Retinal safety of intravitreal rtPA in healthy rats and under excitotoxic conditions

Abstract

Purpose: Intravitreal recombinant tissue plasminogen activator (rtPA) is used off-label for the surgical management of submacular hemorrhage, a severe complication of neovascular age-related macular degeneration. rtPA is approved for coronary and cerebral thrombolysis. However, in ischemic stroke rtPA is known to increase excitotoxic neural cell death by interacting with the N-methyl-D-aspartate (NMDA) receptor. We therefore investigated the retinal toxicity of rtPA in healthy rats and in a model of NMDA-induced retinal excitotoxicity.

Methods: First, rtPA at three different doses (2.16 µg/5 µl, 0.54 µg/5 µl, and 0.27 µg/5 µl) or vehicle (NaCl 0.9%) was injected intravitreally in healthy rat eyes. Electroretinograms (ERGs) were performed at 24 h or 7 days. Annexin V-fluorescein isothiocyanate (FITC)-labeled apoptotic retinal ganglion cells (RGCs) were counted on flatmounted retinas at 24 h or 7 days. Next, NMDA + vehicle or NMDA + rtPA (0.27 µg/5 µl) was injected intravitreally to generate excitotoxic conditions. Apoptotic annexin V-FITC-labeled RGCs and surviving Brn3a-labeled RGCs were quantified on flatmounted retinas and radial sections, 18 h after treatment.

Results: In healthy rat eyes, the number of apoptotic RGCs was statistically significantly increased 24 h after the administration of rtPA at the highest dose (2.16 µg/5 µl; p = 0.0250) but not at the lower doses of 0.54 and 0.27 µg/5 µl (p = 0.36 and p = 0.20), compared to vehicle. At day 7, there was no difference in the apoptotic RGC count between the rtPA- and vehicle-injected eyes (p = 0.70, p = 0.52, p = 0.11). ERG amplitudes and implicit times were not modified at 24 h or 7 days after injection of any tested rtPA doses, compared to the baseline. Intravitreal administration of NMDA induced RGC death, but under these excitotoxic conditions, coadministration of rtPA did not increase the number of dead RGCs (p = 0.70). Similarly, the number of surviving RGCs on the flatmounted retinas and retinal sections did not differ between the eyes injected with NMDA + vehicle and NMDA + rtPA (p = 0.59 and p = 0.67).

Conclusions: At low clinical equivalent doses corresponding to 25 µg/0.1 ml in humans, intravitreal rtPA is not toxic for healthy rat retinas and does not enhance NMDA-induced excitotoxicity. Vitreal equivalent doses ≥200 µg/0.1 ml should be avoided in patients, due to potential RGC toxicity.

Figure 1

Number of apoptotic retinal ganglion cells in flatmounted healthy retinas 24 h after intravitreal administration of rtPA (2.16, 0.54, and 0.27 µg in 5 µl) or vehicle (NaCl 0.9%, 5 µl). There was a statistically significantly higher number of apoptotic cells in the eyes that received 2.16 µg/5 µl compared to vehicle. * p<0.05.

Figure 2

Flatmounted retinas from rats euthanized 24 h after intravitreal administration of rtPA (2.16 µg/5 µl) or vehicle (NaCl 0.9%, 5 µl). Apoptotic retinal ganglion cells labeled with intravitreal annexin V-fluorescein isothiocyanate (FITC) (A–C) or annexin V-FITC and 4',6-diamidino-2-phenylindole (DAPI) (B–D) before the animals were euthanized. A, B: Control eye (NaCl 0.9%, 5 µl). C, D: Eye treated with intravitreal recombinant tissue plasminogen activator (rtPA; 2.16 µg/5 µl) displaying a higher number of annexin V-FITC labeling, colocalizing with retinal ganglion cell nuclei. Green = Annexin-V-FITC staining; Blue = DAPI staining. Bar = 50 µm.

Figure 3

Mixed electroretinogram showing rod- and cone-driven responses 24 h after intravitreal injection of saline solution or three different doses of rtPA (2.16, 0.54, and 0.27 µg in 5 µl) in healthy rats. A: Variation in the a-wave amplitudes (µV) from the preinjection recordings. B: Variation in the b-wave amplitude (µV) from the preinjection recordings. No statistically significant difference was observed between the recombinant tissue plasminogen activator (rtPA)- and vehicle-injected eyes.

Figure 4

Assessment of retinal toxicity 18 h after intravitreal administration of rtPA in rat eyes under excitotoxic conditions. Animals received either N-methyl-D-aspartate (NMDA; 45 nmol/6 µl) + saline solution or NMDA (45 nmol/6 µl) + recombinant tissue plasminogen activator (rtPA; 0.27 µg/5 µl). A: Number of annexin V-positive retinal ganglion cells quantified on flatmounted retinas, showing no difference between the eyes that received NMDA + saline and the eyes that received NMDA + rtPA. B: Number of Brn3a-positive living retinal ganglion cells, quantified on flatmounted retinas, showing no difference between the eyes that received NMDA + saline and the eyes that received NMDA + rtPA. C: Number of living retinal ganglion cells, quantified on the toluidine blue–stained histologic radial retinal sections, as the number of cell nuclei visualized at the level of the retinal ganglion cell layer, showing no difference between the eyes that received NMDA + saline and the eyes that received NMDA + rtPA. D: Toluidine blue–stained retinal radial section illustrating the similar density of retinal ganglion cells in the eyes that received NMDA + saline (left) and the eyes that received NMDA + rtPA (right). GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer. Bar = 20 µm.