Neuroimmune Activation and Microglia Reactivity in Female Rats Following Alcohol Dependence.

The rates of alcohol use disorder among women are growing, yet little is known about how the female brain is affected by alcohol. The neuroimmune system, and specifically microglia, have been implicated in mediating alcohol neurotoxicity, but most preclinical studies have focused on males. Further, few studies have considered changes to the microglial phenotype when examining the effects of ethanol on brain structure and function. Therefore, we quantified microglial reactivity in female rats using a binge model of alcohol dependence, assessed through morphological and phenotypic marker expression, coupled with regional cytokine levels. In a time- and region-dependent manner, alcohol altered the microglial number and morphology, including the soma and process area, and the overall complexity within the corticolimbic regions examined, but no significant increases in the proinflammatory markers MHCII or CD68 were observed. The majority of cytokine and growth factor levels examined were similarly unchanged. However, the expression of the proinflammatory cytokine TNFα was increased, and the anti-inflammatory IL-10, decreased. Thus, female rats showed subtle differences in neuroimmune reactivity compared to past work in males, consistent with reports of enhanced neuroimmune responses in females across the literature. These data suggest that specific neuroimmune reactions in females may impact their susceptibility to alcohol neurotoxicity and other neurodegenerative events with microglial contributions.

Adolescent Intermittent Ethanol Drives Modest Neuroinflammation but Does Not Escalate Drinking in Male Rats.

During adolescence, the brain is highly susceptible to alcohol-induced damage and subsequent neuroimmune responses, effects which may enhance development of an alcohol use disorder (AUD). Neuroimmune reactions are implicated in adolescent alcohol exposure escalating adulthood drinking. Therefore, we investigated whether intermittent alcohol exposure in male, adolescent rats (AIE) escalated adult drinking via two-bottle choice (2BC). We also examined the influence of housing environment across three groups: standard (group-housed with enrichment during 2BC), impoverished (group-housed without enrichment during 2BC), or isolation (single-housed without bedding or enrichment throughout). In the standard group immediately after AIE/saline and after 2BC, we also examined the expression of microglial marker, Iba1, reactive astrocyte marker, vimentin, and neuronal cell death dye, FluoroJade B (FJB). We did not observe an escalation of adulthood drinking following AIE, regardless of housing condition. Further, only a modest neuroimmune response occurred after AIE in the standard group: no significant microglial reactivity or neuronal cell death was apparent using this model, although some astrocyte reactivity was detected in adolescence following AIE that resolved by adulthood. These data suggest that the lack of neuroimmune response in adolescence in this model may underlie the lack of escalation of alcohol drinking, which could not be modified through isolation stress.

Reactive, Adult Neurogenesis From Increased Neural Progenitor Cell Proliferation Following Alcohol Dependence in Female Rats.

Hippocampal neurodegeneration is a consequence of excessive alcohol drinking in alcohol use disorders (AUDs), however, recent studies suggest that females may be more susceptible to alcohol-induced brain damage. Adult hippocampal neurogenesis is now well accepted to contribute to hippocampal integrity and is known to be affected by alcohol in humans as well as in animal models of AUDs. In male rats, a reactive increase in adult hippocampal neurogenesis has been observed during abstinence from alcohol dependence, a phenomenon that may underlie recovery of hippocampal structure and function. It is unknown whether reactive neurogenesis occurs in females. Therefore, adult female rats were exposed to a 4-day binge model of alcohol dependence followed by 7 or 14 days of abstinence. Immunohistochemistry (IHC) was used to assess neural progenitor cell (NPC) proliferation (BrdU and Ki67), the percentage of increased NPC activation (Sox2+/Ki67+), the number of immature neurons (NeuroD1), and ectopic dentate gyrus granule cells (Prox1). On day seven of abstinence, ethanol-treated females showed a significant increase in BrdU+ and Ki67+ cells in the subgranular zone of the dentate gyrus (SGZ), as well as greater activation of NPCs (Sox2+/Ki67+) into active cycling. At day 14 of abstinence, there was a significant increase in the number of immature neurons (NeuroD1+) though no evidence of ectopic neurogenesis according to either NeuroD1 or Prox1 immunoreactivity. Altogether, these data suggest that alcohol dependence produces similar reactive increases in NPC proliferation and adult neurogenesis. Thus, reactive, adult neurogenesis may be a means of recovery for the hippocampus after alcohol dependence in females.

