A labeling strategy for the three-dimensional recognition and analysis of microvascular obstruction in ischemic stroke.

Rationale: Large vessel recanalization in ischemic stroke does not always go along with tissue reperfusion, a phenomenon called "no-reflow". However, knowledge of the mechanism of no-reflow is limited because identifying microvascular obstruction across the cortex and subcortex both in clinical and experimental models is challenging. In this study, we developed a smart three-dimensional recognition pipeline for microvascular obstruction during post-ischemia reperfusion to examine the underlying mechanism of no-reflow. Methods: Transient (60 min) occlusion of the middle cerebral artery (tMCAo) in mice was induced using a filament. Two different fluorophore-conjugated tomato lectins were injected into mice via the tail vein before and after ischemia/reperfusion (I/R), respectively, one to label all blood vessels and the other to label functional blood vessels. Post-I/R microvascular obstruction was visualized using combined iDISCO+-based tissue clearing and optical imaging. Arterioles and capillaries were distinguished using whole-mount immunolabeling with an anti-αSMA antibody. Circulating neutrophils were depleted utilizing an anti-Ly6G antibody. Brain slices were immunostained with the anti-Ly6G antibody to identify co-localized blockage points and neutrophils. MATLAB software was used to quantify the capillary diameters in the ipsilateral brain from the normal and tMCAo mice. Results: Microcirculatory reperfusion deficit worsened over time after I/R. Microvascular obstruction occurred not only in arterioles but also in capillaries, with capillary obstruction associated with local capillary lumen narrowing. In addition, the depletion of circulating neutrophils mitigated reperfusion deficit to a large extent after I/R. The co-localization of blockage points and neutrophils revealed that some neutrophils plugged capillaries with coexisting capillary lumen narrowing and that no neutrophil was trapped in heaps of blockage points. Quantification of the capillary diameter showed that capillary lumen shrunk after I/R but returned to typical measurements when intravascular neutrophils were depleted. Conclusions: According to our findings, both vascular lumen narrowing and neutrophil trapping in cerebral microcirculation are the key causes of microvascular obstruction after I/R. Also, the primary contribution by neutrophils to microvascular obstruction does not occur through microemboli plugging but rather via the exacerbation of capillary lumen narrowing. Our proposed method will help monitor microcirculatory reperfusion deficit, explore the mechanism of no-reflow, and evaluate the curative effect of drugs targeting no-reflow.

Current research and future prospects of IVOCT imaging-based detection of the vascular lumen and vulnerable plaque.

Intravascular optical coherence tomography (IVOCT) is an imaging method that has developed rapidly in recent years and is useful in coronary atherosclerosis diagnosis. It is widely used in the assessment of vulnerable plaque. This review summarizes the main research methods used in recent years for blood vessel lumen boundary detection and segmentation and vulnerable plaque segmentation and classification. This article aims to comprehensively and systematically introduce the research progress on internal tissues of blood vessels based on IVOCT images. The characteristics and advantages of various methods have been summarized to provide theoretical ideas and methods for the reference of relevant researchers and scholars.

Arteriovenous Anastomosis in Human Hand Digital Skin.

While a digital arteriovenous anastomosis (Hoyer-Grosser's organ, Masson's glomus) is a well-known structure, photographic evidence of communication between arterial and venous lumens might not be demonstrated in routine histological or immunohistochemical analysis. Abundant clusters of so-called glomera were found in semi-serial sections of the distal aspect of 14 fingers obtained from 7 donated elderly cadavers. Two to six round or oval clusters were observed in each longitudinal section (over 0.3-0.6 mm in maximum diameter) in subcutaneous tissue 0.5-1.5 mm below the basal layer of the skin, whereas none were often observed in transverse sections. Lumen-to-lumen communication between arteriole and venule at 8 sites in 2 cadavers was identified in these clusters of glomera. The opening in the arteriole was large (50 µm in diameter) at 3 sites in specimens from an 80-year-old man, whereas it was small (10-30 µm) at 5 sites in those from a 91-year-old man. The arterial aspect was tightly surrounded by abundant nerve fibers expressing tyrosine hydroxylase immunoreactivity, whereas the venous part was not. No or little expression of S100 protein immunoreactivity suggested that these nerve fibers were unmyelinated. The morphology at the lumen-to-lumen communication was simple - possibly an end-to-end anastomosis - rather than a sinuous curve of arteriole opening on to a short funnel-shaped venule as seen in the standard textbooks.

Collagen Microfibers Induce Blood Capillary Orientation and Open Vascular Lumen.

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large-sized tissues. Herein, a method based on high water-dispersible collagen microfibers (CMF) to fabricate capillary orientation-controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers-long CMF (CMF-20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers-long CMF (CMF-200). One of the advantages is the prevention of shrinkage on the z-axis of hydrogel-based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF-free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin-induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering.

The invalidity of the Laplace law for biological vessels and of estimating elastic modulus from total stress vs. strain: a new practical method.

