Penetration of Intravitreal Injected Tissue Plasminogen Activator to the Retina - Rats Model Study

Abstract

 

Purpose

 

Tissue plasminogen activator (tPA) is a thrombolytic agent which has the ability to degrade and dissolve fibrin clot. Although the efficacy of intravitreal tPA injections has been shown in clinical practice, the ability of intravitreal Injected tPA to diffuse from the vitreous through the retina and into the subretinal space has been questioned in an experimental models, as tPA conjugated to fluorescein, failed to penetrate the retina. We investigate whether an unconjugated tPA injected into the vitreous could penetrate the neural retina and enter the subretinal space, in a rat model.

 

Methods

 

24 rats eyes were used in the study.14 right eyes were injected with intravitreal tPA (0.75 μg/3 μl), 10 right eyes were injected with intravitreal saline, and served as controls. 3, 24, and 48 hours after tPA injection, animals were euthanized and eyes were taken for cryosections and immunohfluorescence staining. Goat anti tPA, followed by alexafluor 568 donkey anti goat (invitrogene) were used for tPA detection.

 

Results

 

TPA staining was detected in deep retinal layers in all eyes injected with intravitreal tPA. A deeper and more intense staining of tPA was seen after 3 and 24 hours, compared to a decreased staining 48 hours from injection. No staining of tPA was detected in the retina in the eyes injected with saline.

 

Conclusions

 

We demonstrated that an unconjugated tPA at a dose of (0.75 μg / 3 μl) injected into the vitreous penetrates the retina of rats. We speculate that former rabbit model studies that failed to show penetration of tPA to the retina may be explained by the use of conjugated tPA that doesn’t penetrate the retina.  

 

Lack of immunohfluorescence staining in the retina of the eye 3 hours after injected with intravitreal Saline. TPA staining seen here is of vitreous alone. Blue indicate staining of 300 nM 4,6-diamidino-2-phenylidole (Sigma).

 

 

Goat anti tPA, followed by alexafluor 568 donkey anti goat (invitrogene) were used for tPA detection. TPA staining was detected in deep retinal layers in all eyes injected with 0.75 μg/3 μl intravitreal tPA. Here 24 hours after injection.