Morphine-potentiated cognitive deficits correlate to suppressed hippocampal iNOS RNA expression and an absent type 1 interferon response in LP-BM5 murine AIDS.

Opioid use accelerates neurocognitive impairment in HIV/AIDS patients. We assessed the effect of chronic morphine treatment and LP-BM5/murine AIDS (MAIDS) infection on cognition, cytokine production, and type 1 interferon (IFN) expression in the murine CNS. Morphine treatment decreased expression of pro-inflammatory factors (CCL5, iNOS) and reduced cognitive performance in LP-BM5-infected mice, correlating to increased hippocampal viral load and a blunted type 1 IFN response. In the striatum, morphine reduced viral load while increasing IFN-α RNA expression. Our results suggest that differentially regulated type 1 IFN responses may contribute to distinct regional outcomes in the hippocampus and striatum in LP-BM5/MAIDS.

Transitory loss of glia and the subsequent modulation in inflammatory cytokines/chemokines regulate paracellular claudin-5 expression in endothelial cells.

Signaling mechanisms involved in regulating blood-brain barrier (BBB) integrity during central nervous system (CNS) inflammation remain unclear. We show that an imbalance between pro-/anti-inflammatory cytokines/chemokines alters claudin-5 expression. In vivo, gliotoxin-induced changes in glial populations and an imbalance between pro-/anti-inflammatory cytokine/chemokine expression occurred as BBB integrity was compromised. The balance was restored as BBB integrity was re-established. In vitro, TNF-α, IL-6, and MCP-1 induced paracellular claudin-5 expression loss. TNF-α- and IL-6- effects were mediated through the PI3K pathway and IL-10 attenuated TNF-α's effect. This study shows that pro-/anti-inflammatory modulators play a critical role in BBB integrity during CNS inflammation.

Biphasic modulation of paracellular claudin-5 expression in mouse brain endothelial cells is mediated through the phosphoinositide-3-kinase/AKT pathway.

Blood-brain barrier (BBB) integrity is compromised in many central nervous system disorders. Complex astrocyte and vascular endothelial cell interactions that regulate BBB integrity may be disturbed in these disorders. We previously showed that systemic administration of 3-chloropropanediol [(S)-(+)-3-chloro-1,2-propanediol] induces a transitory glial fibrillary acidic protein-astrocyte loss, reversible loss of tight junction complexes, and BBB integrity disruption. However, the intracellular signaling mechanisms that induce BBB integrity marker loss are unclear. We hypothesize that 3-chloropropanediol-induced modulation of tight junction protein expression is mediated through the phosphoinositide-3-kinase (PI3K)/AKT pathway. To test this hypothesis, we used a mouse brain endothelial cell line (bEnd.3) exposed to 3-chloropropanediol for up to 3 days. Results showed early reversible loss of sharp paracellular claudin-5 expression 90, 105, and 120 minutes after 3-chloropropanediol (500 µM) treatment. Sharp paracellular claudin-5 profiles were later restored, but lost again by 2 and 3 days after 3-chloropropanediol treatment. Western blot and immunofluorescence studies showed increased p85-PI3K expression and transitory increased AKT (Thr308) phosphorylation at 15 and 30 minutes after 3-chloropropanediol administration. PI3K inhibitors LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; 2.5-25 µM] and PI-828 [2-(4-morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one; 0.1-10 µM] prevented the 3-chloropropanediol-induced AKT (Thr308) phosphorylation and both early and late loss of paracellular claudin-5. However, AKT inhibitors only prevented the early changes in claudin-5 expression. This mechanistic study provides a greater understanding of the intracellular signaling pathways mediating tight junction protein expression and supports a hypothesis that two independent pathways triggered by PI3K mediate early and late loss of paracellular claudin-5 expression.

Morphine increases hippocampal viral load and suppresses frontal lobe CCL5 expression in the LP-BM5 AIDS model.

Chronic opiate abuse accelerates the development of cognitive deficits in human immunodeficiency virus (HIV)-1 patients. To investigate morphine's effects on viral infection of the central nervous system, we applied chronic morphine treatment to the LP-BM5 murine acquired immunodeficiency syndrome (MAIDS) model. LP-BM5 infection induces proinflammatory cytokine/chemokine production, correlating to increased blood-brain barrier permeability. Morphine treatment significantly increased LP-BM5 viral load in the hippocampus, but not in the frontal lobe. Morphine reduced the chemokine CCL5 to non-infected levels in the frontal lobe, but not in the hippocampus. These data indicate a region-specific mechanism for morphine's effects on virally-induced neurocognitive deficits.

Imaging in vivo astrocyte/endothelial cell interactions at the blood-brain barrier.

