2668134
Prior Binge Ethanol Exposure Potentiates the Microglial Response in a Model of Alcohol-Induced Neurodegeneration
Excessive alcohol consumption results in neurodegeneration which some hypothesize is caused by neuroinflammation. One characteristic of neuroinflammation is microglial activation, but it is now well accepted that microglial activation may be pro- or anti-inflammatory. Recent work indicates that the Majchrowicz model of alcohol-induced neurodegeneration results in anti-inflammatory microglia, while intermittent exposure models with lower doses and blood alcohol levels produce microglia with a pro-inflammatory phenotype. To determine the effect of a repeated binge alcohol exposure, rats received two cycles of the four-day Majchrowicz model. One hemisphere was then used to assess microglia via immunohistochemistry and while the other was used for ELISAs of cytokines and growth factors. A single binge ethanol exposure resulted in low-level of microglial activation; however, a second binge potentiated the microglial response. Specifically, double binge rats had greater OX-42 immunoreactivity, increased ionized calcium-binding adapter molecule 1 (Iba-1+) cells, and upregulated tumor necrosis factor-α (TNF-α) compared with the single binge ethanol group. These data indicate that prior ethanol exposure potentiates a subsequent microglia response, which suggests that the initial exposure to alcohol primes microglia. In summary, repeated ethanol exposure, independent of other immune modulatory events, potentiates microglial activity.
40166754
Epidemiology of DSM-5 Drug Use Disorder: Results From the National Epidemiologic Survey on Alcohol and Related Conditions-III.
IMPORTANCE
Current information on the prevalence and sociodemographic and clinical profiles of individuals in the general population with DSM-5 drug use disorder (DUD) is limited. Given the present societal and economic context in the United States and the new diagnostic system, up-to-date national information is needed from a single uniform data source.


OBJECTIVE
To present nationally representative findings on the prevalence, correlates, psychiatric comorbidity, disability, and treatment of DSM-5 DUD diagnoses overall and by severity level.


DESIGN, SETTING, AND PARTICIPANTS
In-person interviews were conducted with 36,309 adults in the 2012-2013 National Epidemiologic Survey on Alcohol and Related Conditions-III, a cross-sectional representative survey of the United States. The household response rate was 72%; person-level response rate, 84%; and overall response rate, 60.1%. Data were collected April 2012 through June 2013 and analyzed from February through March 2015.


MAIN OUTCOMES AND MEASURES
Twelve-month and lifetime DUD, based on amphetamine, cannabis, club drug, cocaine, hallucinogen, heroin, nonheroin opioid, sedative/tranquilizer, and/or solvent/inhalant use disorders.


RESULTS
Prevalences of 12-month and lifetime DUD were 3.9% and 9.9%, respectively. Drug use disorder was generally greater among men, white and Native American individuals, younger and previously or never married adults, those with lower education and income, and those residing in the West. Significant associations were found between 12-month and lifetime DUD and other substance use disorders. Significant associations were also found between any 12-month DUD and major depressive disorder (odds ratio [OR], 1.3; 95% CI, 1.09-1.64), dysthymia (OR, 1.5; 95% CI, 1.09-2.02), bipolar I (OR, 1.5; 95% CI, 1.06-2.05), posttraumatic stress disorder (OR, 1.6; 95% CI, 1.27-2.10), and antisocial (OR, 1.4; 95% CI, 1.11-1.75), borderline (OR, 1.8; 95% CI, 1.41-2.24), and schizotypal (OR, 1.5; 95% CI, 1.18-1.87) personality disorders. Similar associations were found for any lifetime DUD with the exception that lifetime DUD was also associated with generalized anxiety disorder (OR, 1.3; 95% CI, 1.06-1.49), panic disorder (OR, 1.3; 95% CI, 1.06-1.59), and social phobia (OR, 1.3; 95% CI, 1.09-1.64). Twelve-month DUD was associated with significant disability, increasing with DUD severity. Among respondents with 12-month and lifetime DUD, only 13.5% and 24.6% received treatment, respectively.


CONCLUSIONS AND RELEVANCE
DSM-5 DUD is a common, highly comorbid, and disabling disorder that largely goes untreated in the United States. These findings indicate the need for additional studies to understand the broad relationships in more detail; estimate present-day economic costs of DUDs; investigate hypotheses regarding etiology, chronicity, and treatment use; and provide information to policy makers about allocation of resources for service delivery and research. Findings also indicate an urgent need to destigmatize DUD and educate the public, clinicians, and policy makers about its treatment to encourage affected individuals to obtain help.
21138318
Clinical and pathological features of alcohol-related brain damage
One of the sequelae of chronic alcohol abuse is malnutrition. Importantly, a deficiency in thiamine (vitamin B1) can result in the acute, potentially reversible neurological disorder Wernicke encephalopathy (WE). When WE is recognized, thiamine treatment can elicit a rapid clinical recovery. If WE is left untreated, however, patients can develop Korsakoff syndrome (KS), a severe neurological disorder characterized by anterograde amnesia. Alcohol-related brain damage (ARBD) describes the effects of chronic alcohol consumption on human brain structure and function in the absence of more discrete and well-characterized neurological concomitants of alcoholism such as WE and KS. Through knowledge of both the well-described changes in brain structure and function that are evident in alcohol-related disorders such as WE and KS and the clinical outcomes associated with these changes, researchers have begun to gain a better understanding of ARBD. This Review examines ARBD from the perspective of WE and KS, exploring the clinical presentations, postmortem brain pathology, in vivo MRI findings and potential molecular mechanisms associated with these conditions. An awareness of the consequences of chronic alcohol consumption on human behavior and brain structure can enable clinicians to improve detection and treatment of ARBD.
21511880
Age-related decline in MRI volumes of temporal lobe gray matter but not hippocampus
The effect of normal aging on the volume of the hippocampus and temporal cortex was assessed cross-sectionally with quantitative Magnetic Resonance Imaging (MRI) in 72 healthy men, spanning 5 decades of the adult age range (21 to 70 years). Neither the hippocampal nor cortical white matter volumes were significantly correlated with age. By contrast, left and right temporal lobe gray matter volumes, exclusive of the hippocampal measures, each decreased with age (p < 0.01). Volumes of temporal lobe sulcal CSF and the ventricular system (temporal horns and lateral and third ventricles) significantly increased with age. Measures of verbal and nonverbal working memory showed age-related declines and were related to enlargement of the three ventricular regions, which may be indicative of age-related atrophy of the adjacent cortex but not the hippocampus, at least up to age 70 years.
40302151
Brain gray and white matter volume loss accelerates with aging in chronic alcoholics: a quantitative MRI study.
Magnetic resonance imaging (MRI) was used to study in vivo the brains of 49 patients with chronic alcoholism, 3 to 4 weeks post-withdrawal, and 43 normal healthy controls, all right-handed male veterans between the ages of 23 and 70 years. MRI scans were analyzed using a semi-automated procedure, which allowed the subcortical regions to be segmented into cerebrospinal fluid (CSF) and brain tissue and the cortical regions to be segmented into CSF, gray matter, and white matter. An age regression model was used to examine the effects of alcohol on brain structure, over and above that expected from the normal aging process. The alcoholics exhibited decreased tissue and increased CSF after correcting for aging. In the cortex, there was significant loss of both gray matter and white matter volume. In this sample of alcoholics, no particular cortical region was preferentially affected or spared. Furthermore, brain tissue volume loss increased with advanced age in the alcoholics. In this group of alcoholics there was no relationship between length of illness and age, i.e., the younger alcoholics had as heavy alcohol use histories as did the older alcoholics. Thus, the increased brain tissue loss with advanced age is interpreted as evidence for age-related increase in brain vulnerability to chronic alcohol abuse.
24177321
Binge ethanol exposure in adult rats causes necrotic cell death.
BACKGROUND
Although alcoholics show neurodegeneration after decades of drinking, recent studies with an animal model of binge drinking have found corticolimbic damage after as few as four days. Neurodegeneration can occur through apoptotic or necrotic mechanisms. The goal of this research is to characterize the time course of binge ethanol-induced neurodegeneration and to identify apoptotic or necrotic characteristics of this neurodegeneration.


METHODS
Histologic methods (e.g., amino cupric silver staining, Fluoro-Jade B, hematoxylin and eosin, transmission electron microscopy) were used to quantify the time course of degeneration and to characterize the ultrastructural changes that occur with binge ethanol-induced neurodegeneration.


RESULTS
After 2 days of binge ethanol, significant damage was evident in the olfactory bulb. After 4 days of binge ethanol, there was significant damage in the agranular insular cortex, anterior piriform cortex, perirhinal cortex, lateral entorhinal cortex, and the temporal dentate gyrus. Ultrastructural examination revealed shrunken soma, vacuolated cytoplasm, pyknotic nucleus, and irregularly clumped chromatin consistent with dark cell degeneration, a form of necrotic neuronal death.


