Cell Death Pathways in Astrocytes with a Modified Model of Oxygen-Glucose Deprivation

<div><p>Traditional oxygen-glucose deprivation (OGD) models do not produce sufficiently stable and continuous deprivation to induce cell death in the ischemic core. Therefore, we modified the OGD model to mimic the observed damage in the ischemic core following stroke and utilized this new model to study cell death pathways in astrocytes. The PO₂ and pH levels in the astrocyte culture medium were compared between a physical OGD group, a chemical OGD group and a mixed OGD group. The mixed OGD group was able to maintain anaerobic conditions in astrocyte culture medium for 6 h, while the physical and the chemical groups failed to maintain such conditions. Astrocyte viability decreased and LDH release into in the medium increased as a function of exposure to OGD. Compared to the control group, the expression of active caspase-3 in the mixed OGD group increased within 2 h after OGD, but decreased after 2 h of OGD. Additionally, porimin mRNA levels did not significantly increase during the first 2 h of OGD, while bcl-2 mRNA levels decreased at 1 h. However, both porimin and bcl-2 mRNA levels increased after 2 h of OGD; interestingly, they both suddenly decreased at 4 h of OGD. Taken together, these results indicate that apoptosis and oncosis are the two cell death pathways responsible for astrocyte death in the ischemic core. However, the main death pathway varies depending on the OGD period.</p></div

mRNA levels of bcl-2 and porimin in astrocytes exposed to mixed OGD.

<p>(A) the bcl-2 mRNA relative expression and (B) porimin mRNA relative expression for 1 h, 2 h, 3 h, 4 h and 6 h were altered relative to the rates of expression in the control samples (0 h). (*) indicates a significant difference (P<0.05) from the control group.</p

The PO₂ and pH of the media among the three OGD groups.

<p>PO<b>₂</b> (A) and pH (B) of the media among the three groups measured at different OGD time points. The mixed group kept the PO<b>₂</b> at zero for 6 h in the incubation solution and mimics the ischemic core.</p

LDH leakage induced by mixed OGD.

<p>Mixed OGD induced a significant increase in LDH leakage after 1 h, 2 h, 3 h, 4 h and 6 h (F = 220.7, P = 0.001). (*) indicates a significant difference (P<0.05) from the control group (Ctrl).</p

Representative electron microscopy images of the ultrastructural changes in astrocytes induced by mixed OGD.

<p>(A) Image of a normal astrocyte (6200×). (B,I) Images of typical apoptotic cells (the red arrowhead shows chromatin condensation rendering a curved profile to the nucleus and apoptotic body formation). Apoptotic cells were found in almost all samples, particularly after 1 h (B) and 2 h (C) of OGD. (G–H) Images of typical oncotic-like cells. The co-existence of cellular swelling and vacuolization were found in the 3 h (D) and 4 h (E) OGD samples. After 6 h of OGD, the astrocytes were almost fragmental (F). Scale bars: I: 1 µm; H: 2 µm; D: 10 µm; all others: 5 µm.</p

The viable astrocyte exposed to mixed OGD.

<p>(<b>A</b>) Astrocytes exposed to mixed OGD in the control condition (Ctrl) and for 1 h, 2 h, 3 h, 4 h and 6 h were stained by HE (200×). (B) Counting 5 views for statistical analysis, we found that the amount viable astrocytes decreased as a function of time spent under OGD. (*) indicates a significant difference (P<0.05) from the control group (Ctrl).</p