Laser-Induced Noninvasive Vascular Injury Models in Mice Generate Platelet- and Coagulation-Dependent Thrombi

Abstract

A minimally invasive laser-induced injury model is described to study thrombus development in mice in vivo. The protocol involves focusing the beam of an argon-ion laser through a compound microscope on the vasculature of a mouse ear that is sufficiently thin such that blood flow can be visualized by intravital microscopy. Two distinct injury models have been established. The first involves direct laser illumination with a short, high-intensity pulse. In this case, thrombus formation is inhibited by the GPIIb/IIIa antagonist, G4120. However, the anticoagulants, hirulog, PPACK, and NapC2 have minimal effect. This indicates that thrombus development induced by this model mainly involves platelet interactions. The second model involves low-intensity laser illumination of mice injected with Rose Bengal dye to induce photochemical injury in the region of laser illumination. Thrombi generated by this latter procedure have a slower development and are inhibited by both anticoagulant and anti-platelet compounds.

New insights into the spatiotemporal localization of prothrombinase in vivo

“…[11][12][13] Work using laser-induced injury to the mouse cremasteric arteriole has revealed surprising findings that challenge otherwise accepted ideas about thrombus formation. The findings point to a significant role for other activated cells, in addition to platelets, in supporting fibrin formation.…”

Darkfield orthogonal polarized spectral imaging for studying endovascular laser-tissue interactions in vivo-a preliminary study

“…The acute and chronic inflammatory responses triggered by laser-induced endovascular damage lead to vascular remodeling processes that result in lesional clearance. The exact mechanisms underlying the (endo)vascular responses to laser irradiation have not been extensively elucidated, primarily due to the fact that only few in vivo techniques have been developed to categorically examine these phenomena [14][15][16][17][18]. Moreover, most of these laserinduced vessel wall injury models [14][15][16] focus the laser beam directly on the endothelial layer, creating a damage profile that differs from selective photothermolysis in that the chromophore-containing red blood cells are circumvented as thermal catalysts and heat diffusion is an irrelevant factor.…”

“…The exact mechanisms underlying the (endo)vascular responses to laser irradiation have not been extensively elucidated, primarily due to the fact that only few in vivo techniques have been developed to categorically examine these phenomena [14][15][16][17][18]. Moreover, most of these laserinduced vessel wall injury models [14][15][16] focus the laser beam directly on the endothelial layer, creating a damage profile that differs from selective photothermolysis in that the chromophore-containing red blood cells are circumvented as thermal catalysts and heat diffusion is an irrelevant factor. As a result, the formation of coagula consisting of thermally denatured circulatory cells and plasma proteins [19][20][21] is excluded from the damage profile in these models.…”

Resident Endothelial Cells Surrounding Damaged Arterial Endothelium Reendothelialize the Lesion

“…19 Among various animal models of endothelial damage, 20,21 rose bengal injection with mild laser illumination can selectively injure ECs and is regarded as the model that best resembles thrombosis in the setting of atherosclerosis. [21][22][23] The resultant denuded segment is restricted to the area of laser illumination. 22,23 Other methods of injuring endothelium include laser injury alone, topical application of chemicals (eg, ferric chloride), and mechanical damage, which may lack specificity in injured cell types, may not result in uniform denudation, or may require direct application of the toxic chemicals onto the brain surface.…”

“…[21][22][23] The resultant denuded segment is restricted to the area of laser illumination. 22,23 Other methods of injuring endothelium include laser injury alone, topical application of chemicals (eg, ferric chloride), and mechanical damage, which may lack specificity in injured cell types, may not result in uniform denudation, or may require direct application of the toxic chemicals onto the brain surface. 20,21 To clarify repair processes of endothelial damage and to identify responsible cells for reconstructing endothelium, we damaged ECs with the rose bengal method and evaluated time-lapse images of reendothelialization captured through the cranial window.…”