CONCLUSIONS
Binge drinking causes necrotic neurodegeneration after 2 days of exposure and increased damage after 4 days but does not increase during withdrawal. These studies indicate that binge drinking induced neurodegeneration is necrotic and occurs during ethanol intoxication and not as a result of ethanol withdrawal.
26318057
Intermittent ethanol exposure induces inflammatory brain damage and causes long‐term behavioural alterations in adolescent rats
Adolescent brain development seems to be important for the maturation of brain structures and behaviour. Intermittent binge ethanol drinking is common among adolescents, and this type of drinking can induce brain damage. Because we have demonstrated that chronic ethanol treatment induces inflammatory processes in the brain, we investigate whether intermittent ethanol intoxication enhances cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) in adolescent rats, and whether these mediators induce brain damage and cause permanent cognitive dysfunctions. Adolescent rats were exposed to ethanol (3.0 g/kg) for two consecutive days at 48‐h intervals over 14 days. Levels of COX‐2, iNOS and cell death were assessed in the neocortex, hippocampus and cerebellum 24 h after the final ethanol administration. The following day or 20 days after the final injection (adult stage), animals were tested for different behavioural tests (conditional discrimination learning, rotarod, object recognition, beam‐walking performance) to assess cognitive and motor functions. Our results show that intermittent ethanol intoxication upregulates COX‐2 and iNOS levels, and increases cell death in the neocortex, hippocampus and cerebellum. Furthermore, animals treated with ethanol during adolescence exhibited behavioural deficits that were evident at the end of ethanol treatments and at the adult stage. Administration of indomethacin, a COX‐2 inhibitor, abolishes the induction of COX‐2 and iNOS expression and cell death, preventing ethanol‐induced behavioural deficits. These findings indicate that binge pattern exposure to ethanol during adolescence induces brain damage by inflammatory processes and causes long‐lasting neurobehavioural consequences. Accordingly, administering indomethacin protects against ethanol‐induced brain damage and prevents detrimental ethanol effects on cognitive and motor processes.
34752221
Neuronal degeneration in rat cerebrocortical and olfactory regions during subchronic "binge" intoxication with ethanol: possible explanation for olfactory deficits in alcoholics.
Severe, repetitive ("binge") ethanol intoxication in adult rats (intragastric delivery 3 times daily for 4 days in a modification of the Majchrowicz method) precipitates neuronal degeneration in selected cerebral cortical regions involved in memory and olfaction, confirming the results of Switzer and colleagues (Anat. Rec. 202: 186a, 1982). Neuronal damage was visualized with the de Olmos cupric silver technique for degenerating neurons and processes (argyrophilia), and was quantitated by total counts and densities of argyrophilic cells/fields. The specificity of the degeneration provides a neuropathological basis for the olfactory memory deficits in chronic alcoholics. In highly intoxicated rats, argyrophilia was most extensive among hippocampal dentate gyrus granule cells, pyramidal neurons in layer 3 of the entorhinal cortex, and olfactory nerve terminals in the olfactory bulb. Degenerating pyramidal neurons were also consistently seen in the insular cortex and olfactory cortical regions, such as the piriform and perirhinal cortices. There were few argyrophilic neurons in the CA regions of the hippocampus and none in the cerebellum--regions generally shown to have cell loss in long-term ethanol feeding models--but degenerating mossy fibers in the CA2 region were observed. Degeneration was maximal before the peak period of abstinence symptoms in this model, because argyrophilic densities were no greater 36 hr, compared with 8 hr after the last ethanol dose. High blood ethanol levels were required, because argyrophilia, absent from isocaloric controls, also was only evident in ethanol-intoxicated rats with mean blood ethanol levels for days 2 to 4 above 300 mg/dl; however, it increased substantially between 350 and 550 mg/dl. The resemblance of the argyrophilic distribution to the regional neuropathology that occurs in experimental seizures indicates that the ethanol-induced degeneration may have an excitotoxic basis. Progressive reductions in the seizure threshold (e.g., kindling phenomena that have been documented during binge ethanol intoxication) might be associated with excitotoxic hyperactivity during the repetitive nadirs between high blood and brain ethanol peaks. However, direct toxic actions of ethanol or its metabolites could also be involved. Overall, the model should be useful for studying mechanisms of ethanol-induced selective cortical and olfactory brain damage.
20432690
Upregulated vimentin suggests new areas of neurodegeneration in a model of an alcohol use disorder
Excessive alcohol intake, characteristic of an alcohol use disorder (AUD), results in neurodegeneration as well as cognitive deficits that may recover in abstinence. Neurodegeneration in psychiatric disorders such as AUDs is due to various effects on tissue integrity. Several groups report that alcohol-induced neurodegeneration and recovery include a role for adult neurogenesis. Therefore, the initial purpose of this study was to investigate the effect of alcohol on the temporal profile of neural progenitor cells using the radial glia marker, vimentin, in a model of an AUD. However, striking vimentin expression throughout corticolimbic regions led, instead, to the discovery of a significant gliosis response in this model. Adult male rats were subjected to a 4-day binge model of an AUD and brains harvested for immunohistochemistry at 0, 2, 4, 7, 14, and 28 days following the last dose of ethanol. A prominent increase in vimentin immunoreactivity was apparent at 4 and 7 days post binge that returned to control levels by 14 days in the corticolimbic regions examined. Vimentin-positive cells co-labeled with glial fibrillary acidic protein (GFAP), which suggested that cells were reactive astrocytes. A second experiment supported that increased vimentin was not primarily due to alcohol withdrawal seizures and is more likely due to alcohol-induced cell death. As this gliosis was remarkably distinct in regions where cell death had not previously been reported in this model, adjacent tissue sections were processed for FluoroJade B staining for cell death. FluoroJade B-positive cells were evident immediately following the last ethanol dose as expected, but were significantly elevated in the hippocampal dentate gyrus and CA3 regions and corticolimbic regions from 2 to 7 days post binge. Intriguingly, vimentin labeling of astrogliosis is more widespread than FluoroJade B labeling of cell death, which suggests that 4-day binge ethanol consumption is more damaging than originally realized.
11442834
Alcohol-related dementia: a 21st-century silent epidemic?
Summary Evidence suggests a J-shaped relationship between alcohol consumption and cognitive impairment and other health indicators, with low levels of consumption having better outcomes than abstention or moderate to heavy drinking. Most research to date has focused on the protective effects of drinking small amounts of alcohol. As alcohol consumption is escalating rapidly in many countries, the current cohort of young and middle-aged people may face an upsurge of alcohol-related dementia. The dangers of heavy drinking and its effect on cognition require further attention.
23724017
Alcoholism and dementia.
This article reviews epidemiological, neurological, cognitive, and imaging data on alcohol-induced dementia. Recent studies indicate that "heavy alcohol use" (variously defined) is a contributing factor in 21-24% of cases of dementia. Research difficulties include lack of positive diagnostic criteria, few post-mortem studies, and no accepted pathological mechanism. Sulcal widening and ventricular enlargement (occasionally reversible) are the strongest findings in patients with alcohol-induced dementia. There is evidence for peripheral neuropathy, ataxia, sparing of language, and improved prognosis when patients with alcohol-induced dementia are compared to other dements. Case examples, etiologic theories, and recommendations for research, training, and clinical practice are included.
21143275
Delay discounting of money and alcohol in actively using alcoholics, currently abstinent alcoholics, and controls
Abstract. Rationale: Impulsivity is implicated in alcohol dependence, and discounting of delayed rewards may be an objective indicator of impulsiveness. Objectives: This study evaluated delay discounting functions in alcoholics and controls. It compared discounting rates between different magnitudes ($1000 and $100) and different types (money and alcohol) of rewards. Methods: Active alcoholics (n=19), currently abstinent alcoholics (n=12) and controls (n=15) indicated preferences for immediate versus delayed rewards using a titration procedure that determined indifference points at various delays. Four conditions were presented, and the delayed rewards in the four conditions were $1000, $100, 150 bottles of an alcoholic beverage, and 15 bottles of an alcoholic beverage. Results: In all three groups across all four conditions, hyperbolic discounting functions provided a good fit of the data. Linear contrasts, predicting the most rapid discounting rates in active alcoholics, intermediary rates in currently abstinent alcoholics, and the least rapid rates in controls, were significant for three of the four conditions. Alcohol was discounted more rapidly than money. Conclusions: These data provide further evidence of more rapid discounting of delayed rewards in alcohol abusers compared to controls, and especially steep discounting among current users. Rapid discounting of delayed rewards may be a feature related to addictive disorders. A better understanding of how delaying rewards in time impacts their value may have implications for treatment.
41510737
Sex differences in alcohol-related neurobehavioral consequences.
In this chapter, we review existing research regarding sex differences in alcohol's effects on neurobehavioral functions/processes. Drawn largely from laboratory studies, literature regarding acute alcohol administration and chronic alcohol misuse is explored focusing on commonly employed neuropsychologic domains (e.g., executive function, visuospatial skills, learning and memory, gait and balance), neurophysiologic measures (e.g., electroencephalography and event-related potentials), and structural and functional neuroimaging (e.g., magnetic resonance imaging (MRI), functional MRI, diffusion tensor imaging, positron emission tomography, and magnetic resonance spectroscopy). To provide a historical perspective on the development of these questions, we have included reference to early and more recent research. Additionally, specific biases, knowledge gaps, and continuing controversies are noted.
14422844
Impulsivity, frontal lobes and risk for addiction
Alcohol and substance abuse disorders involve continued use of substances despite negative consequences, i.e. loss of behavioral control of drug use. The frontal-cortical areas of the brain oversee behavioral control through executive functions. Executive functions include abstract thinking, motivation, planning, attention to tasks and inhibition of impulsive responses. Impulsiveness generally refers to premature, unduly risky, poorly conceived actions. Dysfunctional impulsivity includes deficits in attention, lack of reflection and/or insensitivity to consequences, all of which occur in addiction [Evenden JL. Varieties of impulsivity. Psychopharmacology (Berl) 1999;146:348-361.; de Wit H. Impulsivity as a determinant and consequence of drug use: a review of underlying processes. Addict Biol 2009;14:22-31]. Binge drinking models indicate chronic alcohol damages in the corticolimbic brain regions [Crews FT, Braun CJ, Hoplight B, Switzer III RC, Knapp DJ. Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats. Alcohol Clin Exp Res 2000;24:1712-1723] causing reversal learning deficits indicative of loss of executive function [Obernier JA, White AM, Swartzwelder HS, Crews FT. Cognitive deficits and CNS damage after a 4-day binge ethanol exposure in rats. Pharmacol Biochem Behav 2002b;72:521-532]. Genetics and adolescent age are risk factors for alcoholism that coincide with sensitivity to alcohol-induced neurotoxicity. Cortical degeneration from alcohol abuse may increase impulsivity contributing to the development, persistence and severity of alcohol use disorders. Interestingly, abstinence results in bursts of neurogenesis and brain regrowth [Crews FT, Nixon K. Mechanisms of neurodegeneration and regeneration in alcoholism. Alcohol Alcohol 2009;44:115-127]. Treatments for alcoholism, including naltrexone pharmacotherapy and psychotherapy may work through improving executive functions. This review will examine the relationships between impulsivity and executive function behaviors to changes in cortical structure during alcohol dependence and recovery.
24746013
Drug abuse: hedonic homeostatic dysregulation.
Understanding the neurobiological mechanisms of addiction requires an integration of basic neuroscience with social psychology, experimental psychology, and psychiatry. Addiction is presented as a cycle of spiralling dysregulation of brain reward systems that progressively increases, resulting in compulsive drug use and a loss of control over drug-taking. Sensitization and counteradaptation are hypothesized to contribute to this hedonic homeostatic dysregulation, and the neurobiological mechanisms involved, such as the mesolimbic dopamine system, opioid peptidergic systems, and brain and hormonal stress systems, are beginning to be characterized. This framework provides a realistic approach to identifying the neurobiological factors that produce vulnerability to addiction and to relapse in individuals with a history of addiction.
12390139
Alcohol and Neurodegeneration
Studies of alcohol-induced brain damage have clearly indicated that alcohol is neurotoxic. Alcoholics are at increased risk for brain damage from a variety of causes, including poor nutrition, liver disease, and head trauma. Further, alcoholic dementia is the second leading cause of adult dementia in the United States, accounting for approximately 10% of the cases (Alzheimer’s disease is the leading cause, accounting for 40 to 60% of cases). A variety of studies report that 50 to 75% of sober, detoxified, long-term alcohol-dependent individuals suffer from some degree of detectable cognitive impairment with approximately 10% suffering from serious dementia. Although more research is required to precisely delineate the effects of alcohol on various types of brain function, there appears to be a continuum of moderate deficits in the majority of long-term alcoholics, progressing to much more severe deficits of Wernicke’s disease and Wernicke’s encephalopathy with Korsakoff’s amnestic syndrome (12,79). A variety of lifestyle factors, including nutrition, are implicated in the more severe cases. However, all of these on the continuum appear to be related to alcohol consumption and to the amount of alcohol consumed. That is, the more severe cases are associated with more severe and chronic longterm alcoholism (12,79). Alcohol-induced changes in the structure of the adult brain have been studied in both humans and rodents. A variety of postmortem histological analyses, as well as supporting imaging analysis, suggest that chronic alcohol changes brain structure. Computed tomography (CT) and magnetic resonance imaging (MRI) studies of human brain have repeatedly shown enlargement of the cerebral ventricles and sulci in most alcoholics. The enlargement of the ventricles and sulci essentially reflects a shrinking of the brain mass. This is consistent with studies on postmortem brain tissue, where alcoholics have a reduction in total brain weight. Particularly severe alcoholics have reductions in the global cerebral hemisphere and cerebellar brain weights that are significantly reduced compared
15532596
Mechanisms of neurodegeneration and regeneration in alcoholism.
AIMS
This is a review of preclinical studies covering alcohol-induced brain neuronal death and loss of neurogenesis as well as abstinence-induced brain cell genesis, e.g. brain regeneration. Efforts are made to relate preclinical studies to human studies.


