The most upregulated gene identified by microarray in early injury was
Atf3 (3.4-fold,
Table 2), a member of the ATF/CREB family of transcription factors that is inducible by a wide variety of stress signals and upregulated in the whole retina in both experimental glaucoma and after optic nerve transection.
16,20,32 Using qPCR (with more samples), we found a much more dramatic increase in the mRNA level of Atf3 (1492% ± 949%;
P < 0.01) in GCL with early injury (
Fig. 3). As the nerve injury advances, Atf3 mRNA increases in a pattern linear to the injury grades (
r 2 = 0.25;
P < 0.001). Jun family proteins and activator protein 1 (AP-1) components c-Jun and Junb, two early stress responding transcription factors that can dimerize with Atf3,
33,34 showed no change at the mRNA level in the early-injury group. Signal transducers and activators of transcription 3 (Stat3), another early response transcription factor commonly upregulated after nerve injury, including isolated glaucoma model RGC
18 and whole retinas after optic nerve transection,
35 –37 16,32 and shown to act together with Atf3 and cJun in damaged neurons,
38 was not changed either. With advanced injury, however, c-Jun (445% ± 67%;
P = 0.0001), Junb (305% ± 99%;
P < 0.05), and Stat3 (304% ± 33%;
P < 0.0001) mRNAs were all significantly upregulated in GCL, confirming findings in whole retinas and laser-captured RGCs.
16,18 In early-injury GCL, we did not detect any change in the message level for transcription factor hypoxia-inducible factor 1, alpha subunit (Hif1α) which mediates cellular responses to hypoxic stimuli. Also, no change was detected in the mRNA level of erythropoietin (
Epo), a target gene of Hif1α and marker for hypoxia. Neither Hif1α nor Epo was increased in the advanced injury group.