A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage.

Stroke is a major reason for persistent disability due to insufficient treatment strategies beyond reperfusion, leading to oligodendrocyte death and axon demyelination, persistent inflammation and astrogliosis in peri-infarct areas. After injury, oligodendroglial precursor cells (OPCs) have been shown to compensate for myelin loss and prevent axonal loss through the replacement of lost oligodendrocytes, an inefficient process leaving axons chronically demyelinated. Phenotypic screening approaches in demyelinating paradigms revealed substances that promote myelin repair. We established an ex vivo adult organotypic coronal slice culture (OCSC) system to study repair after stroke in a resource-efficient way. Post-photothrombotic OCSCs can be manipulated for 8 d by exposure to pharmacologically active substances testing remyelination activity. OCSCs were isolated from a NG2-CreERT2-td-Tomato knock-in transgenic mouse line to analyze oligodendroglial fate/differentiation and kinetics. Parbendazole boosted differentiation of NG2+ cells and stabilized oligodendroglial fate reflected by altered expression of associated markers PDGFR-α, CC1, BCAS1 and Sox10 and GFAP. In vitro scratch assay and chemical ischemia confirmed the observed effects upon parbendazole treatment. Adult OCSCs represent a fast, reproducible, and quantifiable model to study OPC differentiation competence after stroke. Pharmacological stimulation by means of parbendazole promoted OPC differentiation.

Microglia contributes to remyelination in cerebral but not spinal cord ischemia.

Inflammation after injury of the central nervous system (CNS) is increasingly viewed as a therapeutic target. However, comparative studies in different CNS compartments are sparse. To date only few studies based on immunohistochemical data and all referring to mechanical injury have directly compared inflammation in different CNS compartments. These studies revealed that inflammation is more pronounced in spinal cord than in brain. Therefore, it is unclear whether concepts and treatments established in the cerebral cortex can be transferred to spinal cord lesions and vice versa or whether immunological treatments must be adapted to different CNS compartments. By use of transcriptomic and flow cytometry analysis of equally sized photothrombotically induced lesions in the cerebral cortex and the spinal cord, we could document an overall comparable inflammatory reaction and repair activity in brain and spinal cord between day 1 and day 7 after ischemia. However, remyelination was increased after cerebral versus spinal cord ischemia which is in line with increased remyelination in gray matter in previous analyses and was accompanied by microglia dominated inflammation opposed to monocytes/macrophages dominated inflammation after spinal cord ischemia. Interestingly remyelination could be reduced by microglia and not hematogenous macrophage depletion. Our results show that despite different cellular composition of the postischemic infiltrate the inflammatory response in cerebral cortex and spinal cord are comparable between day 1 and day 7. A striking difference was higher remyelination capacity in the cerebral cortex, which seems to be supported by microglia dominance.

Spatio-temporal data visualization for monitoring of control measures in the prevention of the spread of COVID-19 in Bosnia and Herzegovina.

Aim The damage caused by the COVID-19 pandemic has made the prevention of its further spread at the top of the list of priorities of many governments and state institutions responsible for health and civil protection around the world. This prevention implies an effective system of epidemiological surveillance and the application of timely and effective control measures. This research focuses on the application of techniques for modelling and geovisualization of epidemic data with the aim of simple and fast communication of analytical results via geoportal. Methods The paper describes the approach applied through the project of establishing the epidemiological location-intelligence system for monitoring the effectiveness of control measures in preventing the spread of COVID-19 in Bosnia and Herzegovina. Results Epidemic data were processed and the results related to spatio-temporal analysis of the infection spread were presented by compartmental epidemic model, reproduction number R, epi-curve diagrams as well as choropleth maps for different levels of administrative units. Geovisualization of epidemic data enabled the release of numerous information from described models and indicators, providing easier visual communication of the spread of the disease and better recognition of its trend. Conclusion The approach involves the simultaneous application of epidemic models and epidemic data geovisualization, which allows a simple and rapid evaluation of the epidemic situation and the effects of control measures. This contributes to more informative decision-making related to control measures by suggesting their selective application at the local level.

Modeling of multiply connected sound spaces by the surface coupling approach.

The surface coupling approach is employed to predict the sound field in multiply connected spaces (rooms) with internal noise sources. The coupling is achieved by developing sound pressure and particle velocity at the interfaces between rooms in simple interface functions. Plane surface harmonics can be employed as interface functions where rectangular interfaces are concerned. Using the blocked sound pressure and impedance across the interface surfaces as acoustical descriptors, each room can be identified separately. The acoustical continuity conditions can then be applied to obtain the resulting sound field. Using this approach, the prediction of the sound field created by point sources in three connected parallelepipedic rooms was successfully carried out. The parametric study shows that the normalized harmonics wavenumber equal to 2 times the normalized sound wavenumber enables acceptable sound prediction. Further application was done by predicting sound energy flow of three monopole sources in a rectangular space coupled with a semi-infinite field.

Computation of sound radiation by a driver in a cabinet using a substitute source approach.

Sound radiation by a driver set in a rigid closed cabinet is modeled analytically using the principle of wave superposition. The driver-cabinet assembly is replaced by an array of volumeless substitute sources-monopoles-confined within its surface. The role of substitute sources is to reproduce the sound field exterior to the surface as close to the original field as possible. The frequency dependent positions and strengths of substitute monopoles are optimized by an iterative search procedure aimed at matching the prescribed surface boundary conditions of the original source. The time-consuming optimization of monopole positions is carried out at narrowband center frequencies reducing the computational cost without significant loss of accuracy. The consistency of computed results is verified by checking the power output through the cabinet surface. Modeling is done for anechoic and semi-anechoic conditions. The model has been validated experimentally in a semi-anechoic room with satisfactory results using a mid-range driver set in a closed-box baffle.