The Tri-phasic Role of Hydrogen Peroxide in Blood-Brain Barrier Endothelial cells.

Hydrogen peroxide (H2O2) plays an important role physiologically as the second messenger and pathologically as an inducer of oxidative stress in injury, ischemia and other conditions. However, it is unclear how H2O2 influences various cellular functions in health and disease differentially, particularly in the blood-brain barrier (BBB). We hypothesized that the change in cellular concentrations of H2O2 is a major contributor in regulation of angiogenesis, barrier integrity/permeability and cell death/apoptosis in BBB endothelial cells. Rat brain microvascular endothelial cells were exposed to various concentrations of H2O2 (1 nM to 25 mM). BBB tight junction protein (zonula ocludens-1; ZO-1) localization and expression, cytoskeletal organization, monolayer permeability, angiogenesis, cell viability and apoptosis were evaluated. H2O2 at low concentrations (0.001 µM to 1 µM) increased endothelial cell tube formation indicating enhanced angiogenesis. H2O2 at 100 µM and above induced monolayer hyperpermeability significantly (p < 0.05). H2O2 at 10 mM and above decreased cell viability and induced apoptosis (p < 0.05). There was a decrease of ZO-1 tight junction localization with 100 µm H2O2, but had no effect on protein expression. Cytoskeletal disorganizations were observed starting at 1 µm. In conclusion H2O2 influences angiogenesis, permeability, and cell death/apoptosis in a tri-phasic and concentration-dependent manner in microvascular endothelial cells of the blood-brain barrier.

Quetiapine protects the blood-brain barrier in traumatic brain injury.

BACKGROUND: The integrity of the blood-brain barrier (BBB) is paramount in limiting vasogenic edema following traumatic brain injury (TBI). The purpose of this study was to ascertain if quetiapine, an atypical antipsychotic commonly used in trauma/critical care for delirium, protects the BBB and attenuates hyperpermeability in TBI. METHODS: The effect of quetiapine on hyperpermeability was examined through molecular modeling, cellular models in vitro and small animal models in vivo. Molecular docking was performed with AutoDock Vina to matrix metalloproteinase-9. Rat brain microvascular endothelial cells (BMECs) were pretreated with quetiapine (20 µM; 1 hour) followed by an inflammatory activator (20 µg/mL chitosan; 2 hours) and compared to controls. Immunofluorescence localization for tight junction proteins zonula occludens-1 and adherens junction protein ß-catenin was performed. Human BMECs were grown as a monolayer and pretreated with quetiapine (20 µM; 1 hour) followed by chitosan (20 µg/mL; 2 hours), and transendothelial electrical resistance was measured. C57BL/6 mice (n = 5/group) underwent mild to moderate TBI (controlled cortical impactor) or sham craniotomy. The treatment group was given 10 mg/kg quetiapine intravenously 10 minutes after TBI. The difference in fluorescence intensity between intravascular and interstitium (ΔI) represented BBB hyperpermeability. A matrix metalloproteinase-9 activity assay was performed in brain tissue from animals in the experimental groups ex vivo. RESULTS: In silico studies showed quetiapine thermodynamically favorable binding to MMP-9. Junctional localization of zonula occludens-1 and ß-catenin showed retained integrity in quetiapine-treated cells as compared with the chitosan group in rat BMECs. Quetiapine attenuated monolayer permeability compared with chitosan group (p < 0.05) in human BMECs. In the animal studies, there was a significant decrease in BBB hyperpermeability and MMP-9 activity when compared between the TBI and TBI plus quetiapine groups (p < 0.05). CONCLUSION: Quetiapine treatment may have novel anti-inflammatory properties to provide protection to the BBB by preserving tight junction integrity. LEVEL OF EVIDENCE: level IV.

Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition.

