We carried out in vitro experiments to reveal mechanisms of neuroprotection of LBP underlying OGD. Based on findings that there is heterogeneity of hippocampal neurons in response to ischemia such as highly vulnerable CA1 neurons and resistant CA3 neurons, and cortical neurons in the frontal and parietal cortex are more evenly vulnerable to ischemia (Pulsinelli et al., 1982) we cultured cortical cells for 7 days and identified neurons (MAP2) and astrocytes (GFAP) using immunofluorescent staining. Approximately 95% of cells were neurons and 5% astrocytes (Figure 3A). Next, we investigated the relationship between OGD duration (from 1 h to 12 h) and neuronal death. Our results showed that 20% of neurons died when exposed to OGD for 1 h, whereas 50% of neurons were lost after 4 h OGD (Figure 3B). We then detected the protective effect of LBP on cultured neurons exposed to 4 h OGD. Figure 3C shows the effects of LBP on cortical neuron morphological changes. OGD caused neuronal dendrites to become varicose (Figure 3C3, arrows). However, LBP treatment prevented dendritic degeneration (Figure 3C4) and smooth dendrites were found similar to that of control (Figure 3C1) or control + LBP (Figure 3C2) groups. In addition, we detected neural injury with FJB staining (Schmued and Hopkins, 2000). When exposed to OGD for 4 h, strong FJB staining was evident in injured neurons (Figure 3D3, arrow). However, these injury signals were diminished in OGD+LBP neurons (Figure 3D4) with similar weak staining to that observed in control or control + LBP groups (Figures 3D1,D2). Quantitative analysis showed that the density of FJB positive cells increased to 205.2% ± 10.1 after 4 h exposure to OGD (P < 0.01 vs. control and Con-LBP groups, Figure 3E). However, value dropped to normal levels (107.9 ± 4.9%, P < 0.01 vs. OGD but P > 0.05 vs. control and Con-LBP groups) with LBP treatment. Consistent with FJB detection, neuron mortality significantly increased to 168.9% ± 2.3 after 4 h exposure to OGD (P < 0.01 vs. control and Con-LBP groups, Figure 3E), whereas it decreased to 137.4% ± 4.0 after the administration of LBP (P < 0.01 vs. OGD group). Furthermore, cell viability reduced to 54.2% ± 4.3 in the 4 h OGD group (P < 0.01, vs. control and Con-LBP groups, Figure 3E) but increased to 90.2% ± 9.6 after LBP treatment (P < 0.01, vs. OGD group).