Injury measurements improve interpretation of thrombus formation data in the cremaster arteriole laser‐induced injury model of thrombosis

Abstract

Background: The cremaster arteriole laser-induced injury model is a powerful technique with which to investigate the molecular mechanisms that drive thrombus formation. This model is capable of direct visualization and quantification of accumulation of thrombus constituents, including both platelets and fibrin. However, a large degree of variability in platelet accumulation and fibrin formation is observed between thrombi. Strategies to understand this variability will enhance performance and standardization of the model. We determined whether ablation injury size contributes to variation in platelet accumulation and fibrin formation and, if so, whether incorporating ablation injury size into measurements reduces variation. Methods: Thrombus formation was initiated by laser-induced injury of cremaster arterioles of mice (n = 59 injuries). Ablation injuries within the vessel wall were consistently identified and quantified by measuring the length of vessel wall injury observed immediately following laser-induced disruption. Platelet accumulation and fibrin formation as detected by fluorescently labeled antibodies were captured by digital intravital microscopy. Results: Laser-induced disruption of the vessel wall resulted in ablation injuries of variable length (18-95 µm) enabling interrogation of the relationship between injury severity and thrombus dynamics. Strong positive correlations were observed between vessel injury length and both platelet accumulation and fibrin formation when the data are transformed as area under the curve (Spearman r = .80 and .76, respectively). Normalization of area under the curve measurements by injury length reduced intraclass coefficients of variation among thrombi and improved hypothesis testing when comparing different data sets. Conclusions: Measurement of vessel wall injury length provides a reliable and robust marker of injury severity. Injury length can effectively normalize measurements | 3079 GROVER Et al.

A coagulation defect arising from heterozygous premature termination of tissue factor

“…VSMCs were anti-fibrin-specific antibodies, respectively. Because our prior experiments demonstrated that haploinsufficiency of F3 caused prolonged bleeding following major injury, our analysis accounts for injury size (Figure 6C) (35). The kinetics of thrombus formation diverge when the large injuries for each genotype are compared.…”

Role of Neutrophils and NETs in Animal Models of Thrombosis

“…Classically, one to two pulses are necessary to form a thrombus. The advantages of this thrombosis model are numerous: (1) the ability to monitor thrombus formation in time and space, (2) the ability to generate multiple thrombi in the same animal, (3) the ability to follow the kinetics of thrombus formation in vivo, and (4) the ability to quantify several thrombosis parameters simultaneously [72]. The use of a calcium mobilization reporter compound, Fluo-4-AM, demonstrates that laser injury induces rapid activation of targeted endothelial cells [73].…”

Control of data variations in intravital microscopy thrombosis models

“…However, a well‐experienced experimenter, the same system setup, and multiple thrombi acquired within the same animal can significantly decrease the existing variations discussed here. As shown in the Grover et al study, calculating the AUCs/length in data analysis for the laser‐injury model can further improve the replicability of this widely used technique 22 …”

“…Increasing the n numbers of thrombi, stability of the equipment, and the researcher's experience are the major strategies to control data variability. In this issue of the Journal of Thrombosis and Haemostasis , Grover et al described that laser‐induced disruption of vessel wall resulted in ablation injuries of variable length enabling interrogation of the relationship between injury severity and thrombus dynamics 22 . They found a strong positive correlation between vessel injury length and both platelet and fibrin accumulation at the injury sites when the data are transformed as area under the curve (AUC).…”

“…The tissue may move slightly in three dimensions during laser deliveries, so the ablation site may change both in area and the depth. Using vessel injury length to normalize measurements of platelet accumulation and fibrin formation can certainly reduce the variability caused by differences in power level delivered at the desired ablation sites 22 . Although the calculation will be more complicated, whether the inclusion of the vessel injury area (πr 2 , r = 1/2 × the length) or injury volume (4/3 πr 3 ) in the data normalization can further improve data interpretation may be worthwhile to be considered. The shear stress of blood flow in a vessel has a profound effect on thrombus formation.…”