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 intra-vital 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 and fibrin when the data are transformed as area under the curve (Spearman r = 0.80 and 0.76 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 of platelet accumulation and fibrin formation improving data interpretation and standardization.

Keywords: Animal models; Blood Platelets; Fibrin; Thrombosis.

Figure 1:. Normalizing to injury size reduces platelet and fibrin coefficients of variation.

(A-C) Brightfield images of the cremaster arteriole immediately following laser induced injury were used to identify the region of vascular disruption (white dotted outline). The size of the region of vascular injury in contact with the vessel lumen was then determined (red line) allowing for quantitative measures of injury size to be made. An apparent correlation between the size of vessel injury observed in (D-F) brightfield images and the resultant accumulation of (H-I) platelets and fibrin was detected. (J) Injury size when plotted against the platelet area under the curve (AUC) measurements revealed a strong positive correlation (Spearman rank, P<0.0001). (K) Similarly, when injury size was plotted against the fibrin AUC measurements, a strong positive correlation was also observed (Spearman rank, P<0.0001). Strong positive correlations were also observed between injury size and (L) peak platelet accumulation and (M) peak fibrin accumulation.

Figure 2:. Evaluation of platelet accumulation, fibrin formation, and ablation injury size in genetically identical mice.

(A) Platelet fluorescence (AUC), (B) fibrin fluorescence (AUC), and (C) ablation injury size measurements are shown for the 5 different mice that were used for the evaluation of 59 thrombi presented in Figure 1. Fluorescence values and ablation injury size is plotted for each of the 5 mice to demonstrate the relationship between (D) platelet AUC versus injury size and (E) fibrin AUC and injury size. (F) Platelet fluorescence (corrected AUC) and (G) fibrin fluorescence (corrected AUC) are shown following normalization for injury size. Data represented as individual values with the median.