Abstracts

Rationale:
It has long been established that widespread neuronal injury and neuronal death accompanies status epilepticus (SE). For example, in baboons treated with bicuculline, status epilepticus induces damage to the neocortex, hippocampus, and cerebellum. Similar patterns of cell death have been observed in humans without underlying brain pathology that have died from SE. However, it remains unclear whether the seizure activity itself causes cell death or whether the systemic changes that result from SE, such as hyperpyrexia, hypoxia, acidosis, altered blood flow and hyperglycemia are responsible. In a rat model of SE that leads to chronic epilepsy, the degree of neuronal damage correlates with time since SE, but not with the number of spontaneous seizures, suggesting that seizures themselves are not a primary cause of cell death. Anticonvulsants reduce or alter SE-induced cell death, but they also alter the systemic changes that accompany persistent seizure. In order to develop therapies that minimize neuronal death in patients suffering from SE, it is essential to understand the underlying factors precipitating the cell death.
Method:
In this study, we imaged neuronal survival and activity an in vitro model of post-traumatic epileptogenesis: the organotypic hippocampal slice culture. Slices were prepared from P7 DLX-cre or WT mice that were transduced intracerebroventricularly on P0 with one of two combinations of AAV vectors: DLX-cre mice were injected with FLEX-tdTomato and syn-GCaMP7f, producing pan-neuronal expression of a green calcium sensor and interneuron-specific expression of a red fluorescent protein; WT mice were injected with syn-cre-gfp and syn-jRGECO1b, producing pan-neuronal expression of both a nucleus-localized GFP and a red-shifted calcium sensor. We then used a novel imaging system constructed inside of a tissue culture incubator, to image slices continuously beginning shortly after the injury and continuing through the onset of spontaneous recurrent seizures (after ~7 days in vitro). Every 4 hours, a movie of calcium dynamics with cellular resolution and a field of view spanning the entire epileptic network was acquired. During the 4 hours between recordings, activity for a single slice was continuously monitored using a low-resolution mode of imaging, in which we use a 1-second exposure and average all pixels in the field of view to produce a field potential-like optical recording. Cell survival was monitored using the constitutively fluorescent protein in each case. Individual neurons were detected and tracked over the course of the experiment (or until their fluorescence was quenched during the death process) using the ImageJ plugin Trackmate.
Results:
There is a ~10% loss of interneurons during the first 36h in vitro, well before the onset of the first seizure. After 36h, we observed < 1% loss of interneurons after 3 weeks of status epilepticus, during which slices undergo 30-60s long seizures every 2-4 minutes. Analysis of cell death in all neurons is ongoing, but preliminary evidence suggests a similar lack of ictal cell death in pyramidal cells.
Conclusion:
These results are consistent with an early wave of cell death induced by the “injury” of slice preparation during which many neurons are irreparably damaged. The lack of a second wave of cell death or ongoing cell death associated with weeks of status epilepticus in vitro suggests that SE-induced neuronal death in animal models and human patients is not due to the seizure activity itself, but rather is due to secondary changes such as blood flow, cell volume, oxygen content, or other systemic factors.
Funding:
:NIH 5R01NS034700, 5R37NS077908, 5R01NS040109, CURE W81XWH-15-2-0069