METHODS
The studies described are preclinical rat experiments using a 4-day binge ethanol treatment known to induce physical dependence to ethanol. Neurodegeneration and cognitive deficits following binge treatment mimic the mild degeneration and cognitive deficits found in humans. Various histological methods are used to follow brain regional degeneration and regeneration.


RESULTS
Alcohol-induced degeneration occurs due to neuronal death during alcohol intoxication. Neuronal death is related to increases in oxidative stress in brain that coincide with the induction of proinflammatory cytokines and oxidative enzymes that insult brain. Degeneration is associated with increased NF-kappaB proinflammatory transcription and decreased CREB transcription. Corticolimbic brain regions are most sensitive to binge-induced degeneration and induce relearning deficits. Drugs that block oxidative stress and NF-kappaB transcription or increase CREB transcription block binge-induced neurodegeneration, inhibition of neurogenesis and proinflammatory enzyme induction. Regeneration of brain occurs during abstinence following binge ethanol treatment. Bursts of proliferating cells occur across multiple brain regions, with many new microglia across brain after months of abstinence and many new neurons in neurogenic hippocampal dentate gyrus. Brain regeneration may be important to sustain abstinence in humans.


CONCLUSIONS
Alcohol-induced neurodegeneration occurs primarily during intoxication and is related to increased oxidative stress and proinflammatory proteins that are neurotoxic. Abstinence after binge ethanol intoxication results in brain cell genesis that could contribute to the return of brain function and structure found in abstinent humans.
207067391
Microglial activation is not equivalent to neuroinflammation in alcohol-induced neurodegeneration: The importance of microglia phenotype
Excessive alcohol intake, a defining characteristic of an alcohol use disorder (AUD), results in neurodegeneration in the hippocampus and entorhinal cortex that has been linked to a variety of cognitive deficits. Neuroinflammation is thought to be a factor in alcohol-induced neurodegeneration, and microglia activation is a key but not sole component of an inflammatory response. These experiments investigate the effects of ethanol exposure in a well-accepted model of an AUD on both microglial activation and blood brain barrier disruption (BBB) in order to understand their relationship to classical definitions of inflammation and alcohol-induced neurodegeneration. Following a four-day binge ethanol paradigm, rat hippocampal and entorhinal cortex tissue was examined using three distinct approaches to determine microglia phenotype and BBB disruption: immunohistochemistry, autoradiography, and ELISA. After ethanol exposure, there was an increase in [(3)H]-PK-11195 binding and OX-42 immunoreactivity indicative of microglial activation; however, microglia were not fully activated since both OX-6 and ED-1 immunoreactive microglia were absent. This data was supported by functional evidence as there was no increase in the proinflammatory cytokines IL-6 or TNF-α, but a 26% increase in the anti-inflammatory cytokine, IL-10, and a 38% increase in the growth factor, TGF-β, seven days after exposure. Furthermore, there was no evidence of a disruption of the BBB. These data suggest that the four-day binge model of an AUD, which produces neurodegeneration in corticolimbic regions, does not elicit classical neuroinflammation but instead produces partially activated microglia. Partial activation of microglia following binge ethanol exposure suggest that microglia in this model have beneficial or homeostatic roles rather than directly contributing to neurodegeneration and are a consequence of alcohol-induced-damage instead of the source of damage.
25071602
Adolescent binge alcohol exposure induces long-lasting partial activation of microglia
Accumulating evidence indicates that the adolescent hippocampus is highly susceptible to alcohol-induced structural damage and behavioral deficits. Microglia are vitally important brain constituents needed to support and maintain proper neural function; however, alcohol's effects on microglia have only recently gained attention. The microglial response to alcohol during adolescence has yet to be studied; therefore, we examined hippocampal microglial activation in an adolescence binge alcohol exposure model. Adolescent male Sprague-Dawley rats were administered ethanol 3 times/day for 4 days and were sacrificed 2, 7, and 30 days later. Bromo-deoxy-Uridine was injected 2 days after ethanol exposure to label dividing cells. Microglia morphology was scored using the microglia marker Iba-1, while the extent of microglial activation was examined with ED-1, major histocompatibility complex-II (MHC-II), and tumor necrosis factor (TNF)-α expression. Ethanol induced significant morphological change in hippocampal microglia, consistent with activation. In addition, ethanol increased the number of BrdU+ cells throughout all regions of the hippocampus 2 days after the last dose. Confocal microscopy showed that the proliferating BrdU+ cells in each region were Iba-1+ microglia. Importantly, newly born microglia survived and retained their morphological characteristics 30 days after ethanol exposure. Ethanol did not alter hippocampal ED-1, MHC-II, or TNF-α expression, suggesting that a single period of binge ethanol exposure does not induce a full microglial-driven neuroinflammatory response. These results establish that ethanol triggers partial microglial activation in the adolescent hippocampus that persists through early adulthood, suggesting that alcohol exposure during this unique developmental time period has long-lasting consequences.
34759178
Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function
Damage to the central nervous system (CNS) leads to cellular changes not only in the affected neurons but also in adjacent glial cells and endothelia, and frequently, to a recruitment of cells of the immune system. These cellular changes form a graded response which is a consistent feature in almost all forms of brain pathology. It appears to reflect an evolutionarily conserved program which plays an important role in the protection against infectious pathogens and the repair of the injured nervous system. Moreover, recent work in mice that are genetically deficient for different cytokines (MCSF, IL1, IL6, TNFalpha, TGFbeta1) has begun to shed light on the molecular signals that regulate this cellular response. Here we will review this work and the insights it provides about the biological function of the neuroglial activation in the injured brain.
34919740
Molecular signals for glial activation: pro- and anti-inflammatory cytokines in the injured brain.
Injury to the central nervous system leads to cellular changes not only in the affected neurons but also in adjacent glial cells. This neuroglial activation is a consistent feature in almost all forms of brain pathology and appears to reflect an evolutionarily-conserved program which plays an important role for the repair of the injured nervous system. Recent work in mice that are genetically-deficient for different cytokines (M-CSF, IL-6, TNF-alpha, TGF-beta 1) has begun to shed light on the molecular signals that regulate this cellular response. Here, the availability of cytokine-deficient animals with reduced or abolished neuroglial activation provides a direct approach to determine the function of the different components of the cellular response leading to repair and regeneration following neural trauma.
23213823
Progressive expression of immunomolecules on microglial cells in rat dorsal hippocampus following transient forebrain ischemia
SummaryWe show a differential up-regulation of immunomolecules in the rat dorsal hippocampus accompanying neuronal cell death as a consequence of transient forebrain ischemia (four-vessel occlusion model). Using a panel of monoclonal antibodies (mAbs), we have examined the time course of expression of major histocompatibility complex (MHC) antigens class I (OX-18) and class II (OX-6), leukocyte common antigen (OX-1), CD4 (W3/25) and CD8 (OX-8) antigens, CR3 complement receptor (OX-42), as well as brain macrophage antigen (ED2). The study was performed at time intervals ranging from 1 to 28 days after reperfusion. Throughout all post-ischemic time periods, strongly enhanced immunoreactivity on microglial cells in the CA1 region and dentate hilus and, to a lesser extent, in CA3 was demonstrated with mAb OX-42. MHC class I-positive cells (OX-18) appeared on day 2, whereas cells immunoreactive with OX-1 and W3/25 became evident in the CA1 and hilar regions on post-ischemic day 6. In contrast, MHC class II (Ia) antigen was first detected on indigenous microglia by day 13. In some animals, the OX-8 antibody resulted in the labelling of scattered CD8-positive lymphocytes, but perivascular inflammatory infiltrates were absent. No changes in the expression of ED2 immunoreactivity on perivascular cells could be observed. The results show that following ischemic injury, microglial cells demonstrate a timedependent up-regulation and de novo expression of certain immunomolecules, indicative of their immunocompetence. The findings are compared with those obtained in other models of brain injury.
2941907
Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3.
Two monoclonal antibodies, designated MRC OX-41 and MRC OX-42, have been shown to label subsets of macrophages. Using immunoperoxidase and immunofluorescence analysis, tissue macrophages were shown to be heterogeneous with respect to binding of MRC OX-41 and MRC OX-42 antibodies. Although both antibodies labelled subsets of macrophages, the antibodies also reacted with granulocytes and dendritic cells. The antigens recognized by these antibodies were identified by metabolic and cell surface labelling followed by immunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). MRC OX-41 recognized a surface protein of 110,000-120,000 MW, while MRC OX-42 immunoprecipitated three polypeptides with molecular weights of 160,000, 103,000 and 95,000. The Fab fragment of MRC OX-42 antibody inhibited complement-mediated rosette formation between sensitized erythrocytes and rat macrophages and granulocytes. Membrane molecules with similar biochemical and functional properties to MRC OX-42 antigen have been identified in mouse and man as the receptors for iC3b, and it is probable that MRC OX-42 antibody recognizes the rat homologue of the receptors in these other species.
25694469
Phagocytic activity of macrophages and microglial cells during the course of acute and chronic relapsing experimental autoimmune encephalomyelitis
