Purchase this article with an account.
Verleen K. McSween, Suresh Viswanathan, Joseph A. Bonanno, Tracy T. Nguyen, Shimin Li; Longitudinal Measurement Of In Vivo Fluorescence In Retrogradely Labeled Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1957.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To explore the feasibility of measuring long-term retinal ganglion cell (RGC) intracellular pH in vivo in response to IOP changes using retrograde delivered dextran-linked indicator dyes.
RGCs were retrograde labeled in anesthetized adult male Brown Norway rats by superior colliculi (SC) injection of 10kD Alexa Fluor 790 dextran and the pH sensitive dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) dextran (30 mg/ml, 20 µl). Retinal fluorescence was measured with a confocal scanning laser ophthalmoscope (HRA2, Heidelberg Engineering®) over a 6 week period. The fluorescence of individual cells was analyzed using the MetaMorph image analysis software. In separate animals, 2 weeks following dye injection, RGC fluorescence was measured before, during and after IOP elevations of 60 mm Hg produced by reservoir cannulation of the anterior chamber.
Retinal fluorescence from the retrograde labeling was seen at the optic nerve head as early as 5 hours following the SC injections, labeling of intraretinal axons was seen by day 1 and cell bodies by day 3. The fluorescence intensity of 239 double labeled cells from 6 eyes was tracked over 6 weeks. The intensity of Alexa790 labeling increased by 30% from day 7 to 14 (p<8x10-6) and thereafter gradually reduced by day 28 to 50% of the day 14 value (p<1x10-11). The intensity of BCECF labeling reduced by 20% from day 7 to 14 (p<1x10-11) to reach a plateau. The BCECF/Alexa790 fluorescence ratio increased from 1.4 at day 7 to 2.3 at day 28 (p<1x10-10) and thereafter reduced to 1.6 by day 42 (p<1.3x10-6). Increasing IOP from a baseline of 10 mm Hg to 60 mm Hg for 10 minutes eliminated BCECF but not Alexa 790 fluorescence and BCECF fluorescence was measurable again when IOP was returned to the baseline.
Retrograde labeled RGC fluorescence can be reliably tracked over a 6 week period. Intracellular pH reduces as a result of acute elevation of IOP that is likely to be secondary to ischemia.
This PDF is available to Subscribers Only