Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy

Early Hemorrhagic Transformation of Brain Infarction: Rate, Predictive Factors, and Influence on Clinical Outcome

Background and Purpose— Early hemorrhagic transformation (HT) is a complication of ischemic stroke but its effect on patient outcome is unclear. The aims of this study were to assess: (1) the rate of early HT in patients admitted for ischemic stroke, (2) the correlation between early HT and functional outcome at 3 months, and (3) the risk factors for early HT. Methods— Consecutive patients with ischemic stroke were included in this prospective study in 4 study centers. Early HT was assessed by CT examination performed at day 5±2 after stroke onset. Study outcomes were 3-month mortality or disability. Disability was assessed using a modified Rankin score (≥3 indicating disabling stroke) by neurologists unaware of the occurrence of HT in the individual cases. Outcomes in patients with and without early HT were compared by χ 2 test. Multiple logistic regression analysis was used to identify predictors for HT. Results— Among 1125 consecutive patients (median age 76.00 years), 98 (8.7%) had HT, 62 (5.5%) had hemorrhagic infarction, and 36 (3.2%) parenchymal hematoma. At 3 months, 455 patients (40.7%) were disabled or died. Death or disability was seen in 33 patients with parenchymal hematoma (91.7%), in 35 patients with hemorrhagic infarction (57.4%) as compared with 387 of the 1021 patients without HT (37.9%). At logistic regression analysis, parenchymal hematoma, but not hemorrhagic infarction, was independently associated with an increased risk for death or disability (OR 15.29; 95% CI 2.35 to 99.35). At logistic regression analysis, parenchymal hematoma was predicted by large lesions (OR 12.20, 95% CI 5.58 to 26.67), stroke attributable to cardioembolism (OR 5.25; 95% CI 2.27 to 12.14) or to other causes (OR 6.77; 95% CI 1.75 to 26.18), high levels of blood glucose (OR 1.01; 95% CI 1.00 to 1.01), and thrombolytic treatment (OR 3.54, 95% CI 1.04 to 11.95). Conclusions— Early HT occurs in about 9% of patients. Parenchymal hematoma, seen in about 3% of patients, is associated with an adverse outcome. Parenchymal hematoma was predicted by large lesions attributable to cardioembolism or other causes, high blood glucose, and treatment with thrombolysis

Nanotopography and microconfinement impact on primary hippocampal astrocyte morphology, cytoskeleton and spontaneous calcium wave signalling

Astrocytes' organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation

The glycocalyx affects the mechanotransductive perception of the topographical microenvironment

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features. Graphical Abstract: [Figure not available: see fulltext.]

Micropatterning of substrates for the culture of cell networks by stencil-assisted additive nanofabrication

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown

Airborne pollutants and lacunar stroke. A case cross-over analysis on stroke unit admissions

Particulate air pollution is known to be associated with cardiovascular disease. The relation of particulate air pollution with cerebrovascular disease (CVD) has not been extensively studied, particularly in relation to different subtypes of stroke. A time-series study was conducted to evaluate the association between daily air pollution and acute stroke unit hospitalizations in Mantua, Italy. We analyzed 781 CVD consecutive patients living in Mantua county admitted between 2006-08. Data on stroke types, demographic variables, risk factors were available from the Lombardia Stroke Registry. Daily mean value of particulate matter with a diameter <10 µm (PM(10)), carbon monoxide, nitric oxide, nitrogen dioxide, sulphur dioxide, benzene and ozone were used in the analysis. The association between CVD, ischemic strokes subtypes and pollutants was investigated with a case-crossover design, using conditional logistic regression analysis, adjusting for temperature, humidity, barometric pressure and holidays. Among the 781 subjects admitted 75.7% had ischemic stroke, 11.7% haemorrhagic stroke 12.6% transient ischemic attack. In men admission for stroke was associated with PM(10) [odds ratio (OR) 1.01, 95%; confidence interval (CI) 1.00-1.02; P<0.05]. According to the clinical classification, lacunar anterior circulation syndrome stroke type was related to PM(10) level registered on the day of admission for both genders (OR: 1.01, 95%; CI: 1.00-1.02; P<0.05) while for total anterior circulation syndrome stroke only in men (OR: 1.04, 95%; CI 1.01-1.07; P<0.05).In conclusion, our study confirms that air pollution peaks may contribute to increase the risk of hospitalization for stroke and particulate matter seems to be a significant risk factor, especially for lacunar stroke

Airborne pollutants and lacunar stroke: a case cross-over analysis on stroke unit admissions

Particulate air pollution is known to be associated with cardiovascular disease. The relation of particulate air pollution with cerebrovascular disease (CVD) has not been extensively studied, particularly in relation to different subtypes of stroke. A time-series study was conducted to evaluate the association between daily air pollution and acute stroke unit hospitalizations in Mantua, Italy. We analyzed 781 CVD consecutive patients living in Mantua county admitted between 2006–08. Data on stroke types, demographic variables, risk factors were available from the Lombardia Stroke Registry. Daily mean value of particulate matter with a diameter <10 µm (PM(10)), carbon monoxide, nitric oxide, nitrogen dioxide, sulphur dioxide, benzene and ozone were used in the analysis. The association between CVD, ischemic strokes subtypes and pollutants was investigated with a case-crossover design, using conditional logistic regression analysis, adjusting for temperature, humidity, barometric pressure and holidays. Among the 781 subjects admitted 75.7% had ischemic stroke, 11.7% haemorrhagic stroke 12.6% transient ischemic attack. In men admission for stroke was associated with PM(10) [odds ratio (OR) 1.01, 95%; confidence interval (CI) 1.00–1.02; P<0.05]. According to the clinical classification, lacunar anterior circulation syndrome stroke type was related to PM(10) level registered on the day of admission for both genders (OR: 1.01, 95%; CI: 1.00–1.02; P<0.05) while for total anterior circulation syndrome stroke only in men (OR: 1.04, 95%; CI 1.01–1.07; P<0.05). In conclusion, our study confirms that air pollution peaks may contribute to increase the risk of hospitalization for stroke and particulate matter seems to be a significant risk factor, especially for lacunar stroke