A technical protocol for an experimental ex vivo model using arterially perfused porcine eyes.

Ex vivo ocular perfused models have been described in the past and were applied in different mammalian species as platforms to test drug delivery systems and surgical techniques. However, reproduction of those methods is challenging because extensive and precise description of the protocols used is lacking. In this technical paper we provide a detailed description of all the steps to be followed from the enucleation of porcine eyes to cannulation of the ophthalmic artery and perfusion. This model can contribute to the reduction of use of living animals in ophthalmology research, whereas as opposed to in vitro models, it preserves tissue complexity and integrity.

Method to measure in vivo blood fibrinolytic activity with a (125)I-fibrin coated aorta loop validated with agents which affect blood fibrinolytic activity.

A functional animal model to measure in vivo the blood fibrinolytic activity and pharmacological-induced changes thereof are described. A (125)I-fibrin coated plastic loop is inserted in the rat aorta; the rate of label disappearance (sigmoid curve) is directly registered outside the animal with a gamma scintillation probe. The time needed to let disappear 50% of the removable-labeled fibrin is used as measure for the blood fibrinolytic activity. The direct advantage of this model is the absence of a blood or plasma clot: a thin labeled fibrin layer attached to the inner wall of the loop is in direct contact with the blood and is therefore sensitive to increased or decreased blood fibrinolytic activity. The total experiment needs about 60 min. Experiments with nontreated rats showed that, after an initial lag phase of about 10 min, the labeled fibrin started to disappear from the loop. A sigmoid pattern was obtained showing that about 20-30% of the coated-labeled fibrin is resistant to removal. Registration of the total curve of a nontreated (control or placebo) rat required about 30-40 min. The clinically used thrombolytics (intravenously administered) urokinase and t-PA showed a dose-dependent fibrinolytic activity resulting in increased removal of the bound (125)I-fibrin. Streptokinase was not active, which is in agreement with literature. Tranexamic acid, dexamethasone and endotoxin (inhibitors of fibrinolysis) showed dose-dependent inhibition of removal of the coated fibrin. Retinoic acid was tested as compound, which may enhance the blood fibrinolytic activity; retinoic acid was not found to be significantly active in this model. The disappearance of labeled fibrin is not sensitive to inhibitors of coagulation or platelet aggregation. This technically simple and fast model can thus be used to measure in vivo quantitatively the effects of pharmacological active compounds, which increase or decrease the blood fibrinolytic activity.