Adaptation of the HistoEnder, an open-source 3D printer for automated transmission electron microscopy grid staining.

The HistoEnder, an inexpensive open-source 3D printer published as an automated histological slide stainer, has been adapted for conventional biological transmission electron microscopy (TEM) batch grid staining. Details are presented of the 3D printed apparatus, assembly, G-code programming, and operation on the 3D printer to post-section stains up to 20 grids through aqueous uranyl acetate, distilled water rinses, and lead stains. TEM Results are identical to manual staining with the advantages of automation using the low cost HistoEnder, apparatus, and equipment.

Astrocytes in the Optic Nerve Are Heterogeneous in Their Reactivity to Glaucomatous Injury.

The optic nerve head is thought to be the site of initial injury to retinal ganglion cell injury in glaucoma. In the initial segment of the optic nerve directly behind the globe, the ganglion cell axons are unmyelinated and come into direct contact to astrocytes, suggesting that astrocytes may play a role in the pathology of glaucoma. As in other parts of the CNS, optic nerve head astrocytes respond to injury by characteristic changes in cell morphology and gene expression profile.

Exosome swarms eliminate airway pathogens and provide passive epithelial immunoprotection through nitric oxide.

Background: Nasal mucosa-derived exosomes (NMDEs) harbor immunodefensive proteins and are capable of rapid interepithelial protein transfer.

Objectives: We sought to determine whether mucosal exposure to inhaled pathogens stimulates a defensive swarm of microbiocidal exosomes, which also donate their antimicrobial cargo to adjacent epithelial cells.

Methods: We performed an institutional review board-approved study of healthy NMDE secretion after Toll-like receptor (TLR) 4 stimulation by LPS (12.

Ultrastructural Morphology of the Optic Nerve Head in Aged and Glaucomatous Mice.

Purpose: To study age- and intraocular pressure-induced changes in the glial lamina of the murine optic nerve on the ultrastructural level.

Methods: Naïve C57bl/6 mice at various ages spanning the time between early adulthood (3 months) and senescence (30 months) were used in this study. In addition, the intraocular pressure (IOP) was increased in a group of young mice by injection of microbeads into the anterior chamber.

Exosomes mediate interepithelial transfer of functional P-glycoprotein in chronic rhinosinusitis with nasal polyps.

Objective: P-glycoprotein (P-gp) drives type-2 helper T-cell inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) through unknown posttranslational mechanisms of overexpression. A recent randomized clinical trial demonstrated that inhibition of P-gp was as effective as oral steroids and biologics in treating CRSwNP. Exosomes are 30- to 150-nm vesicles capable of intercellular membrane protein transfer.

Astrocytes in the Optic Nerve Head of Glaucomatous Mice Display a Characteristic Reactive Phenotype.

Purpose: Optic nerve head astrocytes, a subtype of white-matter astrocytes, become reactive early in the course of glaucoma. It was shown recently that in the DBA/2J mouse model of inherited glaucoma optic nerve astrocytes extend new longitudinal processes into the axon bundles before ganglion cell loss becomes apparent. The present study aims at testing whether this behavior of astrocytes is typical of early glaucomatous damage.

Modified Hiraoka TEM grid staining apparatus and technique using 3D printed materials and gadolinium triacetate tetrahydrate, a non-radioactive uranyl acetate substitute.

The Hiraoka transmission electron microscopy (TEM) grid staining apparatus, which allowed multiple grids to be batch stained together on a plate, is no longer commercially available. Because the need for such a device still exists, we developed a modified Hiraoka TEM grid staining apparatus by combining a Leica AC20 grid plate holder with a modified 3D printed grid loading holder and trays lined with Parafilm. This apparatus was then used to test a post-section contrasting method that uses gadolinium triacetate tetrahydrate solution, a non-radioactive uranyl acetate substitute, which produces similar staining results to uranyl acetate when used with lead stain in mouse ocular tissue for TEM.

Heparanase alters arterial structure, mechanics, and repair following endovascular stenting in mice.

Heparan sulfate proteoglycans (HSPGs) are potent regulators of vascular remodeling and repair. Heparanase is the major enzyme capable of degrading heparan sulfate in mammalian cells. Here we examined the role of heparanase in controlling arterial structure, mechanics, and remodeling.

Neointimal formation is reduced after arterial injury in human crp transgenic mice.

Objectives/methods: Elevated CRP levels predict increased incidence of cardiovascular events and poor outcomes following interventions. There is the suggestion that CRP is also a mediator of vascular injury. Transgenic mice carrying the human CRP gene (CRPtg) are predisposed to arterial thrombosis post-injury.

Matrix embedding alters the immune response against endothelial cells in vitro and in vivo.

