Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve ...modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a "nucleus splitting" procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR
and control
mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to
axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed -26.6 ± 9.90% (
< 0.001) and -1.44 ± 7.05% (
= 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (
= 0.008), this slope became insignificant after splitting (
= 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (
= 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.
Proposed radioactive waste repositories require long residence times within deep geological settings for which we have little knowledge of local or regional subsurface dynamics that could affect the ...transport of hazardous species over the period of radioactive decay. Given the role of microbial processes on element speciation and transport, knowledge and understanding of local microbial ecology within geological formations being considered as host formations can aid predictions for long term safety. In this relatively unexplored environment, sampling opportunities are few and opportunistic. We combined the data collected for geochemistry and microbial abundances from multiple sampling opportunities from within a proposed host formation and performed multivariate mixing and mass balance (M3) modeling, spatial analysis and generalized linear modeling to address whether recharge can explain how subsurface communities assemble within fracture water obtained from multiple saturated fractures accessed by boreholes drilled into the crystalline formation underlying the Chalk River Laboratories site (Deep River, ON, Canada). We found that three possible source waters, each of meteoric origin, explained 97% of the samples, these are: modern recharge, recharge from the period of the Laurentide ice sheet retreat (
. ∼12000 years before present) and a putative saline source assigned as Champlain Sea (also
. 12000 years before present). The distributed microbial abundances and geochemistry provide a conceptual model of two distinct regions within the subsurface associated with bicarbonate - used as a proxy for modern recharge - and manganese; these regions occur at depths relevant to a proposed repository within the formation. At the scale of sampling, the associated spatial autocorrelation means that abundances linked with geochemistry were not unambiguously discerned, although fine scale Moran's eigenvector map (MEM) coefficients were correlated with the abundance data and suggest the action of localized processes possibly associated with the manganese and sulfate content of the fracture water.
•Developed a mechanistic TCP model to predict local tumour control for NSCLC.•The TCP model trained clinical outcomes for 3D-CRT, CHART and SABR.•The model is useful for local tumour control ...prediction in high dose radiotherapy.•The impact of clonogenic cell density variations on tumour control was assessed.•60% of the dose was sufficient to maintain a flat TCP a cross the tumour volume.
A fully heterogeneous population tumour control probability (TCP) model, based on the linear-quadratic (LQ) cell survival concept combined with the Poisson statistic, was established to predict local tumour control after one, two and three years. This TCP model was created using data from 16 publications that reported on early-stage non–small-cell lung cancer (NSCLC) treated using either three-dimensional conformal radiation therapy (3D-CRT), continuous hyperfractionated accelerated radiotherapy (CHART) or stereotactic ablative body radiotherapy (SABR). The TCP model was fitted to the clinical outcome data using optimised radiosensitivity values produced by the Nelder-Mead simplex algorithm. The statistical analysis resulted in R2 values of 0.96, 0.96 and 0.97 and wRMSE values of 3.9%, 5.2% and 5.9% for one-, two- and three-year local tumour control rates, respectively. The TCP models for one, two and three years were internally validated using a bootstrap resampling approach. The mean R2 and 95% CI for the bootstrap samples were 0.98 (0.93–0.99), 0.98 (0.95–0.99) and 0.98 (0.96–0.99) for the one-, two- and three-year local tumour control rates, respectively. Variations in the TCP with clonogenic density were then further investigated by introducing a new mathematical model to vary the clonogenic cell and radiation dose distribution across the treated volume. Based on the above model, it was estimated that 60% of the dose was sufficient to maintain the TCP after two years for the areas with lower clonogenic cell density. If externally validated, this lower-dose treatment plan could have beneficial effects on the surrounding healthy tissue without negatively affecting tumour control.
Summary Objective To determine the regional cell density distribution and basal oxygen consumption rates (based on tissue volume and cell number) of temporomandibular joint (TMJ) discs and further ...examine the impact of oxygen tension on these rates. Design TMJ discs from pigs aged 6–8 months were divided into five regions: anterior, intermediate, posterior, lateral and medial. The cell density was determined using confocal laser scanning microscopy. The change in oxygen tension was recorded while TMJ disc explants were cultured in sealed metabolism chambers. The volume based oxygen consumption rate of explants was determined by theoretical curve-fitting of the recorded oxygen tension data with the Michaelis–Menten equation. The rate on a per-cell basis was calculated based on the cell density measurements and volume based rate measured in another group of discs. Results The overall cell density mean, 95% confidence interval (CI) was 51.3 (21.3–81.3) × 106 cells/mL wet tissue. Along the anteroposterior axis, the anterior band had 25.5% higher cell density than the intermediate zone ( P < 0.02) and 29.1% higher than the posterior band ( P < 0.008). Along the mediolateral axes, the medial region had 26.2% higher cell density than the intermediate zone ( P < 0.04) and 25.4% higher than the lateral region ( P < 0.045). The overall volume and cell based maximum oxygen consumption rates were 1.44 (0.44–2.44) μmol/mL wet tissue/h and 28.7 (12.2–45.2) nmol/106 cells/h, respectively. The central regions (intermediate, lateral, and medial) had significantly higher volume based ( P < 0.02) and cell based ( P < 0.005) oxygen consumption rates than the anterior and posterior bands. At high oxygen tension, the oxygen consumption rate remained constant, but dropped as oxygen tension fell below 5%. Conclusions The TMJ disc had higher cell density and oxygen consumption rates than articular cartilage reported in the literature. These results suggest that a steeper oxygen gradient may exist in the TMJ disc and may be vulnerable to pathological events that impede nutrient supply.
The basal forebrain refers to heterogeneous structures located close to the medial and ventral surfaces of the cerebral hemispheres. It contains diverse populations of neurons, including the ...cholinergic cortically projecting cells that show severe loss in Alzheimer’s and related neurodegenerative diseases. The basal forebrain does not display any cytoarchitectural or other structural features that make it easy to demarcate functional boundaries, a problem that allowed different investigators to propose different organizational schemes. The present paper uses novel three-dimensional reconstructions and numerical analyses for studying the spatial organization of four major basal forebrain cell populations, the cholinergic, parvalbumin, calbindin and calretinin containing neurons in the rat. Our analyses suggest that the distribution of these four cell populations is not random but displays a general pattern of association. Within the cholinergic space (i.e. the volume occupied by the cortically projecting cholinergic cell bodies) the three other cell types form twisted bands along the longitudinal axis of a central dense core of cholinergic cells traversing the traditionally defined basal forebrain regions, (i.e. the medial septum, diagonal bands, the substantia innominata, pallidal regions and the bed nucleus of the stria terminalis). At a smaller scale, the different cell types within the cholinergic space occupy overlapping high-density cell clusters that are either chemically uniform or mixed. However, the cell composition of these high-density clusters is regionally specific.
The proposed scheme of basal forebrain organization, using cell density or density relations as criteria, offers a new perspective on structure–function relationship, unconstrained by traditional region boundaries.
The density distributions of red cells from sickle cell disorders and other hematological diseases were determined. In sickle cell anemia there was an increase in the proportion of cells in both the ...heaviest and lightest fractions. In hemoglobin SC disease, a small fraction was shifted to heavier cells. Sickle cell trait blood had a normal density pattern. In hereditary spherocytosis an increase in the number of heavy cells was observed. Thin-layer polyacrylamide gel electrophoresis of red cell pyruvate kinase demonstrated that the pyruvate electrophoretic pattern was related to density distribution.
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