Correction of distortion of histologic sections of arteries.

Histologic sections of arteries can be used to generate three-dimensional (3D) geometric models and identify structural constituents. However, geometric distortions are introduced by fixation, embedding and sectioning; distortions which can, for example, lead to errors in stresses predicted by finite element models. We developed a method to measure and correct for distortions caused by acrylic processing and applied it to intact, healthy porcine coronary arteries. Micro-computed tomography was used to image arteries in the fresh and embedded states. Tissue blocks were sectioned, stained and imaged using a light microscope. Each section contained four registration marks used to determine strains introduced by sectioning and staining. Using these three image sets, 3D geometric models were generated and distortions were measured. Fixation, processing, and embedding resulted in shrinkage of 6.4+/-2.3% axially and 35.4+/-5.0% in mean cross-sectional area (n=5). Shrinkage in a cross section was well characterized by a uniform, equibiaxial strain. Sectioning and staining resulted in additional compressive strains in the sectioning direction of 0.067+/-0.011 and, in the direction perpendicular to sectioning, of 0.023+/-0.005 (n=5). These strains are assumed uniform and form the basis for correcting section geometry. Reconstructions using corrections for sectioning and shrinkage-related distortions had errors of 1.6+/-0.5% (n=5) and 4.0+/-1.7% (n=5), respectively.

Characterizing intramural stress and inflammation in hypertensive arterial bifurcations.

A histology-based methodology was developed and used to determine whether intramural stress and combined monocyte/macrophage density positively correlate within hypertensive bifurcations. Hypertension was induced in Sprague-Dawley rats using Angiotensin II pumps. Analysis focused on mesenteric bifurcations harvested 7 days (n = 4) post implant, but also included normotensive (n = 2) and 21-day hypertensive (n = 1) samples. Mesentery was processed in a manner that preserves morphology, corrects for histology-related distortions and results in reconstructions suitable for finite element analysis. Peaks in intramural stress and monocyte/macrophage density occurred near bifurcations after the onset of hypertension. Cell density peaks occurred in regions where surface curvature is complex and tends to heighten intramural stress. Also, a strong positive correlation between mean stress and mean cell density suggests that they are related phenomena. A point-by-point comparison of stress and cell density throughout each bifurcation did not exhibit a consistent pattern. We offer reasons why this most stringent test did not corroborate our other findings that high intramural stress is correlated with increased inflammation near the center of the bifurcation.

Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy.

Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) "en face" method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of vascular cell adhesion molecule 1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. This innovative en face method allowed simple multicolor profiling, highly sensitive fluorescence quantitation, and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.