Background: Endothelial cell (EC) dysfunction represents the first manifestation of atherosclerotic disease. Restoration of endothelium via seeding or transfection is hampered by local alterations in flow, inflammation, and metabolic activation. Perivascular EC matrix implants are shielded from these forces and still control vascular repair.

Vascular neointimal formation and signaling pathway activation in response to stent injury in insulin-resistant and diabetic animals.

Diabetes and insulin resistance are associated with increased disease risk and poor outcomes from cardiovascular interventions. Even drug-eluting stents exhibit reduced efficacy in patients with diabetes. We now report the first study of vascular response to stent injury in insulin-resistant and diabetic animal models.

Morphometric analysis of particulate debris extracted by four different embolic protection devices from coronary arteries, aortocoronary saphenous vein conduits, and carotid arteries.

Different embolic protection devices have been introduced for endovascular interventions: filters or balloon occlusion and aspiration systems. Despite widening use in a variety of vascular beds and clinical syndromes, little is known about the particulate burden liberated from different vascular beds and caught by different protection devices. We performed histologic and morphometric analyses of particulate debris captured during stenting of degenerated saphenous vein bypass grafts and native coronary arteries during acute myocardial infarction or during elective intervention and carotid arteries to assess the relative performance of different protection devices.

Strut position, blood flow, and drug deposition: implications for single and overlapping drug-eluting stents.

Background: The intricacies of stent design, local pharmacology, tissue biology, and rheology preclude an intuitive understanding of drug distribution and deposition from drug-eluting stents (DES).

Methods And Results: A coupled computational fluid dynamics and mass transfer model was applied to predict drug deposition for single and overlapping DES. Drug deposition appeared not only beneath regions of arterial contact with the strut but surprisingly also beneath standing drug pools created by strut disruption of flow.

Dynamic flow alterations dictate leukocyte adhesion and response to endovascular interventions.

Although arterial bifurcations are frequent sites for obstructive atherosclerotic lesions, the optimal approach to these lesions remains unresolved. Benchtop models of arterial bifurcations were analyzed for flow disturbances known to correlate with vascular disease. These models possess an adaptable geometry capable of simulating the course of arterial disease and the effects of arterial interventions.

Embolic protection with filtering or occlusion balloons during saphenous vein graft stenting retrieves identical volumes and sizes of particulate debris.

Background: Distal embolization of plaque particulate liberated during stenting may cause periprocedural complications. The number, size, and volume of debris released during stenting, however, have not been quantified, rendering embolic protection approaches empiric. We used a novel method of microparticle size assessment to measure volume and characterize individual sizes of particles captured by the PercuSurge GuardWire balloon or a vascular filter during saphenous vein graft stenting.

Liposomal alendronate inhibits systemic innate immunity and reduces in-stent neointimal hyperplasia in rabbits.

Background: Innate immunity is of major importance in vascular repair. The present study evaluated whether systemic and transient depletion of monocytes and macrophages with liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation.

Methods And Results: Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment.

Increased thrombosis after arterial injury in human C-reactive protein-transgenic mice.

Background: C-reactive protein (CRP), an acute-phase reactant long considered merely an innocent bystander in the inflammatory process, is now recognized as a powerful predictor of cardiovascular events. Emerging in vitro evidence suggests that CRP may have direct proinflammatory and prothrombotic effects on monocytes and endothelial cells. To determine whether CRP directly modulates vascular cell function in vivo, we subjected wild-type mice, which do not express CRP, and human CRP-transgenic (CRPtg) mice to 2 models of arterial injury.

Leukocyte recruitment and expression of chemokines following different forms of vascular injury.

Unlabelled: Inflammation plays a central role in restenosis following coronary intervention. Recent human and animal data suggest important differences between the inflammatory responses to simple balloon angioplasty compared with stent implantation. To investigate the mechanisms of these differences, New Zealand white rabbits underwent bilateral iliac artery balloon denudation.

Perivascular endothelial implants inhibit intimal hyperplasia in a model of arteriovenous fistulae: a safety and efficacy study in the pig.

Vascular access complications are a major problem in hemodialysis patients. Native arteriovenous fistulae, historically the preferred mode of access, have a patency rate of only 60% at 1 year. The most common mode of failure is due to progressive stenosis at the anastomotic site.

Systemic inflammation induced by lipopolysaccharide increases neointimal formation after balloon and stent injury in rabbits.

Background: Emerging data indicate that the inflammatory response after mechanical arterial injury correlates with the severity of neointimal hyperplasia in animal models and postangioplasty restenosis in humans. The present study was designed to examine whether a nonspecific stimulation of the innate immune system, induced in close temporal proximity to the vascular injury, would modulate the results of the procedure. Methods and Results- Rabbits subjected to iliac artery balloon injury (balloon denudation with or without stent deployment) were injected twice with a bacterial lipopolysaccharide (LPS) (500 ng/rabbit) before and after surgery.