IH induced oxidative stress in rat primary cerebellar granule cells
Oxidative stress progressively increased with increase in the duration of IH (Figure 1Aa, b). The average fluorescence of the RA (normoxia) 4 day group was set at 100%. In cells stained with HE (to detect O2-·), the average fluorescence of the IH1day-IH4day groups was 123.04 ± 17.64%, 149.11 ± 10.22%, 165.04 ± 1.0%, and 194.01 ± 18.12%, respectively. The fluorescence of the IH4day group was about twice that of the RA4day group. In cells stained with DCFDA (to detect H2O2 and OH·), the average fluorescence of the IH1day-IH4day groups was 193.39 ± 11.37%, 282.52 ± 29.69%, 450.76 ± 9.04%, and 397.27 ± 29.65%, respectively. The fluorescence of the IH3day and IH4day groups was about four times that of the RA4day group (Figure 1Aa, b). The fluorescence of the IH3day group and IH4day group was not significantly different (P = 0.106, LSD test in ANOVA).
IH-induced apoptosis in rat primary cerebellar granule cells
Cell nuclei were visualized by Hoechst dye staining. Increase in the number of apoptotic cells resulted in decrease in the average area of the nuclei. The average area of the nuclei of the RA4day group was set at 100%. Compared to the RA4day group, the RA1day-RA3day groups was 99.63 ± 1.24%, 101.05 ± 1.97%, and 101.07 ± 1.03%, respectively. The IH1day to IH4day groups was 96.85 ± 2.34%, 93.28 ± 2.80%, 89.97 ± 2.04%, and 84.61 ± 2.23%, respectively. The average area of the nuclei among the RA1day-RA4day groups were not significantly different (P = 0.899, ANOVA); however, there were significant difference in the average area of the nuclei between the RA4day from IH2day, IH3day and IH4day groups (Figure 1Bb).
Colocalized TUNEL (green)- and Hoechst nuclear (blue)-stained areas indicate apoptotic cells (Figure 1Ba). The TUNEL (+) ratio in the RA3day and RA4day groups was 4.38 ± 1.59% and 5.27 ± 1.36%, respectively. The TUNEL (+) ratio in the IH3day and IH4day groups was 17.34 ± 1.12% and 18.51 ± 4.46%, respectively. There was a significantly higher TUNEL (+) ratio in the IH3day and IH4day groups than in the RA3day and RA4day groups, respectively (Figure 1Bc).
The IH groups exhibited higher levels of apoptosis, and this trend increased with increased exposure to IH.
IH-induced necrosis in rat primary cerebellar granule cells
Colocalized PI (red)- and Hoechst nuclear (blue)-stained areas indicate necrotic cells and loss of membrane integrity (Figure 1Ca). The PI (+) ratio of the RA1day-RA4day groups was 2.92 ± 0.8%, 2.97 ± 1.55%, 4.39 ± 1.07%, and 2.23 ± 0.6%, respectively. The PI (+) ratio of the IH1day-IH4day groups was 4.22 ± 2.40%, 6.0 ± 2.11%, 13.31 ± 2.12%, and 15.64 ± 3.24%, respectively. The PI (+) ratios among the RA1day-RA4day groups was not significantly different (P = 0.899, ANOVA); however, there were significant differences in the ratios between the RA4day from IH3day and IH4day groups (Figure 1Cb). More necrotic cells were found in the IH groups, and this trend increased with longer durations of IH.
IH-induced cell death can be rescue by inhibitors pretreatment
The average area of the nuclei of cells in the IH3day group was 89.97 ± 2.04%, and that in the IH3day group treated with Phe, 3-AB, and DPQ was 95.34 ± 1.58%, 96.38 ± 1.03%, and 93.44 ± 0.50%, respectively. The average area of the nuclei of cells in the IH4day group was 84.61 ± 2.23%, while that of cells in the IH4day group treated with Phe, 3-AB, and DPQ was 95.33 ± 2.88%, 94.48 ± 2.44%, and 91.90 ± 3.11%, respectively. The average area of the nuclei of cells increased significantly in the IH3day and IH4day groups treated with inhibitors (Figure 2A). The TUNEL (+) ratio of the IH3day and IH4day groups was 17.34 ± 1.12% and 18.51 ± 4.46%, respectively. Furthermore, the ratio of cells treated with Phe in the IH3day and IH4day groups was 10.50 ± 1.84% and 6.26 ± 3.98%, respectively. The ratio of both groups treated with Phe was significantly lower than that of the untreated IH3day and IH4day groups (Figure 2B). Apoptotic cell death decreased in the presence of inhibitors.
