Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue

Ischemic stroke causes cellular alterations in the "neurovascular unit" (NVU) comprising neurons, glia, and the vasculature, and affects the blood-brain barrier (BBB) with adjacent extracellular matrix (ECM). Limited data are available for the zone between the NVU and ECM that has not yet considered for neuroprotective approaches. This study describes ischemia-induced alterations for two main components of the neurovascular matrix adhesion zone (NMZ), i.e., collagen IV as basement membrane constituent and fibronectin as crucial part of the ECM, in conjunction with traditional NVU elements. For spatio-temporal characterization of these structures, multiple immunofluorescence labeling was applied to tissues affected by focal cerebral ischemia using a filament-based model in mice (4, 24, and 72 h of ischemia), a thromboembolic model in rats (24 h of ischemia), a coagulation-based model in sheep (2 weeks of ischemia), and human autoptic stroke tissue (3 weeks of ischemia). An increased fibronectin immunofluorescence signal demarcated ischemia-affected areas in mice, along with an increased collagen IV signal and BBB impairment indicated by serum albumin extravasation. Quantifications revealed a region-specific pattern with highest collagen IV and fibronectin intensities in most severely affected neocortical areas, followed by a gradual decline toward the border zone and non-affected regions. Comparing 4 and 24 h of ischemia, the subcortical fibronectin signal increased significantly over time, whereas neocortical areas displayed only a gradual increase. Qualitative analyses confirmed increased fibronectin and collagen IV signals in ischemic areas from all tissues and time points investigated. While the increased collagen IV signal was restricted to vessels, fibronectin appeared diffusely arranged in the parenchyma with focal accumulations associated to the vasculature. Integrin alpha(5) appeared enriched in the vicinity of fibronectin and vascular elements, while most of the non-vascular NVU elements showed complementary staining patterns referring to fibronectin. This spatio-temporal characterization of ischemia-related alterations of collagen IV and fibronectin in various stroke models and human autoptic tissue shows that ischemic consequences are not limited to traditional NVU components and the ECM, but also involve the NMZ. Future research should explore more components and the pathophysiological properties of the NMZ as a possible target for novel neuroprotective approaches

Solving satisfiability problems by fluctuations: The dynamics of stochastic local search algorithms

Stochastic local search algorithms are frequently used to numerically solve hard combinatorial optimization or decision problems. We give numerical and approximate analytical descriptions of the dynamics of such algorithms applied to random satisfiability problems. We find two different dynamical regimes, depending on the number of constraints per variable: For low constraintness, the problems are solved efficiently, i.e. in linear time. For higher constraintness, the solution times become exponential. We observe that the dynamical behavior is characterized by a fast equilibration and fluctuations around this equilibrium. If the algorithm runs long enough, an exponentially rare fluctuation towards a solution appears.Comment: 21 pages, 18 figures, revised version, to app. in PRE (2003

Damaged Neocortical Perineuronal Nets Due to Experimental Focal Cerebral Ischemia in Mice, Rats and Sheep

As part of the extracellular matrix (ECM), perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons, known to be affected in various neural diseases. Although these structures are considered as important parts of the neurovascular unit (NVU), their role during evolution of acute ischemic stroke and subsequent tissue damage is poorly understood and only a few preclinical studies analyzed PNs after acute ischemic stroke. By employing three models of experimental focal cerebral ischemia, this study was focused on histopathological alterations of PNs and concomitant vascular, glial and neuronal changes according to the NVU concept. We analyzed brain tissues obtained 1 day after ischemia onset from: (a) mice after filament-based permanent middle cerebral artery occlusion (pMCAO); (b) rats subjected to thromboembolic MACO; and (c) sheep at 14 days after electrosurgically induced focal cerebral ischemia. Multiple fluorescence labeling was applied to explore simultaneous alterations of NVU and ECM. Serial mouse sections labeled with the net marker Wisteria floribunda agglutinin (WFA) displayed largely decomposed and nearly erased PNs in infarcted neocortical areas that were demarcated by up-regulated immunoreactivity for vascular collagen IV (Coll IV). Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining along the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulated Coll IV and decomposed PNs accompanied by activated astroglia and altered immunoreactivity for parvalbumin, a calcium-binding protein in fast-firing GABAergic neurons which are predominantly surrounded by neocortical PNs. Furthermore, ischemic neocortical areas in rodents simultaneously displayed less intense staining of WFA, aggrecan, the net components neurocan, versican and the cartilage link protein (CRTL) as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and neuronal nuclei (NeuN). In summary, theconsistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons. These results suggest that PNs could be promising targets of future stroke treatment, and further studies should address their reorganization and plasticity in both stabilizing the acute stroke as well as supportive effects during the chronic phase of stroke

Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of u-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies

Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of α-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons. A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons. A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology