In the present study, our results show that the morphology of the PSD changed, and that levels of major PSD proteins, such as PSD-95, NR2B, NR1, and CaMKII, were all decreased in the CA3 area of organotypic hippocampal slice cultures following OGD/reperfusion.
Brain ischemia triggers complex cellular mechanisms that impair synaptic functions through the breakdown of cellular and structural features, mediated by various excitotoxic signals [35
]. It is well known that the CA1 area of hippocampus is particularly vulnerable to ischemic insult [39
]. However, various biochemical and pathological activities, followed by neuronal cell death, can also be detected in the CA3 area along mossy fibers [41
]. According to our previous studies [31
], neuronal damage expressed with propidium iodide staining was found both in the CA1 area and CA3 area of hippocampal slice cultures following OGD/reperfusion.
Following OGD for 30 min, some swelling of the mitochondria, ER, dendrites, and synaptic terminals was observed, and the thickness of PSDs was notably increased. During reperfusion, swelling of cellular organelles and disorganization of the mitochondria was evident. This finding is in agreement with that of other studies, which showed some ultrastructural abnormalities in postischemic neurons, including disaggregation of polyribosomes and deposition of dark substances [44
Changes in synaptic responses after variable periods of hypoxia or ischemia have also been demonstrated frequently both in vitro
] and in vivo
]. In this study, OGD/reperfusion induced modifications as shown by EPTA staining for PSD in the CA3 area. At 24 h after OGD, the number of PSDs was significantly decreased, and the remaining PSDs became thicker, longer, and more electron-lucent, reflecting the damage to PSDs. This finding is consistent with that of earlier studies, which demonstrated that dramatic ultrastructural changes to PSDs, along with synaptic deficits occurred following transient ischemia [35
]. Kovalenko et al. [37
] reported a rapid increase in the PSD thickness and length, as well as the formation of concave synapses with perforated PSDs in the CA1 stratum radiatum during the first 24 h after an ischemic episode. EPTA primarily stained the synaptic structure and nucleus, but also stained intracellular protein aggregates. After ischemia, EPTA-stained proteins are predominantly accumulated in rat hippocampal CA1 PSDs, a finding which may be attributable to protein unfolding or denaturing exposing their hydrophobic segments during ischemia, and thereby causing interprotein aggregation in the PSDs after ischemia, and resulting in dysfunctional synaptic transmission at the altered synapse. Additionally, unfolding of proteins induced by ischemia may aggregate and change the structure of PSDs, which may lead to loosening of the PSD frame structure, rendering them more diffuse and irregular [56
In PSDs, the heteromeric combinations of NR1 and NR2A-D interact with PSD-95 and CaMKII [22
]. Because this complex plays a central role in the regulation of synaptic function [58
], and is important in linking the synapse to downstream signaling pathways, it could also be involved in the hypoxia/ischemia-induced changes that lead to neuronal damage. Robust morphological alterations to PSD structure after ischemia/reperfusion were accompanied by biochemical changes, such as decreased levels of heat shock cognate protein 70, CaMKII, and protein kinase C in PSDs [55
]. When western blot analysis for PSD-95, NR1, NR2B, and CaMKII was performed to investigate the relationship between OGD/reperfusion-induced structural modifications of PSDs, and the PSD protein complex, levels of all the PSD complex proteins were found to be significantly decreased 24 h following OGD (). Previous studies also reported that ischemia/reperfusion [62
], or perinatal hypoxia [63
] lead to a marked reduction in the mRNA expression of NR1, NR2A, and NR2B, and in the protein expression of PSD-95, total NMDAR levels, and the complexing of PSD-95 with NMDAR subunits within the hippocampal CA1 region, suggesting impaired synaptic performance. Altered PSD-95 expression is centrally involved in the hypoxia-induced changes leading to neuronal injury in the adult brain [55
Aschematic illustration of the interaction of NMDARs with PSD-95 and CaMKII.
Taken together, our results show that OGD/reperfusion can induce significant modifications to PSDs in the CA3 area of organotypic hippocampal slice cultures, both morphologically and biochemically. These changes are likely to be part of the process of neuronal cell death and synaptic dysfunction following OGD/reperfusion.