To study whether Hmgb1 alone mediates similar damage as IR injury, we injected recombinant Hmgb1 (rHmgb1) intravitreally and evaluated the survival of GCL neurons 7 days after treatment. The analysis of rHMGB1-treated animals showed a decrease (83 ± 3%) in cell numbers in the GCL compared with PBS-treated controls (102 ± 5%,
P < 0.01) (
Fig. 3A). Since Hmgb1 as a danger signal alone is expected to trigger inflammation, we investigated the activation of the proinflammatory markers
Ccl2,
Cxcl10,
Il1β,
Nos2,
Tnf, and
Icam1 24 hours after injection of rHMGB1 at the level of RNA expression by quantitative PCR. We observed transcriptional upregulation of the
Tnf and
Il1β cytokines as well as
Ccl2 and
Cxcl10 chemokines (
Fig. 3B). Thus, our results suggest that intravitreal injection of rHmgb1 activates a proinflammatory response in retina. To elucidate in which cell type Hmgb1 exerts its detrimental effect, we turned to cell cultures. We assayed RGC survival in glia RGC cocultures in which RGCs, isolated according to the two-step immunopanning method, were plated directly on a glial monolayer. These cultures were challenged with rHmgb1 or PBS and the number of activated caspase-3/beta III Tubulin-positive RGCs was counted after 24 hours. Treatment with rHmgb1 decreased the number of beta III Tubulin-positive neurons, indicating that RGCs are dying if cultures are treated with rHmgb1 (
Figs. 4A,
4B). We next performed experiments with RGC primary cultures to establish the direct role of Hmgb1 in neuronal vulnerability. RGCs were treated with rHmgb1 or PBS and then assessed for levels of necrotic and apoptotic cells and survival after 24 hours. Quantification of RGC death showed significantly higher cell death in cultures treated with rHmgb1 versus PBS (
Figs. 4C,
4D). To determine whether the observed effects were due to contamination by astrocytes and/or microglia/macrophages, isolated cells were tested by morphology using microscopy (
Fig. 4D), as well as by real-time PCR, to validate the abundance of neuronal marker and to determine the level of contamination by nonneuronal cells such as glia and macrophages. We assessed the relative abundance of the marker genes for RGCs (
Thy1), astrocytes (
S100b), and microglia/macrophage (
Cd11b) cells in RGCs purified by immunopanning. Real-time PCR did not detect contamination with microglia/macrophages (
Cd11b), and astrocytes were detected only with relative abundance of
S100b/
Thy1 marker genes mRNA ranging between 1 and 3% (
Fig. 4E). Thus, the calculated efficiency of RGC purification was 97% to 99%. Importantly, RGC death was significantly higher in glia RGC coculture treated with rHmgb1 (28 ± 2% RGC apoptosis) compared with the primary RGC cultures treated with rHmgb1 (15 ± 2% RGC apoptosis,
P < 0.05) (
Fig. 4). Thus, glia are required for the harmful effect of Hmgb1.