Abstracts

Rationale: The NS-Pten (neuron subset-specific) KO mouse model develops spontaneous seizures beginning as early as 6wks. PTEN is a negative regulator of the mTOR pathway and deletion results in hyperactivation of the pathway. The mTOR pathway is involved in many molecular processes and dysregulation of the pathway has been implicated in a heightened inflammatory response. NS-Pten KO mice have morphological abnormalities, specifically to the granule layer of the dente gyrus (dysplasia), an increase in soma size of neurons, and increases in neuroinflammation found through western blot assay. KO animals have shown deficits in behavioral tasks involved in sociability and learning and memory. This finding is not surprising given that there is a high comorbidity between epilepsy and other cognitive disorders such as Autism Spectrum Disorder (ASD). One overarching theme between epilepsy and disorders such as ASD are increases in inflammation and many suggest this may contribute to severity of the disorder.

Methods: This current study utilized 4, 10, and 15-week-old wildtype (WT) and knockout (KO) mice. For immunofluorescence protocol, mice were perfused with 50mls of 1x PBS followed by 50mls of 4% PFA in PBS. Dorsal hippocampus was located, and tissue was randomly sampled to obtain a total of 6 slices per animal. Slices were stained for Iba1 (microglia) and mounted using a mounting medium containing Dapi. For each animal 3 of the 6 stained slices were imaged on either the left or right hemisphere then total number of microglia was averaged between the 3 for each count. The areas analyzed include total hippocampus, CA1 region, CA2/3 regions, and the dente gyrus. Total number of microglia was counted using a custom semi-automated counting system made by creating a program using macros from ImageJ (NIH). Preliminary results from each group are shown.

Results: Preliminary findings show that across all groups there is a genotype difference that KO animals have an increased total number of microglia in their hippocampus. For 4-week-old mice, all regions of the hippocampus (CA1, CA2/3, dente gyrus) of KO animals were found to have an increase in the number of microglia. For 10-week-old mice, KO animals had more microglia in the CA1, dente gyrus compared to WT mice. For 15-week-old mice, only the dente gyrus had an increase in number of microglia. New did not find sex between the groups.

Conclusions: These data suggest an increase in immune reactivity in KO mice with hyperactive mTOR signaling in the hippocampus in an age-dependent manner. We found that Pten KO mice had an increase in the microglia marker Iba1 in all three time points. Additionally, we found that this increase was in all regions for the 4-week time point, only the CA1 and dente gyrus for the 10-week time point, and only the dente gyrus for the 15-week time point, suggesting that the dente gyrus may be play a critical role in the inflammatory response of the Pten KO mice. Further research is needed to look at other brain areas, as well as characterizing the different stages of microglia in Pten WT and KO mice.

Funding: Please list any funding that was received in support of this abstract.: National Institutes of Health (NIH) [Grant Number: NS088776].