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1ß (IL-1ß) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1ß-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1ß-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1ß treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1ß-induced calpain activity significantly (p < 0.05). IL-1ß had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1ß-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1ß-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.

Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition.

Microvascular hyperpermeability that occurs at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema and elevated intracranial pressure following traumatic brain injury (TBI). At a cellular level, tight junction proteins (TJPs) between neighboring endothelial cells maintain the integrity of the BBB via TJ associated proteins particularly, zonula occludens-1 (ZO-1) that binds to the transmembrane TJPs and actin cytoskeleton intracellularly. The pro-inflammatory cytokine, interleukin-1ß (IL-1ß) as well as the proteolytic enzymes, matrix metalloproteinase-9 (MMP-9) are key mediators of trauma-associated brain edema. Recent studies indicate that melatonin a pineal hormone directly binds to MMP-9 and also might act as its endogenous inhibitor. We hypothesized that melatonin treatment will provide protection against TBI-induced BBB hyperpermeability via MMP-9 inhibition. Rat brain microvascular endothelial cells grown as monolayers were used as an in vitro model of the BBB and a mouse model of TBI using a controlled cortical impactor was used for all in vivo studies. IL-1ß (10 ng/mL; 2 hours)-induced endothelial monolayer hyperpermeability was significantly attenuated by melatonin (10 µg/mL; 1 hour), GM6001 (broad spectrum MMP inhibitor; 10 µM; 1 hour), MMP-9 inhibitor-1 (MMP-9 specific inhibitor; 5 nM; 1 hour) or MMP-9 siRNA transfection (48 hours) in vitro. Melatonin and MMP-9 inhibitor-1 pretreatment attenuated IL-1ß-induced MMP-9 activity, loss of ZO-1 junctional integrity and f-actin stress fiber formation. IL-1ß treatment neither affected ZO-1 protein or mRNA expression or cell viability. Acute melatonin treatment attenuated BBB hyperpermeability in a mouse controlled cortical impact model of TBI in vivo. In conclusion, one of the protective effects of melatonin against BBB hyperpermeability occurs due to enhanced BBB integrity via MMP-9 inhibition. In addition, acute melatonin treatment provides protection against BBB hyperpermeability in a mouse model of TBI indicating its potential as a therapeutic agent for brain edema when established in humans.

Doxycycline Attenuates Lipopolysaccharide-Induced Microvascular Endothelial Cell Derangements.

INTRODUCTION: Lipopolysaccharide (LPS) is known to induce vascular derangements. The pathophysiology involved therein is unknown, but matrix metalloproteinases (MMPs) may be an important mediator. We hypothesized that in vitro LPS provokes vascular permeability, damages endothelial structural proteins, and increases MMP activity; that in vivo LPS increases permeability and fluid requirements; and that the MMP inhibitor doxycycline mitigates such changes. METHODS: Rat lung microvascular endothelial cells were divided into four groups: control, LPS, LPS plus doxycycline, and doxycycline. Permeability, structural proteins ß-catenin and Filamentous-actin, and MMP-9 activity were examined. Sprauge Dawley rats were divided into sham, IV LPS, and IV LPS plus IV doxycycline groups. Mesenteric postcapillary venules were observed. Blood pressure was measured as animals were resuscitated and fluid requirements were compared. Statistical analysis was conducted using Student's t-test and ANOVA. RESULTS: In vitro LPS increased permeability, damaged adherens junctions, induced actin stress fiber formation, and increased MMP-9 enzyme activity. In vivo, IV LPS administration induced vascular permeability. During resuscitation, significantly more fluid was necessary to maintain normotension in the IV LPS group. Doxycycline mitigated all derangements observed. CONCLUSIONS: We conclude that LPS increases permeability, damages structural proteins, and increases MMP-9 activity in endothelial cells. Additionally, endotoxemia induces hyperpermeability and increases the amount of IV fluid required to maintain normotension in vivo. Doxycycline mitigates such changes both in vitro and in vivo. Our findings illuminate the possible role of matrix metalloproteinases in the pathophysiology of lipopolysaccharide-induced microvascular hyperpermeability and pave the way for better understanding and treatment of this process.

Oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals. Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity. Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.