title: Retinal safety of intravitreal rtPA in healthy rats and under excitotoxic conditions.
abstract: 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.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
authors.affiliation: Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland; Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Daruich
authors.affiliation: Inserm U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, Université de Caen Basse-Normandie, GIP Cyceron, Caen, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Parcq
authors.affiliation: Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Kimberley
authors.initials: K
authors.lastname: Delaunay
authors.affiliation: Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Naud
authors.affiliation: Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Le Rouzic
authors.affiliation: Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Picard
authors.affiliation: Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Crisanti
authors.affiliation: Inserm U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, Université de Caen Basse-Normandie, GIP Cyceron, Caen, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Vivien
authors.affiliation: Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Marianne
authors.initials: M
authors.lastname: Berdugo
authors.affiliation: Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland; Inserm, U1138, Team 17, Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.
authors.forename: Francine
authors.initials: F
authors.lastname: Behar-Cohen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D004596:Electroretinography; D058449:Intravitreal Injections; D008297:Male; D009498:Neurotoxins / Q000633:toxicity*; D020318:Rats, Long-Evans; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000009:adverse effects* / Q000494:pharmacology*; D012160:Retina; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000008:administration & dosage / Q000009:adverse effects* / Q000494:pharmacology*
chemical_list: D009498:Neurotoxins; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator