Abstracts

Rationale: Microglia-mediated inflammatory responses are widely known to accompany seizures and epilepsy. Although microglial cells are professional phagocytes less is known about the status of phagocytosis-associated molecules in epilepsy. Recent evidence supports that microglia-mediated synaptic pruning is guided by “eat-me” signals such as the C1q and C3 proteins of the classical complement pathway. Interestingly, it has been shown that the “eat-me” ligand Pros1 along with its microglia receptor MerTK are associated with the elimination of stressed but healthy neurons by phagoptosis. Thus, because neuronal and synaptic decline occur in epilepsy it is possible that aberrant phagocytosis signaling may participate in this process. Therefore, to identify a potential role, we determined the protein levels of C1q, C3, Pros1, MerTK along with the phagocytic receptor Trem2, the “don’t-eat-me” molecule CD47, microgliosis, and astrogliosis in human epileptic brain tissue. In parallel, we determined some of these proteins in hippocampi from epileptic rats. Methods: Cortical brain tissue samples from patients with refractory epilepsy (RE; n=6) and non-epileptic lesions (NE; n=3) were obtained during neurosurgical procedures. The pilocarpine model of status epilepticus (SE) and acquired temporal lobe epilepsy (TLE) was used to generate epileptic rats. SE (1hr) was induced with pilocarpine and stopped with diazepam (n=8). Controls were given saline (n=8). Hippocampi were dissected between 3-5 weeks after SE, when rats were epileptic. Western blotting (WB) was used to determine the protein levels of C1q, C3, MerTK, Trem2, Pros1, CD47, GFAP, IBA1 and the apoptotic marker cleaved-caspase 3. In addition, immunofluorescence was done visualize microglia and dendritic contacts in the human tissues. Results: Densitometry of WB immunoreactive bands showed that the human RE samples had significantly elevated protein levels of C1q (p=0.03), the C3 cleavage product iC3b (p=0.03), and MerTK (p=0.07), while Trem2 (p=0.05) and Pros1 (p=0.01) had decreased protein levels compared to the NE samples. Levels of CD47 (p=0.35), GFAP (p=0.16), IBA1 (p=0.85) and cleaved-caspase 3 (p=0.70) were not different between RE and NE tissues. In the experimental model, we found increased protein levels of MerTK in hippocampi of SE rats compared to controls (p=0.06). Conclusions: These data suggest that altered phagocytosis signaling occurs in both human and experimental models of epilepsy. We speculate that these alterations may contribute to the pathological elimination of neurons and synaptodendritic structures or may impair the elimination of dead cells/cellular debris. Additional studies are underway to determine the time course of SE-induced phagocytosis changes along with their association to epileptogenic synaptic remodeling and seizures in experimental models.  Funding: American Epilepsy Society (ALB); Laboratory Startup Funds, Department of Psychological Sciences, College of Health and Human Sciences, Purdue University (ALB).