Disruption of blood–brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge‐like drinking model

Abstract

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice.

Alcohol intake alters immune responses and promotes CNS viral persistence in mice

“…Kappes et al [50] demonstrated that LCMV infection of the meninges and ependyma precedes infection of neurons and proposed that a critical parameter in the pathogenesis of neurotropic viruses is the rate of immune activation. Thus, EtOH-induced damage to blood brain barrier integrity [53] and delays or impairments in T cell responses, as our data suggests ( e.g ., Figs. 2 and 3), may increase vulnerability to persistent CNS infections [54].…”

The role of neuroimmune signaling in alcoholism

“…(Vetreno and Crews, 2012; Vetreno et al, 2013a). Studies on the post-mortem brains of human alcoholics also find increased microglial markers (He and Crews, 2008; Rubio-Araiz et al, 2016) and increased astrocyte markers (Rubio-Araiz et al, 2016). Post-mortem human alcoholic brains show increased neuroimmune molecules, such as MCP-1, TL2, TLR3, TLR4 and HMGB1 (Crews et al, 2013b; He and Crews, 2008).…”

Effects of Ethanol on Brain Extracellular Matrix: Implications for Alcohol Use Disorder

“…Similar to laminin and fibronectin, collagen‐IV levels are reduced by chronic EtOH exposure. Rubio‐Araiz and colleagues () found that collagen‐IV is decreased in the dorsolateral prefrontal cortex of human alcoholics. The tight junction protein claudin‐5, a component of the BBB, was also decreased, along with an increase in the number of leukocytes infiltrating the brain (Rubio‐Araiz et al., ).…”

“…Laminin is also affected by EtOH exposure in the adult mouse. Mice chronically fed an EtOH diet for 2 weeks, or mice that have intermittently consumed EtOH in a binge‐drinking paradigm for 4 weeks demonstrate decreased laminin protein in the cornus ammonis 1 (CA1) region of the hippocampus (Rubio‐Araiz et al., ; Skrzypiec et al., ). In both of these EtOH exposure paradigms, laminin levels were measured at 6 or 24 hours of EtOH withdrawal, respectively.…”