The EDI monoclonal antibody recognizes an antigen in lysosomal membranes of phagocytes. The expression of this antigen in cells increases during phagocytic activity. Here we describe the expression of ED1‐immunoreactivity during the various stages of both acute (monophasic) and chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. During the first attack of acute and chronic relapsing EAE, ED1‐immunoreactivity was present in macrophages and in cells which displayed morphologic features of activated microglial cells (i.e., cells with thick short processes). At the ultrastructural level these cells were seen to contain phagocytosed myelin structures in lysosomes. ED1‐immunoreactivity in these cells was present in the cytoplasm near lysosomes. During the remission phase of acute EAE and the relapse phase of chronic relapsing EAE, ED1‐positive cells with dendritic morphology not only were present in or nearby lesions, but were also found at sites distant from lesions throughout large parts of the brain. These cells had a morphology comparable to microglial cells in normal brain. A major difference between animals which were in remission and animals which on day 25 were suffering from a relapse, was that the latter showed the presence of lesions with darkly stained round ED1‐positive macrophages and activated microglial cells. These results indicate that during a relapse, newly recruited bloodborne macrophages infiltrate the brain and, together with activated lymphocytes and microglial cells, recommence a new demyelination process. © 1994 Wiley‐Liss, Inc.
3034541
Regulation and function of class II major histocompatibility complex, CD40, and B7 expression in macrophages and microglia: Implications in neurological diseases
The ability of microglia, the brain’s resident macrophage, to present antigen through the class II major histocompatibility complex (MHC) to T cells allows these normally quiescent cells to play a critical role in shaping the outcome of many neurological diseases. The expression of class II MHC antigens and the costimulatory molecules CD40 and B7 on microglia and infiltrating macrophages is regulated through a complex network of cytokines in the inflamed brain. In this review, we describe the molecular mechanisms underlying class II MHC, CD40 and B7 regulation in microglia and macrophages. Our focus is on the cis-elements in the promoters of their genes and the transcription factors activated by cytokines that bind them. The functional implications of aberrant class II MHC, CD40 and B7 expression by microglia and macrophages as related to the diseases of Multiple Sclerosis and Alzheimer’s Disease are discussed.
347264
Microglia-specific localisation of a novel calcium binding protein, Iba1.
Recently it has been shown that mRNA of Iba1 (ionized calcium binding adaptor molecule 1), which was a novel calcium binding protein cDNA-cloned by our group, is specifically expressed in microglia in cultures of rat brain cells [Imai et al. Biophys. Biochem. Res. Commun., 224 (1996) 855-862]. In the present study, immunocytochemical and immunohistochemical examinations demonstrated that Iba1 protein is expressed in microglia alone both in cultured brain cells and in the brain, respectively. In a mixed cell culture of embryonic rat brain, immunocytochemically positive for Iba1 protein were the microglia but it was not detectable in neurons, astroglia, or oligodendroglia. Immunohistochemical staining of adult rat brain sections showed Iba1 protein to be specifically localised in ramified microglia. In addition, immunohistochemical staining and immunoblot analysis of activated microglia in the facial nucleus after facial nerve axotomy shows that expression of Iba1 protein was upregulated and peaked at 7 days. These results indicated that localisation of Iba1 protein is restricted to microglia both in vitro and in vivo, and that Iba1 protein plays a role in regulating the function of microglia, especially in the activated microglia.
3801897
Stereological estimation of total microglia number in mouse hippocampus
Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimer's disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.
6823210
Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator
A stereological method for obtaining estimates of the total number of neurons in five major subdivisions of the rat hippocampus is described. The new method, the optical fractionator, combines two recent developments in stereology: a three‐dimensional probe for counting neuronal nuclei, the optical disector, and a systematic uniform sampling scheme, the fractionator. The optical disector results in unbiased estimates of neuron number, i.e., estimates that are free of assumptions about neuron size and shape, are unaffected by lost caps and over‐projection, and approach the true number of neurons in an unlimited manner as the number of samples is increased. The fractionator involves sampling a known fraction of a structural component. In the case of neuron number, a zero dimensional quantity, it provides estimates that are unaffected by shrinkage before, during, and after processing of the tissue. Because the fractionator involves systematic sampling, it also results in highly efficient estimates. Typically only 100–200 neurons must be counted in an animal to obtain a precision that is compatible with experimental studies. The methodology is compared with those used in earlier works involving estimates of neuron number in the rat hippocampus and a number of new stereological methods that have particular relevance to the quantitative study of the structure of the nervous system are briefly described in an appendix.
23935936
Chronic exposure to ethanol alters neurotrophin content in the basal forebrain-cortex system in the mature rat: effects on autocrine-paracrine mechanisms.
Neurotrophins are broadly expressed in the mammalian forebrain: notably in cerebral cortex and the basal forebrain (e.g., the septal and basal nuclei). These factors promote neuronal survival and plasticity, and have been implicated as key players in learning and memory. Chronic exposure to ethanol causes learning and memory deficits. We tested the hypothesis that ethanol affects neurotrophin expression and predicted that these changes would be consistent with alterations in retrograde or autocrine/paracrine systems. Mature rats were fed a liquid diet containing ethanol daily for 8 or 24 weeks. Weight-matched controls were pair-fed an isocaloric, isonutritive diet. Proteins from five structures (parietal and entorhinal cortices, hippocampus, and the basal and septal nuclei) were studied. ELISAs were used to determine the concentration of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). All three neurotrophins were detected in each structure examined. Ethanol treatment significantly (p < 0.05) affected neurotrophin expression in time- and space-dependent manners. NGF content was generally depressed by ethanol exposure, whereas NT-3 content increased. BDNF concentration was differentially affected by ethanol: it increased in the parietal cortex and the basal forebrain and decreased in the hippocampus. With the exception of NGF in the septohippocampal system, the ethanol-induced changes in connected structures were inconsistent with changes that would be predicted from a retrograde model. Thus, the present data (a) support the concept that neurotrophins act through a nonretrograde system (i.e., a local autocrine/paracrine system), and (b) that chronic exposure to ethanol disrupts these regulatory mechanisms.
22128438
Repeated episodic exposure to ethanol affects neurotrophin content in the forebrain of the mature rat
Chronic exposure to ethanol can cause deficits in learning and memory. It has been suggested that withdrawal is potentially more damaging than the ethanol exposure per se. Therefore, we explored the effect of repeated episodic exposure to ethanol on key regulators of cortical activity, the neurotrophins. Rats were exposed to ethanol via a liquid diet for 3 days per week for 6-24 weeks. Control rats were pair-fed an isocaloric liquid diet or ad libitum fed chow and water. The concentrations of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were determined using enzyme-linked immunosorbant assays (ELISAs). Five telencephalic structures were examined: parietal cortex, entorhinal cortex, hippocampus, the basal nucleus, and the septal nuclei. All five areas expressed each of the three neurotrophins; BDNF was most abundant and NGF the least. The parietal cortex was susceptible to ethanol exposure, NGF and BDNF content increased, and NT-3 content fell, whereas no changes were detectable in the entorhinal cortex. In the hippocampus, the amount all three neurotrophins increased following episodic ethanol exposure. Neurotrophin content in the two segments of the basal forebrain was affected; NGF and NT-3 content in the basal forebrain was reduced and NGF and BDNF content in the septal nuclei was increased by ethanol exposure. In many cases where ethanol had an effect, the change was transient so that by 24 weeks of episodic exposure, no significant changes were evident. Thus, the effects of ethanol are site- and time-dependent. This pattern differs from changes caused by chronic ethanol exposure, hence, neurotrophins must be vulnerable to the effects of withdrawal. Furthermore, the ethanol-induced changes do not appear to fit a model consistent with retrograde regulation, rather they suggest that neurotrophins act through autocrine/paracrine systems.
8482279
Caloric Restriction Suppresses Microglial Activation and Prevents Neuroapoptosis Following Cortical Injury in Rats
Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI.
6417123
Moderate Dietary Restriction Reduces p53-Mediated Neurovascular Damage and Microglia Activation After Hypoxic Ischemia in Neonatal Brain
Background and Purpose— Neurovascular damage, including neuronal apoptosis and blood–brain barrier (BBB) damage, and microglia activation account for the hypoxic-ischemia (HI) susceptibility in neonatal brain. The p53 upregulation is involved in apoptosis, endothelial cell damage, and microglia activation. We hypothesized that underweight induced by dietary restriction (DR) protects against HI in rat pups by attenuating p53-mediated neurovascular damage. Methods— Male rat pups were grouped as normal litter (NL) size (12 pups/dam), DR (18 pups/dam), and extreme DR (24 pups/dam) from postnatal day 1 and subjected to HI on postnatal day 7. Immunohistochemistry and immunoblotting were used to determine p53, phospho-murine double minute-2, caspases, BBB damage and microglia activation, and immunofluorescence to determine the cellular distribution of p53. Pharmacological approaches were used to regulate p53. Results— The NL, DR, and extreme DR pups had similar TUNEL-positive cells and caspases on postnatal day 7 and comparable learning performance at adulthood. After HI, the DR-HI, but not extreme DR-HI, pups had significantly lower p53, higher phospho-murine double minute-2, lower cleaved caspases, less BBB damage and microglia activation, and less brain volume loss than NL-HI pups. In NL-HI pups, p53 expression was located mainly in the neurons, endothelial cells, and microglia. The p53 blockage by pifithrin-&agr; in NL-HI pups decreased apoptosis, BBB damage, and microglia activation, and was neuroprotective. In contrast, upregulating p53 by nutlin-3 in DR-HI pups increased apoptosis, BBB damage, and microglia activation, and worsened brain damage. Conclusions— Moderate DR, but not extreme DR, reduces p53-mediated neurovascular damage after HI and confers long-term protection in neonatal brain.
39542001
Brain microglia constitutively express beta-2 integrins.
