Timothy L Jackson1, John Marshall. 1. Rayne Institute, Academic Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom. june.spacey@kcl.ac.uk
Abstract
PURPOSE: To evaluate the role of fluorescent antibodies as a means of enhancing the detection of retinal breaks during vitrectomy for rhegmatogenous retinal detachment. METHODS: In ex vivo studies, unfixed, porcine retinal flatmounts were incubated with Cy3 anti-GFAP. Experiments were repeated in the presence of excess soluble GFAP and after surface excimer laser ablation through the internal limiting membrane, into the Müller cell foot processes. Tissue was also incubated with trypan blue, and cross-species immunoreactivity was determined in bovine, rabbit, and human retina. In vivo studies were conducted in a porcine model of rhegmatogenous retinal detachment. Cy3 anti-GFAP was injected into the vitreous cavity of eyes with retinal breaks and then rinsed from the eye. Barrier filters were fitted to the operating microscope to allow intraoperative visualization of tissue stained with Cy3. Excitation endoillumination was provided by a 532-nm diode-pumped laser. RESULTS: In ex vivo studies, retinal flatmounts exposed to Cy3 anti-GFAP showed minimal surface fluorescence, but exposed glial elements at the cut edge of the flatmount stained brightly, as did those exposed by excimer ablation of the Müller cell membrane. Blocking studies confirmed that binding was antigen specific. Trypan blue colocalized to the cut edge of retinal flatmounts. All species showed high levels of immunoreactivity except rabbit. In vivo studies demonstrated selective intraoperative staining of retinal breaks with a high level of specificity. CONCLUSIONS: Intraoperative vital staining of retinal breaks is possible in an animal model of retinal detachment. Ex vivo studies indicate that this occurs because the Cy3 anti-GFAP selectively binds the intermediate filaments of glial cells with damaged or destroyed cell membranes.
PURPOSE: To evaluate the role of fluorescent antibodies as a means of enhancing the detection of retinal breaks during vitrectomy for rhegmatogenous retinal detachment. METHODS: In ex vivo studies, unfixed, porcine retinal flatmounts were incubated with Cy3 anti-GFAP. Experiments were repeated in the presence of excess soluble GFAP and after surface excimer laser ablation through the internal limiting membrane, into the Müller cell foot processes. Tissue was also incubated with trypan blue, and cross-species immunoreactivity was determined in bovine, rabbit, and human retina. In vivo studies were conducted in a porcine model of rhegmatogenous retinal detachment. Cy3 anti-GFAP was injected into the vitreous cavity of eyes with retinal breaks and then rinsed from the eye. Barrier filters were fitted to the operating microscope to allow intraoperative visualization of tissue stained with Cy3. Excitation endoillumination was provided by a 532-nm diode-pumped laser. RESULTS: In ex vivo studies, retinal flatmounts exposed to Cy3 anti-GFAP showed minimal surface fluorescence, but exposed glial elements at the cut edge of the flatmount stained brightly, as did those exposed by excimer ablation of the Müller cell membrane. Blocking studies confirmed that binding was antigen specific. Trypan blue colocalized to the cut edge of retinal flatmounts. All species showed high levels of immunoreactivity except rabbit. In vivo studies demonstrated selective intraoperative staining of retinal breaks with a high level of specificity. CONCLUSIONS: Intraoperative vital staining of retinal breaks is possible in an animal model of retinal detachment. Ex vivo studies indicate that this occurs because the Cy3 anti-GFAP selectively binds the intermediate filaments of glial cells with damaged or destroyed cell membranes.