The belts of endothelial tight junctions, which impede diffusion between blood and brain, were reduced to fragmentary, small junctions in subcultured brain endothelium. When cocultured with the capillaries' nearest neighbor, the astrocytes, these endothelial tight junctions were enhanced in length, width, and complexity, as seen by en face views of the cell membranes with freeze-fracture electron microscopy. Gap junctions, common in brain endothelium in vitro but absent in mature brain capillaries in wiwo, were markedly diminished in area from among the enhanced tight junctions of the cocultures. Thus, astrocytes in vitro play a role in the formation, extent, and configuration of the junctional complexes in brain endothelium, whose diffusion barrier may likewise be influenced by astrocytes in viva.Brain capillaries have zonular tight junctions (TJs) impermeable to hydrophilic solutes (Reese and Kamovsky, 1967;Brightman and Reese, 1969). In freeze-fracture replicas of TJs, the constituent strands are continuous, anastomose extensively without free ends, and are devoid of gap junctions (Connel and Mercer, 1974;Dermietzel, 1975;Shivers, 1979;Nagy et al., 1984). The ability of brain endothelial cells to form this barrier depends on the environment in which they grow (Stewart and Wiley, 198 1). The astrocyte, as the cell immediately ensheathing brain capillaries, is the most likely candidate to affect the capillaries' passive permeability by modulating the formation and maintenance of the TJs between their endothelial cells. To test this notion, we have cultured beef brain microvessel endothelium alone or together with rat astroglial cultures, and examined the endothelial TJ with freeze-fracture electron microscopy. In thin plastic sections, tight junctions appear as fused spots between 2 cell membranes, and it is very difficult to derive any quantitative information from these cross-sectional views of the TJs. The freeze-fracture technique displays the en face vista of the interior of the cell membrane (Branton, 1966), where the TJs appear as strands of particles in a 2-dimensional plane. Furthermore, the length and width of the TJ strands can be measured morphometrically. Preliminary results of the present work have been published as an abstract (Tao-Cheng et al., 1986).Received Dec. 9, 1986; revised Feb. 24, 1987; accepted Apr. 1, 1987. We Bowman et al. (1983). The endothelial culture medium consisted of equal volumes of minimum essential medium and nutrient F12 (Gibco) with 5% plasma-derived horse serum (P. D. Bowman et al., personal communication), 10 mM HEPES, 13 mM sodium bicarbonate, 100 ILLg/ml heparin, 20 &ml endothelial cell growth supplement (Sigma), 100 pg/ml penicillin, 100 &ml streptomycin, and 2.5 &ml amphotericin B. After the cells were confluent at 7-20 d in vitro, 0.05% trypsin and 0.02% EDTA in Puck's saline solution without Ca*+ or Mg*+ were used to dissociate the cells for subculturing. In the present study, only the subcultures were examined because (1) primary cultures require laborious pro...

Background: Dementia results from heterogeneous diseases of the brain. Mixed disease forms are increasingly recognized. Methods: We performed a survey within brain banks of BrainNet Europe to estimate the proportion of mixed disease forms underlying dementia and age- and gender-specific influences. Results: Data collected in 9 centres from 3,303 individuals were analysed. The proportion of patients with mixed diagnoses among all cases with Alzheimer disease (AD), vascular pathology (VP), argyrophilic grain dementia (AGD), and synucleinopathies, such as Lewy body dementia (LBD), Parkinson disease (PD) and synuclein pathology only in the amygdala, was 53.3%. Mixed pathology was more frequently reported with LBD, PD, AGD, and VP than with AD. The percentage of mixed diagnoses for AGD and VP significantly differed between centres. In patients younger than 75 years, synucleinopathies, and pure forms of AD, VP, and AGD were more frequent in men. Above 75 years of age, more women had pure AD and pure AGD. Conclusions: The most obvious neuropathological alteration should not terminate the diagnostic procedure since copathology is likely to be found. Neuropathological interpretation of AGD and VP has not been sufficiently established in a consensus. Pure forms of synucleinopathies are unlikely sole substrates for dementia.