Localization of beta-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively CD11a (LFA-1), CD11b (Mac-1, CR3), CD11c (P150, 95; CR4), and CD18 (beta-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor Fc gamma RI. The intensity of expression of each of these antigens was enhanced on reactive microglia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9-12% of the total glial population in gray matter and 7.5-9% in white matter.
12592856
Distinct cell proliferation events during abstinence after alcohol dependence: Microglia proliferation precedes neurogenesis
Excessive alcohol intake characteristic of Alcohol Use Disorders (AUDs) produces neurodegeneration that may recover with abstinence. The mechanism of regeneration is unclear, however neurogenesis from neural stem/progenitor cells is a feasible mechanism of structural plasticity. Therefore, a timecourse of cell proliferation was examined in a rat model of an AUD and showed a striking burst in cell proliferation at 2 days of abstinence preceding the previously reported neurogenic proliferation at 7 days. New cells at 2 days, assessed by bromo-deoxy-uridine incorporation and endogenous markers, were observed throughout hippocampus and cortex. Although the majority of these new cells did not become neurons, neurogenesis was not altered at this specific time point. These new cells expressed a microglia-specific marker, Iba-1, and survived at least 2 months. This first report of microglia proliferation in a model of an AUD suggests that microgliosis could contribute to volume recovery in non-neurogenic regions during abstinence.
39920076
Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury
Microglial activation has been reported to promote neurotoxicity and also neuroprotective effects. A possible contributor to this dichotomy of responses may be the degree to which proximal neurons are injured. The aim of this study was to determine whether varying the severity of neuronal injury influenced whether microglia were neuroprotective or neurotoxic. We exposed cortical neuronal cultures to varying degrees of hypoxia thereby generating mild (<20% death, 30min hypoxia), moderate (40–60% death, 2 h hypoxia), or severe (>70% death, 6 h hypoxia) injuries. Twenty‐four hours after hypoxia, the media from the neuronal cultures was collected and incubated with primary microglial cultures for 24 h. Results showed that the classic microglial proinflammatory mediators including inducible nitric oxide synthase, tumor necrosis factor α, and interleukin‐1‐β were upregulated only in response to mild neuronal injuries, while the trophic microglial effectors brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor were upregulated in response to all degrees of neuronal injury. Microglia stimulated with media from damaged neurons were co‐cultured with hypoxic neurons. Microglia stimulated by moderate, but not mild or severe damage were neuroprotective in these co‐cultures. We also showed that the severity‐dependent phenomenon was not related to autocrine microglial signaling and was dependent on the neurotransmitters released by neurons after injury, namely glutamate and adenosine 5′‐triphosphate. Together our results show that severity of neuronal injury is an important factor in determining microglial release of “toxic” versus “protective” effectors and the resulting neurotoxicity versus neuroprotection. © 2007 Wiley‐Liss, Inc.
34503958
C3-dependent mechanism of microglial priming relevant to multiple sclerosis
Microglial priming predisposes the brain to neurodegeneration and affects disease progression. The signal to switch from the quiescent to the primed state is unknown. We show that deleting the C3 convertase regulator complement receptor 1-related protein y (Crry) induces microglial priming. Mice that were double-knockout for Crry and either C3 or factor B did not show priming, demonstrating dependence on alternative pathway activation. Colocalization of C3b/iC3b and CR3 implicated the CR3/iC3b interaction in priming. Systemic lipopolysaccharide challenge overactivated primed microglia with florid expression of proinflammatory molecules, which were blocked by complement inhibition. Relevance for neurodegenerative disease is exemplified by human multiple sclerosis (MS) and by experimental autoimmune encephalomyelitis (EAE), a model of MS. In human MS, microglial priming was evident in perilesional white matter, in close proximity to C3b/iC3b deposits. EAE was accelerated and exacerbated in Crry-deficient mice, and was dependent on C activation. In summary, C3-dependent microglial priming confers susceptibility to other challenges. Our observations are relevant to progression in MS and other neurological diseases exacerbated by acute insults.
23810724
Priming effect of morphine on the production of tumor necrosis factor-alpha by microglia: implications in respiratory burst activity and human immunodeficiency virus-1 expression.
Opiates alter a variety of functional activities of the somatic immune system; within the central nervous system, however, their effects on immune responses are unknown. In the present study, we investigated the effect of morphine on the release of tumor necrosis factor (TNF)-alpha from murine neonatal microglia. Microglial cell cultures did not release TNF-alpha when incubated with morphine alone; however, an enhanced (P < .01) release of TNF-alpha was observed when cultures were first primed with morphine for 24 h and then stimulated with lipopolysaccharide. A bell-shaped dose-response curve was observed for the priming effect of morphine; maximal enhancement of TNF-alpha release (310 +/- 15% of control) was detected at a concentration of 10(-10) M morphine. Pretreatment of microglia for 30 min with opioid receptor antagonists (i.e. naloxone and beta-funaltrexamine) completely blocked the priming effect of morphine. In addition, morphine treatment amplified (P < .01) the priming effect of lipopolysaccharide on phorbol myristate acetate-triggered superoxide anion production by microglial cell cultures, and this effect was abrogated (P < .01) by anti-TNF-alpha antibody. Furthermore, culture supernatants derived from microglial cell cultures that had been treated with morphine before stimulation with lipopolysaccharide had an increased capacity to upregulate human immunodeficiency virus-1 expression in the latently infected promonocytic clone U1. This effect was also blocked by anti-TNF-alpha antibody. These findings suggest that morphine primes microglia for enhanced production of TNF-alpha which could alter several functional activities of these cells within the brain.
14035076
Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system
Recent studies suggest that activation of the peripheral immune system elicits a discordant central (i.e., in the brain) inflammatory response in aged but otherwise healthy subjects compared with younger cohorts. A fundamental difference in the reactive state of microglial cells in the aged brain has been suggested as the basis for this discordant inflammatory response. Thus, the aging process appears to serve as a “priming” stimulus for microglia, and upon secondary stimulation with a triggering stimulus (i.e., peripheral signals communicating infection), these primed microglia release excessive quantities of proinflammatory cytokines. Subsequently, this exaggerated cytokine release elicits exaggerated behavioral changes including anorexia, hypersomnia, lethargy, decreased social interaction, and deficits in cognitive and motor function (collectively known as the sickness behavior syndrome). Whereas this reorganization of host priorities is normally adaptive in young subjects, there is a propensity for this response to be maladaptive in aged subjects, resulting in greater severity and duration of the sickness behavior syndrome. Consequently, acute bouts of cognitive impairment in elderly subjects increase the likelihood of poor self‐care behaviors (i.e., anorexia, weight loss, noncompliance), which ultimately leads to higher rates of hospitalization and mortality.
12137537
Microglial priming in neurodegenerative disease
Under physiological conditions, the number and function of microglia—the resident macrophages of the CNS—is tightly controlled by the local microenvironment. In response to neurodegeneration and the accumulation of abnormally folded proteins, however, microglia multiply and adopt an activated state—a process referred to as priming. Studies using preclinical animal models have shown that priming of microglia is driven by changes in their microenvironment and the release of molecules that drive their proliferation. Priming makes the microglia susceptible to a secondary inflammatory stimulus, which can then trigger an exaggerated inflammatory response. The secondary stimulus can arise within the CNS, but in elderly individuals, the secondary stimulus most commonly arises from a systemic disease with an inflammatory component. The concept of microglial priming, and the subsequent exaggerated response of these cells to secondary systemic inflammation, opens the way to treat neurodegenerative diseases by targeting systemic disease or interrupting the signalling pathways that mediate the CNS response to systemic inflammation. Both lifestyle changes and pharmacological therapies could, therefore, provide efficient means to slow down or halt neurodegeneration.
13785713
Are binge drinkers more at risk of developing brain damage?
Alcoholism is often associated with brain damage and cognitive deficits. Because drinking patterns can include periods of alcohol consumption followed by abstinence, binge drinking may enhance the possibility of brain damage. Chronic administration of ethanol leads to upregulation of N-methyl-D-aspartate (NMDA) and calcium receptors and increased release of glucocorticoids. NMDA-mediated mechanisms and glucocorticoid actions on the hippocampus are associated with brain damage. Thus, ethanol withdrawal may make the brain more vulnerable to damage from these mechanisms, especially with binge drinking. Therapeutic adjuncts for treating ethanol withdrawal, including NMDA, calcium, and glucocorticoid antagonists, may eventually prove useful in preventing further brain damage in alcoholism.
44252068
A comparison of continuous and episodic drinkers using the MCMI, MMPI, and ALCEVAL-R.
This study was designed to identify clinically meaningful differences between continuous and episodic drinkers who were entering inpatient treatment (N = 125) on the basis of information from intake administrations of the revised Alcohol Evaluation Instrument, the Million Clinical Multiaxial Inventory, and the Minnesota Multiphasic Personality Inventory. Separate discriminant analyses based upon (a) the MCMI and ALCEVAL-R and (b) MMPI and ALCEVAL-R yielded correct classification rates of 86% and 85%, respectively. Both analyses suggest that continuous drinkers may be characterized by greater confused and disorganized thinking, agitation, and cynicism and distrust in interpersonal relationships than are episodic drinkers. In contrast, episodic drinkers appear to have somewhat more socially conforming attitudes that may inhibit daily social drinking. Despite this, episodic drinking was associated with a higher divorce rate (71% vs. 32%) and less occupational stability.
140209874
Recognition of a bacterial adhesin by an integrin: Macrophage CR3 (α
 M
 β
 2, 
 
