Figure 1.
Enhanced survival of RGCs after 4-s ON-crush with OSM treatment.
RGCs were retrogradely labeled with Fluorogold (FG). One week after FG labeling, the left eye of an animal received ON-crush (4-s) and treated either with OSM (3 µg in 2 µl, C, F, I) or PBS (2 µl, B, E, H) immediately after ON-crush, while the right eye was untreated and used as control (A, G, D). Two weeks after ON-crush, retinas were harvested for RGC counts. Rod shaped and brightly labeled microglial cells were excluded from RGC counts. Flat-mounted retinas stained with antibodies against CD11b confirmed that those cells were CD11b positive (J, K, L) and thus they were microglias, not RGCs. Quantitative data show a significant loss of RGCs after ON-crush in both PBS- and OSM-treated eyes (Mean+SEM, n = 6, M). However, the remaining RGCs in the OSM-treated eyes were significantly more than in the PBS-treated (J). Scale bars: A–C, 1 mm; D–F, 100 µm; G–I, 100 µm; J–K, 50 µm. Triple asterisks indicate P<0.001.
Figure 2.
RGCs were retrogradely labeled with Fluorogold and ON-crush (1-s) was performed one week later. At least 48% of RGCs survived 1 week after ON-crush at all eccentricities, and at least 24% survived 2 weeks after ON-crush (mean+SEM, n = 5). The difference between 1-s and 4-s ON-crush on RGC survival at 2 weeks was significant (P<0.01, 2 way ANOVA). In comparison, only 14% RGCs left 2 weeks after 4-s ON-crush (n = 6).
Figure 3.
Effect of OSM or CNTF treatment on PERG after ON-crush.
OSM (3 µg in 2 µl) or CNTF (3 µg in 2 µl) was delivered intravitreally immediately after 1-s ON-crush. Eyes of control animals received PBS (2 µl). PERG was recorded before ON-crush as baseline and at 8, 15, 22 days after ON-crush. A: grand-average PERG waveforms recorded in PBS-, OSM-, and CNTF-treated eyes before and at different times after ON-crush. B: mean PERG amplitude as a function of time in PBS-, OSM-, and CNTF-treated groups. Eight days after ON-crush, the PERG amplitude was in the noise range (hatched area) in the PBS-treated group. However, in both OSM- and CNTF-treated groups, the PERG amplitudes were significantly higher (P = 0.003) than that of the PBS-treated group. By day 15 and 22, the PERG amplitudes were at or close to the noise range in all groups. Error bars represent the SEM (n = 6).
Figure 4.
Increase in STAT3 phosphorylation in Müller cells after OSM treatment.
Eyes were intravitreally injected with either PBS (2 µl) or OSM (3 µg in 2 µl), and retinas were harvested 1 hour later. Cryosections (16 µm) were stained for phosphorylated STAT3 (pSTAT3). Although no specific pSTAT3 staining was detected in the PBS treated control retina (A), a substantial increase in pSTAT3 immunoreactivity was seen in the OSM-treated retinas (B, C). The increased pSTAT3 is not co-localized with the RGC marker NeuN (B), but well co-localized with the Müller cell marker glutamine synthetase (C). RPE, retinal pigment epithelium; OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 50 µm.