Background: The loss of RGCs expressing Thy-1 after optic nerve injury has an initial phase of rapid decline followed by a longer phase with slower reduction rate. This study used longitudinal retinal imaging of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) to determine how the α2-adrenergic agonist brimonidine influences loss of Thy1 promoter activation. Methods. Baseline images of the fluorescent retinal neurons in 30 Thy1-CFP mice were obtained using a modified confocal scanning laser ophthalmoscope. Next, brimonidine (100 ug/kg, IP) was administered either one time immediately after optic nerve crush, or immediately after optic nerve crush and then every 2 days for four weeks. A control group received a single saline injection immediately after optic nerve crush. All animals were imaged weekly for four weeks after optic nerve crush. Loss of fluorescent retinal neurons within specific retinal areas was determined by counting. Results: At one week after optic nerve crush, the proportion of fluorescent retinal neurons retaining fluorescence was 44±7% of baseline in control mice, 51±6% after one brimonidine treatment, and 55±6% after brimonidine treatment every other day (P<0.05 for both brimonidine treatment groups compared to the control group). Subsequently, the number of fluorescent retinal neurons in the group that received one treatment differed insignificantly from the control group. In contrast, the number of fluorescent retinal neurons in the group that received repeated brimonidine treatments was greater than the control group by 28% at two weeks after crush and by 32% at three weeks after crush (P<0.05 at both time points). Rate analysis showed that brimonidine slowed the initial rate of fluorescent cell decline in the animals that received multiple treatments (P<0.05). Differences in the rate of loss among the treatment groups were insignificant after the second week. Conclusion: Repeated brimonidine treatments protect against loss of fluorescence within fluorescent retinal neurons of Thy1-CFP mice after optic nerve crush. As most of fluorescent retinal neurons in this system are RGCs, these findings indicate that repeated brimonidine treatments may protect RGC health following optic nerve crush. © 2013 Dai et al.; licensee BioMed Central Ltd.