The goal of this chapter is to highlight methods used to demonstrate in vivo changes in astrocyte expression at the blood-brain barrier (BBB). Loss of BBB integrity is seen in many acute and chronic disease conditions. However, despite the importance of the BBB to homeostasis and correct functioning of the CNS, the nature of factors responsible for the induction and maintenance of BBB properties in development and the adult remains unclear. We have studied the role of astrocytes in modulating BBB integrity in two in vivo models using a gliotoxin (3-chloropropanediol), and under hypoxic stress. 3-chloropropanediol-induced astrocytic loss within the inferior colliculus leads to loss of endothelial tight junction protein expression and loss of BBB integrity. As glial fibrillary acidic protein (GFAP)-immunopositive astrocytes repopulated the lesion, tight junction protein expression returned to paracellular domains and BBB integrity was restored. Under hypoxic conditions, increased GFAP expression was seen with changes in tight junction protein expression and loss of BBB integrity. These studies suggest a critical role for glial/endothelial interactions in regulating BBB integrity in health and disease.

Glia-induced reversible disruption of blood-brain barrier integrity and neuropathological response of the neurovascular unit.

The blood-brain barrier (BBB) is the regulated interface that mediates selective transcellular transport of nutrients and essential components from the blood into the brain parenchyma. Many neurodegenerative diseases including stroke, multiple sclerosis, rheumatoid arthritis, and AIDS dementia exhibit loss of BBB integrity. Despite the increasing body of evidence for the involvement of glia in maintaining the BBB, few studies have addressed glial/endothelial/extracellular matrix interactions. A chemically induced astrocyte lesion provides a noninvasive model to study reversible BBB dysfunction in vivo. Blood-brain barrier integrity was assessed with fluorescent dextran tracers (3-70 kDa) and magnetic resonance imaging, in parallel with confocal and electron microscopy imaging of the neurovascular unit. These studies demonstrated modified tight-junction protein expression with loss of vascular integrity. We propose that adherens junction proteins and extracellular matrix remodeling provide a temporary size-selective barrier, whereas astrocyte and microglia activation direct tight-junction proteins to paracellular domains and restore BBB integrity. Morphological comparisons were made with the area postrema, a circumventricular organ with a naturally porous BBB. Further studies into cellular mechanisms of glial/endothelial/extracellular matrix interactions may identify novel glial-based therapeutic targets and innovate therapies for modulating diseases in which gliosis and raised levels of pro-inflammatory mediators are central components.

Protein kinase C activation modulates reversible increase in cortical blood-brain barrier permeability and tight junction protein expression during hypoxia and posthypoxic reoxygenation.

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-θ distribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538) and aPKC-ζ (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 (θ and ζ) expression, and phosphorylation. This study supports the hypothesis that nPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.

Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress.

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% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 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.

Microvascular P-glycoprotein expression at the blood-brain barrier following focal astrocyte loss and at the fenestrated vasculature of the area postrema.

The multidrug transporter, P-glycoprotein, expressed at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but its relationship to astrocyte expression is unclear. We have studied P-glycoprotein expression in the inferior colliculus after a temporary loss of blood-brain barrier integrity following chemically induced astrocyte loss and at the fenestrated vascular endothelium of the area postrema. Male Fisher F344 rats given 3-chloropropanediol showed astrocyte loss from 12 to 24 h until the lesion was repopulated 8-28 days later. In non-dosed tissue, P-glycoprotein expression was seen the entire length of platelet endothelial cell adhesion molecule immunoreactive vessels. Within 6 h of dosing, a significant (p<0.05) reduction in the total length of P-glycoprotein immunoreactive vasculature was evident. By 48 h, P-glycoprotein immunoreactivity was heavily fragmented. The total length of P-glycoprotein immunoreactive vessels became minimal at 4 days (p<0.001) but was still present in many vessels. From 6 to 28 days, P-glycoprotein immunoreactivity returned across the inferior colliculus, in parallel with astrocytic repopulation of the lesion, and by 28 days resembled that seen in control tissue. The area postrema showed GFAP immunoreactive astrocytes but which made limited contact with the vasculature, while the platelet endothelial cell adhesion molecule immunoreactive vasculature showed no expression of P-glycoprotein. These findings provide evidence supporting a link between GFAP-astrocyte and P-glycoprotein expression in the mature brain vasculature in vivo.

Antioxidants attenuate MK-801-induced cortical neurotoxicity in the rat.

