Vascular contributions to Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia and is characterized by progressive neurodegeneration and cognitive decline. Understanding the pathophysiology underlying AD is paramount for the management of individuals at risk of and suffering from AD. The vascular hypothesis stipulates a relationship between cardiovascular disease and AD-related changes although the nature of this relationship remains unknown. In this review, we discuss several potential pathological pathways of vascular involvement in AD that have been described including dysregulation of neurovascular coupling, disruption of the blood brain barrier, and reduced clearance of metabolite waste such as beta-amyloid, a toxic peptide considered the hallmark of AD. We will also discuss the two-hit hypothesis which proposes a 2-step positive feedback loop in which microvascular insults precede the accumulation of Aß and are thought to be at the origin of the disease development. At neuroimaging, signs of vascular dysfunction such as chronic cerebral hypoperfusion have been demonstrated, appearing early in AD, even before cognitive decline and alteration of traditional biomarkers. Cerebral small vessel disease such as cerebral amyloid angiopathy, characterized by the aggregation of Aß in the vessel wall, is highly prevalent in vascular dementia and AD patients. Current data is unclear whether cardiovascular disease causes, precipitates, amplifies, precedes, or simply coincides with AD. Targeted imaging tools to quantitatively evaluate the intracranial vasculature and longitudinal studies in individuals at risk for or in the early stages of the AD continuum could be critical in disentangling this complex relationship between vascular disease and AD.

Toll-Like Receptor 4 Signaling in Focal Cerebral Ischemia: a Focus on the Neurovascular Unit.

A robust innate immune activation leads to downstream expression of inflammatory mediators that amplify tissue damage and consequently increase the morbidity after stroke. The Toll-like receptor 4 (TLR4) pathway is a major innate immune pathway activated acutely and chronically after stroke. Hence, understanding the intricacies of the temporal profile, specific control points, and cellular specificity of TLR4 activation is crucial for the development of any novel therapeutics targeting the endogenous innate immune response after focal cerebral ischemia. The goal of this review is to summarize the current findings related to TLR4 signaling after stroke with a specific focus on the components of the neurovascular unit such as astrocytes, neurons, endothelial cells, and pericytes. In addition, this review will examine the effects of focal cerebral ischemia on interaction of these neurovascular unit components.

HMGB1 is a Potential Mediator of Astrocytic TLR4 Signaling Activation following Acute and Chronic Focal Cerebral Ischemia.

Limited, and underutilized, therapeutic options for acute stroke require new approaches to treatment. One such potential approach involves better understanding of innate immune response to brain injury such as acute focal cerebral ischemia. This includes understanding the temporal profile, and specificity, of Toll-like receptor 4 (TLR4) signaling in brain cell types, such as astrocytes, following focal cerebral ischemia. This study evaluated TLR4 signaling, and downstream mediators, in astrocytes, during acute and chronic phases post transient middle cerebral artery occlusion (MCAO). We also determined whether high mobility group box 1 (HMGB1), an endogenous TLR4 ligand, was sufficient to induce TLR4 signaling activation in astrocytes in vivo and in vitro. We injected HMGB1 into normal cortex, in vivo, and stimulated cultured astrocytes with HMGB1, in vitro, and determined TLR4, and downstream mediator, expression by immunohistochemistry. We found that expression of TLR4, and downstream mediators, such as inducible nitric oxide synthase (iNOS), occurs in penumbral astrocytes in acute and chronic phases after focal cerebral ischemia, but was undetectable in cortical astrocytes in the contralateral hemisphere. In addition, cortical injection of recombinant HMGB1 led to a trend towards an almost 2-fold increase in TLR4 expression in astrocytes surrounding the injection site. Consistent with these results, in vitro stimulation of the DI TNC1 astrocyte cell line, with recombinant HMGB1, led to increased TLR4 and iNOS message levels. These findings suggest that HMGB1, an endogenous TLR4 ligand, is an important physiological ligand for TLR4 signaling activation, in penumbral astrocytes, following acute and chronic ischemia and HMGB1 amplifies TLR4 signaling in astrocytes.

Sur1-Trpm4 Cation Channel Expression in Human Cerebral Infarcts.