 
 CD11b
 
 
 CD18
 
 
 ) binds filamentous hemagglutinin of Bordetella pertussis
Abstract During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 ( α M β 2 , CD11bCD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097–1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.
14669705
Complement-Mediated Microglial Clearance of Developing Retinal Ganglion Cell Axons
In many parts of the developing vertebrate nervous system, axons are pruned to establish mature patterns of connectivity. In this issue of Neuron, Schafer et al. (2012) show that microglia may play a role in developmental axon pruning in the thalamus by engulfing presynaptic retinal ganglion cell terminals via a C3- and CR3-dependent mechanism.
6029896
Critical Role of TLR4 Response in the Activation of Microglia Induced by Ethanol1
Microglial cells are the primary immune effector cells in the brain and play a pivotal role in the neuroinflammatory processes associated with a variety of neurological and pathological disorders. Alcohol consumption induces brain damage, although the neuropathological processes are poorly understood. We previously suggested that ethanol promotes inflammatory processes in the brain, up-regulating inflammatory mediators and signaling pathways associated with IL-1RI/TLR4 receptors. In the present study we investigate whether ethanol induces microglia activation by stimulating TLR4 response and whether this response causes neuronal death and contributes to ethanol-induced neuroinflammatory damage. We demonstrate that ethanol activates microglía and stimulates NF-κB, MAPKs, and MyD88-independent (IFN regulatory factor-3, IFN-β) pathways to trigger the production of inflammatory mediators, causing neuronal death. The inflammatory response induced by ethanol is completely abrogated in microglia of TLR4-deficient mice (TLR4−/−), thus supporting the role of these receptors in microglia activation and neuronal death. In accord with the in vitro findings, acute ethanol administration induces microglia activation (CD11b+ cells) in cerebral cortex of TLR4+/+ mice, but not in TLR4−/− mice. Taken together, our results not only provide the first evidence of the critical role of the TLR4 response in the ethanol-induced microglia activation, but also new insight into the basic mechanisms participating in ethanol-induced neuroinflammatory damage.
11063914
Activated microglia are implicated in cognitive deficits, neuronal death, and successful recovery following intermittent ethanol exposure
Microglia function as the primary immune effector cells in the brain and play a pivotal role in the neuroinflammatory processes which are critical component of neurodegenerative diseases. Alcohol abuse has been considered as one of the common reasons for neurodegeneration although the causative factors are poorly understood. Here, we investigated whether activated microglia were implicated in neurodegeneration and cognitive dysfunctions in adult rats after intermittent alcohol abuse. Rats were given orally a priming dose of 5 g/kg ethanol and then 3g/kg every 8h for 4 days, followed by a 3-day ethanol-withdrawal period. These 4 days of ethanol treatments were repeated four times intermittently to simulate the binge drinking of human alcoholics. Neurodegeneration and microglial activation were detected by Fluoro-Jade B staining, Golgi staining, immunohistochemistry and ELISA, respectively, while cognitive function was assessed by Morris water maze and novel object recognition. The results showed that microglial activation and inflammatory cytokine expression were obvious in the parietal association cortex, entorhinal cortex and hippocampus accompanied by neurodegeneration following ethanol treatment. Moreover, learning and memory abilities also declined following ethanol treatments. However, the hypertrophied microglia disappeared accompanied by the decrease of inflammatory cytokines levels on day 4, and ramified microglial proliferated significantly on day 14 after ethanol withdrawal, along with a recovery from neuronal damage and cognitive impairment. Thus, the present study indicated that activated microglia might be involved in neurodegeneration and cognitive dysfunctions induced by intermittent ethanol exposure, and neurotrophic microglia appear to have a contribution to the recovery during abstinence.
83924230
BHT blocks NF-κB activation and ethanol-induced brain damage
Background: Binge ethanol administration causes corticolimbic brain damage that models alcoholic neurodegeneration. The mechanism of binge ethanol-induced degeneration is unknown, but is not simple glutamate–N-methyl-d-aspartate (NMDA) excitotoxicity. To test the hypothesis that oxidative stress and inflammation are mechanisms of binge ethanol-induced brain damage, we administered 4 antioxidants, e.g., butylated hydroxytoluene (BHT), ebselen (Eb), vitamin E (VE), and blueberry (BB) extract, during binge ethanol treatment and assessed various measures of neurodegeneration. 
 
 
 
Methods: Adult Sprague–Dawley rats were treated with intragastric ethanol 3 times per day (8–12 g/kg/d) alone or in combination with antioxidants or isocaloric diet for 4 days. Animals were killed, and brains were perfused and extracted for histochemical silver stain determination of brain damage, markers of neurogenesis, or other immunohistochemistry. Some animals were used for determination of nuclear factor κ B (NF-κB)–DNA binding by electrophoretic mobility shift assay (EMSA) or for reverse transcription–polymerase chain reaction (RT-PCR) of cyclooxygenase 2 (COX2). 
 
 
 
Results: Binge ethanol induced corticolimbic brain damage and reduced neurogenesis. Treatment with BHT reversed binge induced brain damage and blocked ethanol inhibition of neurogenesis in all regions studied. Interestingly, the other antioxidants studied, e.g., Eb, VE, and BB, did not protect against binge-induced brain damage. Binge ethanol treatment also caused microglia activation, increased NF-κB–DNA binding and COX2 expression. Butylated hydroxytoluene reduced binge-induced NF-κB–DNA binding and COX2 expression. 
 
 
 
Conclusions: Binge-induced brain damage and activation of NF-κB–DNA binding are blocked by BHT. These studies support a neuroinflammatory mechanism of binge ethanol-induced brain damage.
15873098
Microglia derived from aging mice exhibit an altered inflammatory profile
Microglia play a critical role in neurodegenerative diseases and in the brain aging process. Yet, little is known about the functional dynamics of microglia during aging. Thus, using young and aging transgenic mice expressing enhanced‐green fluorescent protein (EGFP) under the promoter of the c‐fms gene for macrophage‐colony stimulating factor receptor, we evaluated invivo‐induced inflammatory responses of EGFP‐expressing microglia sorted by flow cytometry. Aging microglia were characterized by the presence of lipofuscin granules, decreased processes complexity, altered granularity, and increased mRNA expression of both pro‐inflammatory (TNFα, IL‐1β, IL‐6) and anti‐inflammatory (IL‐10, TGFβ1) cytokines. Following lipopolysaccharide (LPS) challenge (1 mg/kg, 3 h), aging microglia exhibit increased basal expression of TNFα, IL‐1β, IL‐6, and IL‐10. Yet, the fold‐over‐basal LPS response remained constant across age, implying that the inflammatory machinery in aging microglia is functional and adjusted to the basal state. Gender differences were not overall observed across the treatments (age, LPS). The low but sustained production of pro‐inflammatory cytokines by aging microglia may have a profound impact in the brain aging process. © 2007 Wiley‐Liss, Inc.
1895197
Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment
BackgroundCytokines and alcohol share a common modulation of inflammation and hormones as well as being implicated in multiple diseases, but the mechanisms are poorly understood. The purpose of this study was to investigate the interaction of liver, serum and brain cytokines as well as whether ethanol would potentiate endotoxin (Lipopolysaccharide, LPS) responses once ethanol had cleared.MethodsMale C57BL/6J mice were treated intragastrically with water (control) or ethanol (5 g/kg, i.g., 25% ethanol, w/v), with volumes matched, for 1 day or daily for 10 days. Mice were then injected intraperitoneally with saline (control) or LPS (3 mg/kg, i.p.) in saline 24 hrs after the last dose of ethanol. Gene expression and protein synthesis of proinflammatory cytokines and anti-inflammatory cytokine, oxidative enzymes, microglial activation and inhibition of neurogenesis were examined using real-time PCR, ELISA, and immunohistochemistry.ResultsLPS increased proinflammatory cytokines (TNFα, MCP-1, IL-1β) several fold in liver, brain and serum at 1 hr. Ethanol is known to increase liver cytokines and alter the risk of multiple chronic diseases. Ten daily doses of ethanol increased brain and liver TNFα, and pretreatment with ethanol potentiated LPS-induced increases in TNFα, MCP-1, IL-1βin liver, serum and brain. Proinflammatory cytokine levels in liver and serum returned to basal levels within a day, whereas brain proinflammatory cytokines remained elevated for long periods. IL-10, an anti-inflammatory cytokine, is reduced in brain by ethanol and LPS, while brain proinflammatory cytokines remain increased, whereas liver IL-10 is increased when proinflammatory cytokines have returned to control levels. Activation of brain microglia indicated by morphological changes, reduced neurogenesis and increased brain expression of COX-2 and gp91phox NADPH oxidase subunit mRNA were found in the 10 daily doses of ethanol-pretreated LPS group.ConclusionAcute increases in serum cytokines induce long lasting increases in brain proinflammatory cytokines. Ten daily doses of ethanol exposure results in persistent alterations of cytokines and significantly increases the magnitude and duration of central and peripheral proinflammatory cytokines and microglial activation. Ethanol induced differential anti-inflammatory cytokine IL-10 responses in liver and brain could cause long lasting disruption of cytokine cascades that could contribute to protection or increased risk of multiple chronic diseases.
40036267
Measurement of serum, liver, and brain cytokine induction, thiamine levels, and hepatopathology in rats exposed to a 4-day alcohol binge protocol.
BACKGROUND
 In rodent and human studies, ethanol (EtOH) exposure is associated with elevated brain levels of the magnetic resonance spectroscopy (MRS) signal representing choline-containing compounds (Cho). One interpretation of elevated brain Cho is that it is a marker of neuroinflammation, and some evidence suggests that EtOH exposure promotes neuroinflammation. This study aimed to determine whether binge EtOH exposure (intragastric 3 g/kg 25% EtOH every 8 hours for 4 days) would induce the expression of certain cytokines in blood, liver, or brain, thereby supporting the neuroinflammation hypothesis of elevated Cho.


METHODS
Ten of 18 wild-type male Wistar rats (~322 g at baseline) were exposed to EtOH and attained average blood alcohol levels of ~315 mg/dl across 4 days. Blood for cytokine immunoassays was collected at baseline, after 5 doses of EtOH (binge), and immediately preceding euthanasia either 4 or 24 hours after the last dose of EtOH. Blood was additionally assayed for the levels of thiamine and liver enzymes; liver histopathology was performed postmortem; and tissue from liver and 6 brain regions was assayed for the potential induction of 7 cytokines.


RESULTS
There were no group effects on the levels of thiamine or its phosphate derivatives, thiamine monophosphate or thiamine diphosphate. ANOVAs of liver enzyme levels indicated that only alkaline phosphatase (ALP) levels were higher in the EtOH group than in control group at binge; ALP elevations, however, are difficult to explain in the absence of changes in the levels of additional liver enzymes. Postmortem liver pathology provided evidence for minimal microvesicular lipidosis and portocentric fibrosis in the EtOH group. Group effects on the levels of the measured cytokines in the blood (TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-13, and GRO/CXCL1) were not significant. Similarly, postmortem evaluation of liver cytokines did not reveal group effects. Postmortem evaluation of the 7 cytokines in 6 brain regions (anterior cerebellar vermis, cingulate cortex, frontal cortex, hippocampus, hypothalamus, striatum) also failed to identify group effects.