Oxidative stress has been implicated in the pathogenesis of several neurodegenerative diseases and may result from excessive free radical production due to increased local metabolism. Non-competitive N-methyl-D-aspartate (NMDA) antagonists (MK-801 and phencyclidine) increase glucose metabolism in many brain areas and induce cytoplasmic vacuoles, heat shock protein and necrotic cell death in neurones of the rodent posterior cingulate and retrosplenial cortex. We have investigated the effect of several antioxidants with differing properties on MK-801-induced neuronal loss. Free radical scavengers (dimethyl sulfoxide (DMSO) and alpha-tocopherol) and spin traps (N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-(diethoxyphosphoryl)-5-methyl-1-pyrrole N-oxide (DEPMPO)), produced marked attenuation of MK-801-induced neuronal necrosis in the rat posterior cingulate and retrosplenial cortex. Further, administration of DMSO could be delayed by up to 4 h after MK-801 dosing and still achieve between 80 and 86% reduction in neuronal loss. We also show that MK-801 administration rapidly induced a four-fold and prolonged increase in cerebral blood flow in the posterior cingulate. This elevated regional blood flow was only transiently reduced by DMSO administration. The anterior cingulate, a region which undergoes no neuronal loss, showed only a two-fold increase in regional blood flow following MK-801 administration. These results support a hypothesis that oxidative stress plays a role in MK-801-induced neuronal necrosis since pathological changes can be attenuated by several antioxidants.

Basal forebrain cholinergic lesions reduce heat shock protein 72 response but not pathology induced by the NMDA antagonist MK-801 in the rat cingulate cortex.

Non-competitive N-methyl-D-aspartate (NMDA) antagonists, in addition to their neuroprotective potential, possess neurotoxic properties and induce seizures and psychosis. MK-801 induces cytoplasmic vacuoles and heat shock protein in pyramidal neurones in the rodent posterior cingulate and retrosplenial cortex. The mechanism of this neurotoxicity is unclear, involving many neurotransmitter systems. The aim of this study was to investigate the role of cholinergic pathways from the nucleus basalis of Meynert in mediating MK-801-induced neurotoxicity. Cholinergic projections from the nucleus basalis of Meynert were lesioned by focal injection of 192-IgG-saporin (80 ng), which after 7 days reduced the number of cholinergic cell bodies by 70% in the lesioned nucleus compared to the uninjected nucleus. Following a unilateral cholinergic lesion, MK-801 (5 mg/kg s.c.) induced expression of hsp72 mRNA (6 h) and HSP72 protein immunoreactivity (24 h) was reduced by 42 and 60%, respectively in the ipsilateral compared to the contralateral posterior cingulate. Despite this apparent protective effect, the unilateral cholinergic lesion did not affect the degree of neuronal vacuolation (6 h), necrosis (24 h) or the large and prolonged increase in cerebral blood flow which occurred over the first 9h following MK-801 administration. These results demonstrate that cholinergic neurones in the nucleus basalis of Meynert play an important role in the heat shock response to NMDA antagonist-induced neurotoxicity but also reveal an unexpected divergence between the heat shock response and the pathophysiological response. This suggests that other cholinergic pathways or non-cholinergic mechanisms are responsible for the pathological changes induced by MK-801.

Reversible disruption of tight junction complexes in the rat blood-brain barrier, following transitory focal astrocyte loss.

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).

Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact.

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.

Microglial activation and increased synthesis of complement component C1q precedes blood-brain barrier dysfunction in rats.

A reliable way to visualise the state of microglial activation is to monitor the microglial gene expression profile. Microglia are the only CNS resident cells that synthesise C1q, the recognition sub-component of the classical complement pathway, in vivo. C1q biosynthesis in resting ramified microglia is often low, but it increases dramatically in activated microglia. In this study, the expression of C1q was used to monitor microglial activation at all stages of 3-chloropropanediol-induced neurotoxicity, a new model of blood-brain barrier (BBB) breakdown. In rats, 3-chloropropanediol produces very focused lesions in the brain, characterised by early astrocyte swelling and loss, followed by neuronal death and barrier dysfunction. Using in situ hybridisation, immunohistochemistry, and real-time RT-PCR, we found that increased C1q biosynthesis and microglial activation precede BBB dysfunction by at least 18 and peak 48 h after injection of 3-chloropropanediol, which coincides with the onset of active haemorrhage. Microglial activation is biphasic; an early phase of global activation is followed by a later phase in which microglial activation becomes increasingly focused in the lesions. During the early phase, expression of the pro-inflammatory mediators interleukin-1beta (IL1beta), tumour necrosis factor alpha (TNFalpha) and early growth response-1 (Egr-1) increased in parallel with C1q, but was restricted to the lesions. Expression of C1q (but not IL1beta, TNFalpha or Egr-1) remains high after BBB function is restored, and is accompanied by late up-regulation of the C1q-associated serine proteases, C1r and C1s, suggesting that microglial biosynthesis of the activation complex of the classical pathway may support the removal of cell debris by activation of complement.