The PI (+) ratio of the IH3day group was 13.31 ± 2.12%, while that of the IH3day group treated with Phe, 3-AB, and DPQ was 5.66 ± 1.22%, 3.51 ± 0.71%, and 4.27 ± 0.67%, respectively. The PI (+) ratio of the IH4day group was 15.64 ± 3.24%, while that of the IH4day group treated with Phe, 3-AB, and DPQ was 4.21 ± 0.98%, 3.13 ± 1.38%, and 5.06 ± 2.62%, respectively. The ratio in the IH3day and IH4day groups treated with inhibitors decreased significantly (Figure 2C). Necrotic cell death decreased in the presence of inhibitors. These data suggested that reduction in oxidative stress or PARP inhibition resulted in the decrease in apoptosis and necrosis.
Caspase-3 activation was not involved in IH-induced cell death
Caspase-3 cleaved the substrate FITC-DEVD-FMK, resulting in its fluorescence. The fluorescence of the RA4day group was set at 100%. The fluorescence of the IH1day-IH4day groups was 85.69 ± 32.78%, 92.24 ± 16.57%, 96.87 ± 13.30%, and 96.70 ± 22.12%, respectively. There were no significant differences in fluorescence among the RA4day and IH1day-IH4day groups. Cells treated with H2O2 served as the positive control, and their fluorescence was 451 ± 11.0%. (Figure 3Aa, b). Whole cell proteins of the RA4day and IH1day-IH4day groups were extracted for Western blotting analysis, with β-actin as the internal control. Results did not indicate the activation of caspase-3 or fragmentation of PARP, which was cleaved by caspase-3 in the caspase-dependent apoptotic pathway (Figure 3B). Therefore, IH did not induce caspase-3 activation.
IH-induced cell death was correlated with AIF nuclear-translocation
Cells were co-stained with AIF and VDAC, a mitochondrial marker. The nucleus and mitochondria were labeled as N and M, respectively (Figure 3Ca). The ratio of fluorescence of the nucleus/mitochondria (N/M) was measured by AIF immunostaining. Increase in the N/M ratio was indicative of increased AIF translocation to the nucleus. The N/M ratio of the RA4day group was 0.21 ± 0.0065%, while that of the IH4day group was 0.41 ± 0.0109%. The N/M ratio of the IH4day group was significantly higher than that of the RA4day group. Inclusion of the PARP inhibitor 3-AB in the IH4day group decreased the N/M ratio to 0.30 ± 0.0047%, which was significantly different from the ratio of the untreated IH4day group (Figure 3Cb). Cells of the RA4day and IH4day groups were subjected to subcellular fractionation, and immunoblotting was performed on the cytosolic and mitochondrial fractions (Figure 3D). β-actin and VDAC were used as cytosolic and mitochondrial loading control, respectively. The amount of AIF in the mitochondria of the cells in the IH4day group seemed to be less than that in the RA4day group, indicating that AIF was released from the mitochondria and translocated to the nucleus. The amount of cytochrome c expressed in the mitochondria of the cells in the RA4day and IH4day groups was the same. Cytochrome c and activated caspase-3 were not detected in the cytosol. Similar to the above observation (Figure 3A, B), since cytochrome c was not released from the mitochondria into the cytosol, it did not induce the activation of caspase-3.
PARP inhibition abrogates calpain's activation
Exposure cells to IH4 day resulted in elevated calpain expression which was blocked by using PARP inhibitor 3-AB (Figure 4A). The quantitative data of Figure 4A showed that calpain-positive ratio in the IH4day group was 1.65 ± 0.063% fold higher than RA4day group, and decreased to 1.08 ± 0.03% fold in the IH4day treated with 3-AB group (Figure 4B). We validated the up-regulation of calpain by western blot. IH elicited an increased expression of calpain proteins that was diminished in IH4day treated with 3-AB group (Figure 4C).