CONCLUSIONS
A single 4-day bout of binge EtOH exposure alone was insufficient to induce the expression of 7 cytokines in blood, liver, or 6 brain regions of wild-type Wistar rats. Alternative interpretations for elevations in brain Cho in response to a 4-day binge EtOH treatment are therefore necessary and may include induction of cytokines not measured herein or other noninflammatory mechanisms.
24630520
Systemic infections and inflammation affect chronic neurodegeneration
It is well known that systemic infections cause flare-ups of disease in individuals with asthma and rheumatoid arthritis, and that relapses in multiple sclerosis can often be associated with upper respiratory-tract infections. Here we review evidence to support our hypothesis that in chronic neurodegenerative diseases such as Alzheimer's disease, with an ongoing innate immune response in the brain, systemic infections and inflammation can cause acute exacerbations of symptoms and drive the progression of neurodegeneration.
14462070
The Role of Microglia in the Healthy Brain
Microglia were recently shown to play unexpected roles in normal brain development and adult physiology. This has begun to dramatically change our view of these resident “immune” cells. Here, we briefly review topics covered in our 2011 Society for Neuroscience minisymposium “The Role of Microglia in the Healthy Brain.” This summary is not meant to be a comprehensive review of microglia physiology, but rather to share new results and stimulate further research into the cellular and molecular mechanisms by which microglia influence postnatal development, adult neuronal plasticity, and circuit function.
25711981
Brain‐Derived Neurotrophic Factor in Neuronal Survival and Behavior‐Related Plasticity
Neurotrophins are critical to the development and maintenance of the mammalian central nervous system. Among them is brain‐derived neurotrophic factor (BDNF), whose synthesis and release is targeted by activation of glutamate receptors. Perturbation of this process probably underlies neurodegenerative and psychiatric disorders. A naturally occurring variation in humans, in the form of a common single‐nucleotide polymorphism in the pro region of the polypeptide at codon 66 (Val66→Met), affects processing of the pro‐BDNF polypeptide and its activation‐dependent release. This variant is associated with differences in the volume of the hippocampal formation and with anxiety and depression‐related phenotypes. Convergent findings supporting a role for BDNF in alterations to hippocampal structure and behavior are found in a “humanized” BDNF transgenic mouse. Also, recent human genetic studies have supported a role of BDNF signaling in addictive behaviors by allele‐, genotype‐, and haplotype‐based association of the TrkB gene, which encodes the cognate receptor for BDNF, with alcohol dependence. A better understanding of the influence of BDNF‐mediated pathways in cell survival and plasticity will aid in developing new approaches to restoring normal function in disease states.
2687946
Ethanol-BDNF interactions: still more questions than answers.
Brain-derived neurotrophic factor (BDNF) has emerged as a regulator of development, plasticity and, recently, addiction. Decreased neurotrophic activity may be involved in ethanol-induced neurodegeneration in the adult brain and in the etiology of alcohol-related neurodevelopmental disorders. This can occur through decreased expression of BDNF or through inability of the receptor to transduce signals in the presence of ethanol. In contrast, recent studies implicate region-specific up-regulation of BDNF and associated signaling pathways in anxiety, addiction and homeostasis after ethanol exposure. Anxiety and depression are precipitating factors for substance abuse and these disorders also involve region-specific changes in BDNF in both pathogenesis and response to pharmacotherapy. Polymorphisms in the genes coding for BDNF and its receptor TrkB are linked to affective, substance abuse and appetitive disorders and therefore may play a role in the development of alcoholism. This review summarizes historical and pre-clinical data on BDNF and TrkB as it relates to ethanol toxicity and addiction. Many unresolved questions about region-specific changes in BDNF expression and the precise role of BDNF in neuropsychiatric disorders and addiction remain to be elucidated. Resolution of these questions will require significant integration of the literature on addiction and comorbid psychiatric disorders that contribute to the development of alcoholism.
18553639
Bacterial lipopolysaccharide induces a dose-dependent activation of neuroglia and loss of basal forebrain cholinergic cells in the rat brain.
In a rat model of neuroinflammation induced with a low-dose infusion lipopolysaccharide (5.0 ng/hr, LPS), we reported that brain arachidonic acid (ARA, 20:4 n-6), but not docosahexaenoic acid (DHA, 22:6n-3), metabolism is increased compared to control rats. To further characterize the impact LPS has on the induction of injury in this model, we quantified the dose-dependent activation of neuroglia and the loss of cholinergic cells in rats subjected to increasing doses of LPS. In this study, we found that LPS produced a statistically significant and linear dose-dependent increase in the percentage of activated CD11b-positive microglia ranging from 26% to 82% following exposure to doses ranging between 0.05 and 500 ng/hr, respectively. The percentage of activated GFAP-positive astrocytes also increased linearly and significantly from 35% to 91%. Significant astroglial scaring was evident at the lateral ventricular boarder of rats treated with 50 and 500 ng/hr LPS, but not evident in control treated rats or rats treated with lower doses of LPS. A dose-dependent decrease in the numbers of ChAT-positive cells in the basal forebrain of LPS-treated rats was found at higher doses of LPS (5, 50, and 500 ng/hr) but not at lower doses. The numbers of ChAT-positive cells within individual regions of the basal forebrain (medial septum and diagonal bands) and the composite basal forebrain were similar in their response. These data demonstrate that extremely low doses of LPS are sufficient to induce significant neuroglia activation while moderate doses above 5.0 ng/hr are required to induce cholinergic cell loss.
15909831
Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice
The immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations. The technique comprises two phases: (1) slides preparation and stages involved for the reaction; (2) interpretation and quantification of the obtained expression. Immunohistochemistry is an important tool for scientific research and also a complementary technique for the elucidation of differential diagnoses which are not determinable by conventional analysis with hematoxylin and eosin. In the last couple of decades there has been an exponential increase in publications on immunohistochemistry and immunocytochemistry techniques. This review covers the immunohistochemistry technique; its history, applications, importance, limitations, difficulties, problems and some aspects related to results interpretation and quantification. Future developments on the immunohistochemistry technique and its expression quantification should not be disseminated in two languages–-that of the pathologist and another of clinician or surgeon. The scientific, diagnostic and prognostic applications of this methodology must be explored in a bid to benefit of patient. In order to achieve this goal a collaboration and pooling of knowledge from both of these valuable medical areas is vital
5134443
Neuro‐inflammation induced in the hippocampus of ‘binge drinking’ rats may be mediated by elevated extracellular glutamate content
The neuropathological and immune changes induced in the brain by ‘binge drinking’ have been investigated in a rat model. Evidence of neuro‐inflammation was identified in the ‘binge drinking’ rat model of alcohol abuse after 3 weeks of administration of 2 or 3 g/kg ethanol (EtOH), three times per day for two consecutive days, followed by 5 days of abstinence: Firstly, alveolar macrophages, isolated from these animals, showed significant increases in inducible nitric oxide synthase, as assayed by nitrite release, both before and after lipopolysaccaharide stimulation. Secondly, significant numbers of activated microglia were present in the dentate gyrus region of the hippocampus of the ‘binge drinking’ model, after major histocompatibility complex class II staining, by comparison with the control. Microdialysis studies in the ventral hippocampus identified a significant increase in the basal extracellular concentration of glutamate, in both the 2 and 3 g/kg administered ‘binge drinking’ rats. In contrast, no changes in the hippocampal extracellular concentrations, of GABA and taurine, or the dopamine and serotonin metabolites were observed under basal conditions. A further dose of EtOH induced a significant decrease in the concentrations of both 3,4‐dihydroxyphenylacetic acid and 5‐hydroxyindoleacetic acid, whereas glutamate, taurine and GABA levels were unaffected. There was no evidence that EtOH preference was initiated by the ‘binge drinking’ regimen. Our results suggest that the possible toxicity associated with ‘binge drinking’ maybe directed by the elevated glutamate levels, which in turn, activate phagocytic cells to release their inflammatory cytokines and chemokines, ultimately leading to neuro‐inflammation.
24357290
Calorie restriction increases lipopolysaccharide-induced neuropeptide Y immunolabeling and reduces microglial cell area in the arcuate hypothalamic nucleus
Calorie restriction (CR) increases longevity and elicits many health promoting benefits including delaying immunosenescence and reducing the incidence of age-related diseases. Although the mechanisms underlying the health-enhancing effects of CR are not known, a likely contributing factor is alterations in immune system functioning. CR suppresses lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines, blocks LPS-induced fever, and shifts hypothalamic signaling pathways to an anti-inflammatory bias. Furthermore, we have recently shown that CR attenuates LPS-stimulated microglial activation in the hypothalamic arcuate nucleus (ARC), a brain region containing neurons that synthesize neuropeptide Y (NPY), an orexigenic neuropeptide that is upregulated by a CR diet and has anti-inflammatory properties. To determine if increased NPY expression in the ARC following CR was associated with changes in microglial activation, a set of brain sections from mice that were exposed to 50% CR or ad libitum feeding for 28 days before being injected with LPS were immunostained for NPY. The density of NPY-immunolabeling was assessed across the rostrocaudal extent of the ARC and hypothalamic paraventricular nucleus (PVN). An adjacent set of sections were immunostained for ionized calcium-binding adapter molecule-1 (Iba1) and immunostained microglia in the ARC were digitally reconstructed to investigate the effects of CR on microglial morphology. We demonstrated that exposure to CR increased NPY expression in the ARC, but not the PVN. Digital reconstruction of microglia revealed that LPS increased Iba1 intensity in ad libitum fed mice but had no effect on Iba1 intensity in CR mice. CR also decreased the size of ARC microglial cells following LPS. Correlational analyses revealed strong associations between NPY and body temperature, and body temperature and microglia area. Together these results suggest that CR-induced changes in NPY are not directly involved in the suppression of LPS-induced microglial activation, however, NPY may indirectly affect microglial morphology through changes in body temperature.
206962673
Mifepristone pretreatment reduces ethanol withdrawal severity in vivo.
BACKGROUND
Prolonged ethanol (EtOH) intake may perturb function of the hypothalamic-pituitary-adrenal axis in a manner that promotes dependence and influences EtOH withdrawal severity. Prior in vivo and in vitro studies suggest that corticosteroids, in particular, may be elevated during EtOH intoxication and withdrawal, suggesting that intracellular glucocorticoid receptors (GRs) may promote the development of EtOH dependence.