Thrombin receptor protease-activated receptor 4 is a key regulator of exaggerated intimal thickening in diabetes mellitus.

BACKGROUND: Diabetes mellitus predisposes to thrombotic and proliferative vascular remodeling, to which thrombin contributes via activation of protease-activated receptor (PAR) 1. However, the use of PAR-1 inhibitors to suppress remodeling may be limited by severe bleeding. We recently reported upregulation of an additional thrombin receptor, PAR-4, in human vascular smooth muscle cells exposed to high glucose and have now examined PAR-4 as a novel mediator linking hyperglycemia, hypercoagulation, and vascular remodeling in diabetes mellitus. METHODS AND RESULTS: PAR-4 expression was increased in carotid atherectomies and saphenous vein specimens from diabetic versus nondiabetic patients and in aorta and carotid arteries from streptozotocin-diabetic versus nondiabetic C57BL/6 mice. Vascular PAR-1 mRNA was not increased in diabetic mice. Ligated carotid arteries from diabetic mice developed more extensive neointimal hyperplasia and showed greater proliferation than arteries from nondiabetic mice. The augmented remodeling response was absent in diabetic mice deficient in PAR-4. At the cellular level, PAR-4 expression was controlled via the mRNA stabilizing actions of human antigen R, which accounted for the stimulatory actions of high glucose, angiotensin II, and H2O2 on PAR-4 expression, whereas cicaprost via protein kinase A activation counteracted this effect. CONCLUSIONS: PAR-4 appears to play a hitherto unsuspected role in diabetic vasculopathy. The development of PAR-4 inhibitors might serve to limit mainly proliferative processes in restenosis-prone diabetic patients, particularly those patients in whom severe bleeding attributed to selective PAR-1 blockade or complete thrombin inhibition must be avoided or those who do not require anticoagulation.

Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes.

Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1 U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1 µM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [(3)H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10 µM for SFLLRN or SLIGRL, 0.1 U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [(3)H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed concept of bimodal PAR function in cardiomyocytes remains to be defined, particularly in vivo where hemodynamic and other regulatory factors may counteract or mask the direct cellular actions described here.

Nrf2 induces interleukin-6 (IL-6) expression via an antioxidant response element within the IL-6 promoter.

IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-κB, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.

[Puumala and Dobrava viruses in the northeastern and central regions of Bosnia]. / Puumala i Dobrava virusi na podrucju sjeveroistocne i srednje Bosne.

UNLABELLED: Bosnia and Herzegovina has been known as a highly endemic region for Hantavirus infections for more than 50 years. Previous studies have shown that at least two different hantaviruses, the murine Dobrava (DOB) and avricoline Puumala (PUU) viruses, each carried by a different rodent species, have been circulating in the area. However, there is little information on rodent population density fluctuations in Bosnia over the past years as well as on the ratio of Puumala to Dobrava infection in humans. THE AIMS OF OUR STUDY WERE: to identify the rodent species which may serve as hantavirus reservoirs in the north-east and central Bosnia; to assess the geographical distribution, density and population dynamics of rodent species in the area; to assess the influence of climatic conditions on the size of rodent population; and to determine the ratio of Puumala to Dobrava infection in humans. METHODS: The epidemiologic and epizootic study in the north-east and central Bosnia was conducted during the 8-year period (1995-2003). The average yearly and monthly temperatures, air humidity and precipitation during the study period were analyzed. A total of 381 small rodents were caught during the epidemic years (1995 and 2002), and in-between the epidemic periods (1999 and 2000). The animals were caught by live-traps and identified by morphometric methods. The density of animals was estimated by counting the number of holes per 1000 m2. Sera of 311 patients with clinical signs and symptoms of hemorrhagic fever with renal syndrome (HFRS) were tested for the presence of antibodies reactive to the Dobrava, Puumala and Seoul viruses by using indirect immunofluorescence test (IIF), and IgG and IgM ELIS. Sera of 84 patients were tested using only IIF, and 227 sera were tested by IIF and -capture IgM ELIS tests. RESULTS: During the epidemic years, the average monthly temperatures in February were by 4.3 times higher than the average temperatures during the nonepidemic years, which may have influenced the early reproduction of rodents and development of "mouse years". The rodents were identified as: Apodemus flavicollis (n = 139), Apodemus sylvaticus (n = 89), Apodemus agrarius (n = 4), Clethrionomys glareolus (n = 117), Sorex araneus (n = 5), Pytimus subterraneus (n = 23), Mus musculus (n = 1), Mycrotus arvalis (n = 1) and Rattus norvegicus (n = 2). Clethrionomys glareolus was predominant in the regions with the altitude higher than 1160 meters and Apodemus species in the regions with the altitude lower than 670 meters. The rodent population density changes seasonally and cyclically. During the epidemic years, the rodent population density was marked as very high, whereas during the nonepidemic years it was designated from low to moderate. Well-known natural hosts of Hantaviruses (A. flavicolis and C. glareolus) are most widely spread species of small rodents, and the increase in their population is closely related with outbreaks of epidemics of HVBS-a. Puumala virus caused HVBS-a in 49.84% (155/311); Dobrava virus in 23.15% (72/311) of cases, whereas Hantaviruses serotype was not identified in 27.00% (84/311) of cases. Infections caused by Puumala virus were more frequent than the infections caused by Dobrava virus during both epidemic and nonepidemic periods. The proportion of humans infected with Puumala and Dobrava viruses correlated with the number of natural hosts of Hantaviruses in the areas of HVBS outbreaks. The study of the prevalence of hantavirus antibodies in the populations of rodents and humans, which had been under way, should elucidate these relationships.