There are strong medical motivations to measure changes in material properties of tubular organs, in vivo and in vitro. The current approach estimates hoop stress from intraluminal pressure using the Laplace law and identifies 'elastic modulus' as the slope of a curve fitted hoop stress plotted against strain data. We show that this procedure is fundamentally flawed because muscle and other soft tissue are closely incompressible, so that the total stress includes a volume-preserving material-dependent hydrostatic response that invalidates the method. Furthermore, we show that the Laplace law incorrectly estimates total stress in biological vessels. However, the great need to estimate elastic modulus leads us to develop an alternative practical method, based on shear stress-strain, i.e. insensitive to nonelastic response from incompressibility, but that uses the same measurement data as the current (incorrect) method. The individual material parameters in the underlying (unknown) constitutive relation combine into an effective shear modulus that is a true measure of elastic response, unaffected by incompressibility and without reference to the Laplace law. Furthermore, our effective shear modulus is determined directly as a function of deformation, rather than as the slope of a fitted curve. We validate our method by comparing effective shear moduli against exact shear moduli for four theoretical materials with different degrees of nonlinearity and numbers of material parameters. To further demonstrate applicability, we reanalyse an in vivo study with our new method and show that it resolves an inconsistent change in modulus with the current method.

Generation of Multi-Scale Vascular Network System within 3D Hydrogel using 3D Bio-Printing Technology.

Although 3D bio-printing technology has great potential in creating complex tissues with multiple cell types and matrices, maintaining the viability of thick tissue construct for tissue growth and maturation after the printing is challenging due to lack of vascular perfusion. Perfused capillary network can be a solution for this issue; however, construction of a complete capillary network at single cell level using the existing technology is nearly impossible due to limitations in time and spatial resolution of the dispensing technology. To address the vascularization issue, we developed a 3D printing method to construct larger (lumen size of ~1mm) fluidic vascular channels and to create adjacent capillary network through a natural maturation process, thus providing a feasible solution to connect the capillary network to the large perfused vascular channels. In our model, microvascular bed was formed in between two large fluidic vessels, and then connected to the vessels by angiogenic sprouting from the large channel edge. Our bio-printing technology has a great potential in engineering vascularized thick tissues and vascular niches, as the vascular channels are simultaneously created while cells and matrices are printed around the channels in desired 3D patterns.

Blood and lymphatic vascular tube formation in mouse.

The blood and lymphatic vasculatures are essential for nutrient delivery, gas exchange and fluid homeostasis in all tissues of higher vertebrates. They are composed of a hierarchical network of vessels, which are lined by vascular or lymphatic endothelial cells. For blood vascular lumen formation to occur, endothelial cell cords polarize creating apposing apical cell surfaces, which repulse each other and give rise to a small intercellular lumen. Following cell shape changes, the vascular lumen expands. Various junctional proteins, polarity complexes, extracellular matrix binding and actin remodelling molecules are required for blood vascular lumen formation. In contrast, little is known regarding the molecular mechanisms leading to lymphatic vascular tube formation. Current models agree that lymphatic vessels share a blood vessel origin, but they differ in identifying the mechanism by which a lymphatic lumen is formed. A ballooning mechanism was proposed, in which lymph sacs are connected via their lumen to the cardinal veins. Alternatively, a mechanism involving budding of streams of lymphatic endothelial cells from either the cardinal veins or both the cardinal veins and the intersomitic vessels, and subsequent assembly and lumenisation was recently described. Here, we discuss what is currently known about the molecular and cellular machinery that guides blood and lymphatic vascular tube formation in mouse.

Essential role of SH3-domain GRB2-like 3 for vascular lumen maintenance in zebrafish.

OBJECTIVE: Studying the underlying molecular mechanisms for maintaining stereotyped vascular lumen diameters should help toward a comprehensive understanding of vascular homeostasis and function. We aimed to determine the role of SH3-domain GRB2-like 3 (Sh3gl3) and its interacting pathways in dorsal aorta (DA) maintenance in zebrafish. APPROACH AND RESULTS: Sh3gl3 and its binding partner, Cbl-interacting protein of 85K (Cin85), together regulate endocytosis and were expressed in the developing vasculature. Morpholino knockdown of either gene resulted in shrinkage of the DA lumen, although artery/vein specification and the initial formation of vascular lumens were unaffected. In addition, sh3gl3 and cin85 morpholinos exerted a synergistic effect in causing the vascular phenotypes. To identify the signaling pathways in which Sh3gl3/Cin85 may participate, we screened several candidate inhibitors for their ability to induce similar circulatory defects. Chemical inhibition of the epidermal growth factor receptor and the phosphatidylinositol 3-kinase/Akt cascade led to a loss of circulation and shrunken DA in zebrafish embryos. Furthermore, inhibition of the epidermal growth factor receptor/phosphatidylinositol 3-kinase pathway showed a functional cooperation with Sh3gl3 deficiency in impairing DA lumens. CONCLUSIONS: These results identify 2 new factors, Sh3gl3 and Cin85, which are essential for DA lumen maintenance, and suggest that endocytosis, possibly involving epidermal growth factor receptor and phosphatidylinositol 3-kinase, is implicated in Sh3gl3/Cin85 function.