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Early transient presence of implanted bone marrow stem cells reduces lesion size after cerebral ischaemia in adult ratsAims: Previous studies on the therapeutic time window for intravascular administration of bone marrow stem cells (BMSCs) after stroke have shown that early intervention (from 3 h after onset) in the middle cerebral artery occlusion (MCAO) rat model is the most effective approach to reduce ischaemic lesion size. We have confirmed these observations but noticed that 2 weeks after transplantation, almost none of the grafted BMSCs could be detected in or around the lesion. The present experiments aimed to assess the fate and kinetics of intravascularly injected BMSCs shortly after administration in correlation to the development of the ischaemic lesion after MCAO. Methods: We administered a syngeneic suspension of complete (haematopoietic and mesenchymal) BMSCs via the carotid artery to rats at 2 h after MCAO onset. We examined the distribution and tissue location of BMSCs within the first 24 h after arterial administration by perfusion-fixating rats and performing immunohistochemical analysis at different time points. Results: The vast majority (>95%) of BMSCs appeared to become trapped in the spleen shortly after injection. Six hours after implantation, together with the appearance of activated microglia, the first BMSCs could be detected in and around the lesion; their number gradually increased during the first 12 h after implantation but started to decrease at 24 h. The implanted BMSCs were surrounded by activated and phagocytotic microglia. Conclusion: Our results show that ischaemic lesion size reduction can already be achieved by the early transient presence at the lesion site of intravascularly implanted BMSCs, possibly mediated via activated microglia.

Synchronous brain activity in motor cortex in perception or in complex cognitive processing has been the subject of several studies. The advanced analysis of cerebral electro-physiological activity during the course of planning (PRE) or execution of movement (EXE) in a high temporal resolution could reveal interesting information about the brain functional organization in patients following stroke damage. High-power (128 channels) electroencephalography registration was carried out on 8 healthy subjects and on a patient with stroke with capsular lacuna in the right hemisphere. For activation of motor cortex, the finger tapping paradigm was used. In this preliminary study, we tested a theoretical graph approach to characterize the task-related spectral coherence. All of the obtained brain functional networks were analyzed by the connectivity degree, the degree distribution, and efficiency parameters in the Theta, Alpha, Beta, and Gamma bands during the PRE and EXE intervals. All the brain networks were found to hold a regular and ordered topology. However, significant differences (P < 0.01) emerged between the patient with stroke and the control subjects, independently of the neural processes related to the PRE or EXE periods. In the Beta (13-29 Hz) and Gamma (30-40 Hz) bands, the significant (P < 0.01) decrease in globaland local-efficiency in the patient's networks, reflected a lower capacity to integrate communication between distant brain regions and a lower tendency to be modular. This weak organization is principally due to the significant (P < 0.01 Bonferroni corrected) increase in disconnected nodes together with the significant increase in the links in some other crucial vertices.

Highlights-Neutrophil extracellular traps (NETs) modify the structure and stability of fibrin.-NET content of thrombi varies at different locations (brain, heart, peripheral arteries).-DNA and histones in thrombi correlate with age and systemic inflammatory markers.-The amount of fibrin is similar at all examined arterial locations.-Thicker fibrin fibers are formed in coronaries than in brain and peripheral arteries. 3 Abstract Introduction-The ultrastructure and cellular composition of thrombi has a profound effect on the outcome of acute ischemic stroke (AIS), coronary (CAD) and peripheral artery disease (PAD). Activated neutrophils release a web-like structure composed mainly of DNA and citrullinated histones, called neutrophil extracellular traps (NET) that modify the stability and lysability of fibrin. Here, we investigated the NET-related structural features of thrombi retrieved from different arterial localizations and their interrelations with routinely available clinical data. Patients and methods-Thrombi extracted from AIS (n=78), CAD (n=66) or PAD (n=64) patients were processed for scanning electron microscopy, (immune)stained for fibrin, citrullinated histone H3 (cH3) and extracellular DNA. Fibrin fiber diameter, cellular components, DNA and cH3 were measured and analyzed in relation to clinical parameters. Results-DNA was least present in AIS thrombi showing a 2.5-fold lower DNA/fibrin ratio than PAD, whereas cH3 antigen was unvaryingly present at all locations. The NET content of thrombi correlated parabolically with systemic inflammatory markers and positively with patients' age. The median platelet content was lower in PAD (2.2%) than in either AIS (3.9%) or CAD (3.1%) and thrombi from smokers contained less platelets than non-smokers. Fibrin fibers were significantly thicker in male patients with CAD (median fiber diameter 76.3 nm) compared to AIS (64.1 nm) or PAD (62.1 nm) and their diameter correlated parabolically with systemic inflammatory markers.Conclusions-The observed NET-related variations in thrombus structure shed light on novel determinants of thrombus stability that eventually affect both the spontaneous progress and therapeutic outcome of ischemic arterial diseases.

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