Blood-brain barrier (BBB) breakdown is a feature of cerebral ischaemia, multiple sclerosis, and other neurodegenerative diseases, yet the relationship between astrocytes and the BBB integrity remains unclear. We present a simple in vivo model in which primary astrocyte loss is followed by microvascular damage, using the metabolic toxin 3-chloropropanediol (S-alpha-chlorohydrin). This model is uncomplicated by trauma, ischaemia, or primary immune involvement, permitting the study of the role of astrocytes in vascular endothelium integrity, maintenance of the BBB, and neuronal function. Male Fisher F344 rats given 3-chloropropanediol show astrocytic damage and death at 4-24 h in symmetrical brainstem and midbrain nuclear lesions, while neurons show morphological changes at 24-48 h. Fluorescent 10 kDa dextran tracers show the BBB leaking from 24 h, progressing to petechial haemorrhage after 48-72 h, with apparent repair after 6 days. BBB breakdown, but not the earlier astrocytic death, is accompanied by a delayed increase in blood flow in the inferior colliculus. An ED1 inflammatory response develops well after astrocyte loss, suggesting that inflammation may not be a factor in starting BBB breakdown. This model demonstrates that the BBB can self-repair despite the apparent absence of direct astrocytic-endothelial contact. The temporal separation of pathological events allows pharmacological intervention, and the mild reversible ataxia permits long-term survival studies of repair mechanisms.

Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood-brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O 2 ) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-h distribution, increased nPKC-h and aPKC-f protein expression, and activation by phosphorylation of nPKC-h (Thr538) and aPKC-f (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (h and f) expression, and phosphorylation. This study supports the hypothesis that nPKC-h and aPKC-f signaling mediates TJ protein disruption resulting in increased BBB permeability.

Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O2, 60 min), Hx (6% O2, 60 min), or hypoxia/reoxygenation (H/R, 6% O2, 60 min followed by 21% O2, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with ‘TJ-associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.

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Breakdown of the blood-brain barrier is a feature of acute and chronic neurodegenerative changes, yet the relationship between astrocytes and the mature barrier remains unclear. We studied this role of astrocytes in vivo using a gliotoxin and evaluated changes in three vascular tight junction markers. Male Fisher F344 rats given systemic 3-chloropropanediol showed astrocytic loss in the inferior colliculus from 12-24 h until the lesion was repopulated 8-28 days later. Within 6 h of astrocyte loss, microvessels in this area began to demonstrate a loss of the normal paracellular localization of the transmembrane proteins occludin and claudin-5 and cytoplasmic zonula occludens-1, which correlated with focal vascular leak of dextran (10 kDa) and fibrinogen. Platelet endothelial adhesion molecule-1 staining revealed that there was no loss of the endothelial lining. Between 4-8 days, severe downregulation of tight junction protein expression was observed, which subsequently returned over the same time period as astrocytes repopulated the lesion. Unexpectedly, dextran and fibrinogen leak from vessels had ceased at 6 days, well before the return of occludin and claudin-5 to appropriate paracellular domains. Control nonvulnerable cortical tissue showed no change in astrocyte morphology and tight junction expression over the same time course. Our data supports a primary role for astrocytic contact in the expression of occludin, claudin-5, and zonula occludens-1 in the mature brain vasculature in vivo. However, barrier integrity to dextran (10 kDa) and fibrinogen can be restored in the absence of astrocytes and tight junction proteins (occludin, claudin-5, and zonula occludens-1).

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Pain is a complex phenomenon involving both a peripheral innate immune response and a CNS response as well as activation of the hypothalamic-pituitary-adrenal axis. The peripheral innate immune response to injury involves the rapid production and local release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-/alpha), interleukin-1 (IL-1) and IL-6. Recent studies into the CNS response to peripheral chronic inflammatory pain strongly implicates a role for glia, and local synthesis of proinflammatory cytokines and growth factors. A characteristic feature of CNS inflammation is gliosis, in which inflammatory mediators activate glial cells (e.g. astrocytes and microglia, macrophages and leukocytes) which have been shown to induce and maintain hyperalgesia. In addition, inflammatory pain induces changes in blood-brain barrier (BBB) permeability and alters transport of clinically relevant drugs used to treat pain into the brain. Despite the increasing body of evidence for the involvement of glia in chronic pain and the role of glia in maintaining the BBB, few studies have addressed glial/endothelial interactions and the mechanisms by which glia may regulate the BBB during inflammatory pain. Further studies into the cellular mechanisms of glial/endothelial interactions may identify novel therapeutic targets for reversing chronic inflammatory induced BBB dysfunction and innovate therapies for modulating the severity of chronic inflammatory pain.

Blood-brain barrier (BBB) dysfunction is a feature of many neurodegenerative disorders. The mechanisms and interactions between astrocytes, extracellular matrix and vascular endothelial cells in regulating the mature BBB are poorly understood. We have previously shown that transitory GFAP-astrocyte loss, induced by systemic administration of 3-chloropropanediol, leads to reversible disruption of tight junction complexes and BBB integrity to a range of markers. However, early restoration of BBB integrity to dextran (10-70 kDa) and fibrinogen was seen in the absence of paracellular tight junction proteins claudin-5 and occludin. In the present study we show that in the GFAP-astrocyte lesioned rat inferior colliculus, paracellular expression of adherens junction proteins (VE-cadherin and β-catenin) was maintained in vascular endothelial cells that lacked paracellular claudin-5 expression and which showed reversible post-translational occludin modification. Claudin-1 expression paralleled the loss and recovery of claudin-5, while claudin -3 or -12 immunoreactivity was not detected. In addition, the extracellular matrix, as visualized by laminin and fibronectin, underwent extensive reversible remodeling and perivascular CD169 macrophages become abundant throughout the lesioned inferior colliculus. At a time that GFAP-astrocytes repopulated the lesion area and tight junction proteins were returned to paracellular domains, the extracellular matrix and leukocyte profiles normalized and resembled profiles seen in control tissue. This study supports the hypothesis that a combination of paracellular adherens junctional proteins, remodeled basement membrane and the presence perivascular leukocytes provide a temporary barrier to limit extravasation of macromolecules and potentially neurotoxic substances into the brain parenchyma until tight junction proteins are restored to paracellular domains.