The nonselective monovalent cation channel transient receptor potential melastatin 4 (Trpm4) is transcriptionally upregulated in neural and vascular cells in animal models of brain infarction. It associates with sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which have critical roles in cytotoxic edema, cell death, blood-brain barrier breakdown, and vasogenic edema. We examined Trpm4 expression in postmortem brain specimens from 15 patients who died within the first 31 days of the onset of focal cerebral ischemia. We found increased Trpm4 protein expression in all cases using immunohistochemistry; transcriptional upregulation was confirmed using in situ hybridization of Trpm4 messenger RNA. Transient receptor potential melastatin 4 colocalized and coassociated with Sur1 within ischemic endothelial cells and neurons. Coexpression of Sur1 and Trpm4 in necrotic endothelial cells was also associated with vasogenic edema indicated by upregulated perivascular tumor necrosis factor, extravasation of serum immunoglobulin G, and associated inflammation. Upregulated Trpm4 protein was present up to 1 month after the onset of cerebral ischemia. In a rat model of middle cerebral artery occlusion stroke, pharmacologic channel blockade by glibenclamide, a selective inhibitor of sulfonylurea receptor, mitigated perivascular tumor necrosis factor labeling. Thus, upregulated Sur1-Trpm4 channels and associated blood-brain barrier disruption and cerebral edema suggest that pharmacologic targeting of this channel may represent a promising therapeutic strategy for the clinical management of patients with cerebral ischemia.

The Immune Response to Acute Focal Cerebral Ischemia and Associated Post-stroke Immunodepression: A Focused Review.

It is currently well established that the immune system is activated in response to transient or focal cerebral ischemia. This acute immune activation occurs in response to damage, and injury, to components of the neurovascular unit and is mediated by the innate and adaptive arms of the immune response. The initial immune activation is rapid, occurs via the innate immune response and leads to inflammation. The inflammatory mediators produced during the innate immune response in turn lead to recruitment of inflammatory cells and the production of more inflammatory mediators that result in activation of the adaptive immune response. Under ideal conditions, this inflammation gives way to tissue repair and attempts at regeneration. However, for reasons that are just being understood, immunosuppression occurs following acute stroke leading to post-stroke immunodepression. This review focuses on the current state of knowledge regarding innate and adaptive immune activation in response to focal cerebral ischemia as well as the immunodepression that can occur following stroke. A better understanding of the intricate and complex events that take place following immune response activation, to acute cerebral ischemia, is imperative for the development of effective novel immunomodulatory therapies for the treatment of acute stroke.

A new role for downstream Toll-like receptor signaling in mediating immediate early gene expression during focal cerebral ischemia.

To better understand the role of downstream Toll-like receptor (TLR) signaling during acute cerebral ischemia, we performed cDNA microarrays, on brain RNA, and cytokine arrays, on serum, from wild type (WT), MyD88-/- and TRIF-mutant mice, at baseline and following permanent middle cerebral artery occlusion (pMCAO). The acute stress response pathway was among the top pathways identified by Ingenuity Pathway Analysis of microarray data. We used real-time polymerase chain reaction to confirm the expression of four immediate early genes; EGR1, EGR2, ARC, Nurr77, in this pathway, and insulin degrading enzyme (IDE). Compared to WT, baseline immediate early gene expression was increased up to10-fold in MyD88-/- and TRIF-mutant mice. However, following pMCAO, immediate early gene expression remained unchanged, from this elevated baseline in these mice, but increased up to 12-fold in WT. Furthermore, expression of IDE, which also degrades ß-amyloid, decreased significantly only in TRIF-mutant mice. Finally, sE-Selectin, sICAM, sVCAM-1, and MMP-9 levels were significantly decreased only in MyD88-/- compared with WT mice. We thus report a new role for downstream TLR signaling in immediate early gene expression during acute cerebral ischemia. We also show that the TRIF pathway regulates IDE expression; a major enzyme that clears ß-amyloid from the brain.

Disruption of downstream MyD88 or TRIF Toll-like receptor signaling does not protect against cerebral ischemia.

