that cell cycle induction in postmitotic neurons in mice produces Aβ and tau pathologies in the absence of APP or tau mutations (Park et al., 2007, J Neurosci). Our recent work examines the microglial and astrocytic responses in these mice. SV40T expression was restricted to postmitotic neurons in our mice using the tet-off system. Our inducible transgenic mice express SV40T under the regulation of TRE promoter (Tag mice). Tag mice were paired with Camk2a-tTA transgenic mice (Off mice) and maintained on doxycycline (dox) diet. The offspring were maintained on dox diet until 30 days of age, at which point they were continuously maintained on standard diet. Tag mice were used as SV40T transgene controls. Animals were examined at 2, 4, 6, and 12 months of age. Vibratome sectioned brains were immunofluorescent labeled for Iba1, CD45, CD68, MHCII and GFAP. Cellular senescence was assessed using anti-p16 antibody. Microglia and astrocyte activations are observed as early as 2 months of age after neuronal cell cycle activation. Tag/Off mice demonstrated increase in GFAP immunolabeling compared to Tag mice, indicative of astrocyte activation. Tag/Off mice also showed increased Iba1 immunostaining compared to Tag mice. A subset of Iba1-positive microglia showed co-labeling with CD45, CD68, or MHCII antibodies, demonstrating activated microglia. At 12-months of age, microglia demonstrates blebbing and p16 immunopositivity, both hallmarks of senescent phenotype. Our Tag/Off mice demonstrate early and persistent activation of microglia and astrocytes. Additionally, these mice develop senescent microglia phenotype along with Aβ and tau pathologies following chronic and persistent neuronal cell cycle activation. Our mice simultaneously display the major pathological features of AD in the absence of APP or tau mutations, and thereby may represent a sporadic AD mouse model.

Polyphenolic Compounds Ameliorate Stress-induced Depression by Preventing NLRP3 Inflammasome Priming (P19-011-19)

ObjectivesChronic stress activates danger-associated molecular patterns (DAMPs), which then stimulate the NLRP3 inflammasome. NLRP3 activation triggers the released of the pro-inflammatory cytokine IL‐1β. The activity of the NLRP3 inflammasome propagates pro-inflammatory signaling cascades implicated in the onset of depression. In previous studies conducted in our lab, polyphenolic compounds were found to ameliorate stress induced depression in mouse models. However, the mechanism by which they do so has not been identified. This study examined the effect of administering polyphenols on DAMP signaling in enriched mice microglia. MethodsThis study examined the effect of administering polyphenols on DAMP signaling in mice microglia. To recapitulate stress-induced depression, mice underwent chronic unpredictable stress (CUS). Microglia were isolated at various time points throughout the CUS protocol. We also assessed long-term persistent changes after CUS and susceptibility to subthreshold unpredictable stress (US) re-exposure. ResultsInterestingly, the development of US - induced depression and anxiety depended upon a previous exposure to CUS. We found that CUS caused robust upregulation of IL-1β mRNA in enriched microglia, an effect that persists for up to 4 weeks following CUS exposure. Following the subthreshold US re-exposure, we observed the upregulation of pro- IL-1β as well as pro-receptor for advanced glycation end products (RAGE). Toll-like receptor 4 (TLR-4) was not. We also observed an increase in RAGE mRNA expression when mice were exposed to US prior to the start of the CUS paradigm. Importantly, a primary exposure to US, was sufficient to increase RAGE mRNA expression. We found that polyphenol administration improved CUS-induced depressive-like phenotypes and also reversed neuroinflammation in mice. Treatment with dietary polyphenols prevented upregulation of IL-1β, RAGE mRNA, which reflects the ability of polyphenols that may have begun following the primary exposure to US. ConclusionsTaken all together, the results provide evidence of the role of polyphenols in preventing persistent microglial activation, which has been shown to result in reduced long term vulnerability to depressive-like behaviors following expose to chronic stress. Funding SourcesThis study was supported by a P50 CARBON Center grant from the NCCIH and ODS.