METHODS
Adult male Sprague-Dawley rats were subjected to a 4-day binge-like EtOH administration regimen (3 to 5 g/kg/i.g. every 8 hours designed to produce peak blood EtOH levels (BELs) of <300 mg/dl). Subgroups of animals received s.c. injection of the GR antagonist mifepristone (20 or 40 mg/kg in peanut oil at 0800 hours on each of the 4 days prior to withdrawal). BELs were assessed at 0900 and 1500 hours on Days 2 (D2) and 4 (D4) of the regimen. BEL, blood corticosterone levels (BCLs), and EtOH withdrawal-associated behavioral abnormalities were assessed 10 to 12 hours after the final EtOH administration.


RESULTS
Daily mean EtOH doses for D1 to D4 of the regimen were 14.4, 9.9, 7.1, and 8.6 g/kg, respectively. The EtOH gavage regimen produced mean BELs of 255 mg/dl at 0900 on D2 and 156.2 mg/dl at 0900 on D4 of the regimen. Withdrawal from the EtOH exposure regimen, beginning 10 hours after the last EtOH administration, produced significant elevations in BCL and behavioral abnormalities including tremors, stereotypy, and "wet dog shakes." Mifepristone administration did not alter food intake or weight during the 4-day regimen, nor were there drug-dependent differences in BEL or BCL on withdrawal day. Although mifepristone produced no significant changes in behavior of EtOH-naïve animals, pretreatment with mifepristone (40 mg/kg) significantly reduced the severity of EtOH withdrawal.


CONCLUSIONS
Findings suggest that activation of GRs promotes neuroadaptation to binge-like EtOH exposure, contributing to the development of EtOH dependence. Further, GRs may represent therapeutic targets to be exploited in reducing the severity of EtOH withdrawal.
2234062
Microglia serve as a neuroimmune substrate for stress-induced potentiation of CNS pro-inflammatory cytokine responses
Prior exposure to a stressor can potentiate CNS pro-inflammatory immune responses to a peripheral immune challenge. However, the neuroimmune substrate(s) mediating this effect has not been determined. The present investigation examined whether microglia serve as this neuroimmune substrate given that microglia are the primary immune effector cell in the CNS. The effect of inescapable shock (IS) on glial activation (MHC II, CD11b, Iba-1, and GFAP) and regulatory markers (CD200) in vivo, and microglia pro-inflammatory responses (interleukin-1beta; IL-1beta) to lipopolysaccharide (LPS) ex vivo, were assessed in rat hippocampus. IS upregulated the microglia activation marker MHC II 24h post-IS, while the astroglia marker GFAP was unaffected. IS also downregulated the neuronal glycoprotein CD200, which functions to hold microglia in a quiescent state. Moreover, IS potentiated the pro-inflammatory response to LPS ex vivo 24h post-IS in isolated hippocampal microglia. Finally, the behavioral controllability of shock was manipulated and the effect of escapable (controllable) shock was comparable to the effect of IS on hippocampal microglia responses to LPS ex vivo. The present results suggest that stress can activate microglia, thereby sensitizing the pro-inflammatory reactivity of microglia to immunogenic stimuli.
38340146
Similar withdrawal severity in adolescents and adults in a rat model of alcohol dependence.
Alcohol use during adolescence leads to increased risk of developing an alcohol use disorder (AUD) during adulthood. Converging evidence suggests that this period of enhanced vulnerability for developing an AUD may be due to the adolescent's unique sensitivity and response to alcohol. Adolescent rats have been shown to be less sensitive to alcohol intoxication and withdrawal susceptibility; however, age differences in ethanol pharmacokinetics may underlie these effects. Therefore, this study investigated alcohol intoxication behavior and withdrawal severity using a modified Majchrowicz model of alcohol dependence that has been shown to result in similar blood ethanol concentrations (BECs) despite age differences. Adolescent (postnatal day, PND, 35) and adult rats (PND 70+) received ethanol according to this 4-day binge paradigm and were observed for withdrawal behavior for 17h. As expected, adolescents showed decreased sensitivity to alcohol-induced CNS depression as evidenced by significantly lower intoxication scores. Thus, adolescents received significantly more ethanol each day (12.3+/-0.1g/kg/day) than adults (9.2+/-0.2g/kg/day). Despite greater ethanol dosing in adolescent rats, both adolescent and adult groups had comparable peak BECs (344.5+/-10.2 and 338.5+/-7.8mg/dL, respectively). Strikingly, withdrawal severity was similar quantitatively and qualitatively between adolescent and adult rats. Further, this is the first time that withdrawal behavior has been reported for adolescent rats using this model of alcohol dependence. A second experiment confirmed the similarity in BECs at various time points across the binge. These results demonstrate that after consideration of ethanol pharmacokinetics between adults and adolescents by using a model that produces similar BECs, withdrawal severity is nearly identical. This study, in combination with previous reports on ethanol withdrawal in adolescents and adults, suggests only a BEC-dependent effect of ethanol on withdrawal severity regardless of age.
20655972
The effects of repeated oral gavage on the health of male CD-1 mice
Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.
36066820
Stress-induced microglial activation may facilitate the progression of neurodegenerative disorders.
Microglial activation has been associated with various clinical disorders. In particular, activated microglia have been reported in various brain regions, including the hippocampus, substantia nigra, striatum, and cerebral cortex, in which significant neurodegeneration occurs in various neurodegenerative disorders. Our previous study demonstrated that acute stress, restraint combined with water immersion, substantially induces massive microglial activation in the brain, particularly in the hippocampus, hypothalamus, thalamus, and periaquaductal gray. Furthermore, several studies have also demonstrated that exposure of animals to stress induces microglial activation in the hippocampus and striatum, and also induces the proliferation of microglial cells. Although stress has been shown to contribute to the neurodegenerative changes in the brain, the precise mechanism of this effect remains to be elucidated. Here, we propose that stress-induced microglial activation may be involved in the progression of neurodegenerative changes. Reduction of stress-induced microglial activation may prevent further progression of neurodegenerative disorders.
41205984
Cognitive deficits and CNS damage after a 4-day binge ethanol exposure in rats
Impairments of learning and memory are common neuropsychological sequelae of chronic alcohol abuse. Alcoholics often have impairments of anterograde memory, including spatial memory dysfunction, and a tendency toward response perseveration. This study was designed to assess the effects of binge ethanol exposure on neurodegeneration and cognitive function. Rats were given ethanol three times daily for 4 days. Silver staining revealed neurodegeneration in the olfactory bulb, piriform cortex, perirhinal cortex, entorhinal cortex, and dentate gyrus. After withdrawal, behavioral testing in the Morris water maze revealed significant differences in reversal learning between treatment groups. Ethanol-treated animals required more trials to learn the reversal task, entered the previously trained quadrant more often, and spent more time there than controls. [3H]PK-11195 binding, an index of CNS damage, was elevated in the piriform cortex of ethanol-treated animals. Thus, binge ethanol exposure resulted in neurodegeneration of a corticolimbic circuit with common excitatory inputs from the olfactory bulb and was associated with perseverative responding on a spatial learning task. These studies suggest that a single binge drinking episode could cause neurodegeneration and cognitive dysfunction in humans. The perseverative nature of the behavioral deficit could be related to both cognitive dysfunction and the behavioral components of the addiction process.
27416930
Induction of innate immune genes in brain create the neurobiology of addiction
Addiction occurs through repeated abuse of drugs that progressively reduce behavioral control and cognitive flexibility while increasing limbic negative emotion. Recent discoveries indicate neuroimmune signaling underlies addiction and co-morbid depression. Low threshold microglia undergo progressive stages of innate immune activation involving astrocytes and neurons with repeated drug abuse, stress, and/or cell damage signals. Increased brain NF-κB transcription of proinflammatory chemokines, cytokines, oxidases, proteases, TLR and other genes create loops amplifying NF-κB transcription and innate immune target gene expression. Human post-mortem alcoholic brain has increased NF-κB and NF-κB target gene message, increased microglial markers and chemokine-MCP1. Polymorphisms of human NF-κB1 and other innate immune genes contribute to genetic risk for alcoholism. Animal transgenic and genetic studies link NF-κB innate immune gene expression to alcohol drinking. Human drug addicts show deficits in behavioral flexibility modeled pre-clinically using reversal learning. Binge alcohol, chronic cocaine, and lesions link addiction neurobiology to frontal cortex, neuroimmune signaling and loss of behavioral flexibility. Addiction also involves increasing limbic negative emotion and depression-like behavior that is reflected in hippocampal neurogenesis. Innate immune activation parallels loss of neurogenesis and increased depression-like behavior. Protection against loss of neurogenesis and negative affect by anti-oxidant, anti-inflammatory, anti-depressant, opiate antagonist and abstinence from ethanol dependence link limbic affect to changes in innate immune signaling. The hypothesis that innate immune gene induction underlies addiction and affective disorders creates new targets for therapy.
4853038
Current hypotheses on the mechanisms of alcoholism.
Chronic use of alcohol results in progressive changes to brain and behavior that often lead to the development of alcohol dependence and alcoholism. Although the mechanisms underlying the development of alcoholism remain to be fully elucidated, diminished executive functioning due to hypoactive prefrontal cortex executive control and hyperactive limbic system anxiety and negative emotion might contribute mechanistically to the shift from experimental use to alcoholism and dependence. In the chapter that follows, behavioral deficits associated with cortical dysfunction and neurodegeneration will be related to the behavioral characteristics of alcoholism (e.g., diminished executive function, impulsivity, altered limbic modulation). We will provide evidence that alterations in cyclic AMP-responsive element binding protein (CREB: neurotrophic) and NF-κB (neuroimmune) signaling contribute to the development and persistence of alcoholism. In addition, genetic predispositions and an earlier age of drinking onset will be discussed as contributing factors to the development of alcohol dependence and alcoholism. Overall chronic ethanol-induced neuroimmune gene induction is proposed to alter limbic and frontal neuronal networks contributing to the development and persistence of alcoholism.