Toll-like receptor (TLR) signaling plays an important role in cerebral ischemia, but downstream signaling events, which can be organ-specific, are incompletely understood. We thereby investigated involvement of the MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling pathways in 2 in vitro and 2 in vivo models of cerebral ischemia. For in vitro studies, we used a model of oxygen-glucose deprivation (OGD) followed by flow cytometric analysis to determine:1) viability of PC12 cells following knock-down with MyD88 siRNA compared to negative control siRNA and 2) viability, apoptosis and necrosis of cortical neurons from MyD88 null (-/-) , TRIF mutant, and wild type (WT) mice. In addition, in vivo, 1) We examined CA1 neuronal survival 7 days after global forebrain ischemia and 2) infarct volumes 24h after Middle Cerebral Artery Occlusion (MCAO) in all 3 types of mice. OGD: 1) There were no differences in either percent viability of PC12 cells transfected with MyD88 compared to negative control siRNA or 2) in percent viability, apoptosis and necrosis of cortical neurons from MyD88-/-,TRIF mutant and WT mice. Global ischemia: neuronal survival was similar in all 3 groups of mice. Finally, MCAO: infarct volumes were not statistically different among all 3 groups of mice: MyD88-/-, 23.9±9.9 mm(3), TRIF mutant, 26.7±5.8 mm(3) and WT, 17.9±8.4mm(3). These findings show that disruption of MyD88 or TRIF signaling does not confer protection in brain ischemia and suggests the possibility of additional or alternate downstream adaptors during TLR signaling in cerebral ischemia.

Causes and severity of ischemic stroke in patients with symptomatic intracranial arterial stenosis.

BACKGROUND AND PURPOSE: There are limited data on the causes and severity of subsequent stroke in patients presenting initially with TIA or stroke attributed to intracranial arterial stenosis. METHODS: We evaluated the location, type (lacunar vs nonlacunar), cause, and severity of stroke in patients who had an ischemic stroke endpoint in the Warfarin Aspirin Symptomatic Intracranial Disease (WASID) trial. RESULTS: Of the 569 patients enrolled in the WASID trial, 106 patients (18.6%) had an ischemic stroke during a mean follow-up of 1.8 years. Stroke occurred in the territory of the symptomatic artery in 77 (73%) of 106 patients. Among the 77 strokes in the territory, 70 (91%) were nonlacunar and 34 (44%) were disabling. Stroke out of the territory of the symptomatic artery occurred in 29 (27%) of 106 patients. Among these 29 strokes, 24 (83%) were nonlacunar, 14 (48%) were attributed to previously asymptomatic intracranial stenosis, and 9 (31%) were disabling. CONCLUSIONS: Most subsequent strokes in patients with symptomatic intracranial artery stenosis are in the same territory and nonlacunar, and nearly half of the strokes in the territory are disabling. The most commonly identified cause of stroke out of the territory was a previously asymptomatic intracranial stenosis. Penetrating artery disease was responsible for a low number of strokes.

Expression of IL-12 in CNS and lymphoid organs of mice with experimental allergic encephalitis.

EAE is a Th1 cell-mediated inflammatory autoimmune demyelinating disease of the central nervous system. IL-12 is a 70 kd heterodimeric cytokine, capable of regulating a wide range of immune functions. In view of its crucial role in the development of Th1 immune responses, we studied the expression of IL-12 p40 in the CNS and lymphoid organs of mice with EAE. RT-PCR analysis showed an increase in the expression of IL-12 p40 in brain and spinal cord during the acute paralytic phase of EAE and that decreased upon clinical recovery. The expression of p40 mRNA was also increased in spleen, lymph node and liver along with an elevated levels of circulating serum IL-12 during the height of disease. In vivo administration of rIL-12 increased the proliferative response and IFN-gamma production of MBP sensitized T cells and that was decreased following treatment with anti-IL-12 antibody. The expression of IL-12 in the target and lymphoid organs of animals with EAE, the induction of a Th1 type immune response following immunization with neuronal antigens and the inhibition of clinical disease upon treatment with anti-IL-12 antibody, suggest the crucial role of IL-12 in the pathogenesis of EAE.