Authors :
Presenting Author: Sol Ah Kim, BS – Ewha Womans University
Yunseo Choi, PhD – EWHA Womans University; Gyu Hyun Kim, researcher – Korea Brain Research Institute; YuJaung Kim, PhD – EWHA Womans University; Dong Hyun Kwak, A.B – Princeton University; Hyang Woon Lee, MD, PhD – EWHA Womans University; Kea Joo Lee, PhD – Korea Brain Research Institute; Su Min Shin, researcher – Korea Brain Research Institute
Rationale:
In temporal lobe epilepsy (TLE), the role of neuron-glia interaction generating seizures in hippocampal circuit has been suggested. However, an association with comorbid behavioral abnormalities in TLE has not yet been established. This study investigated microstructural alterations of neurons and glial cells in hippocampal circuits, and their relationship between seizures and coexisting behavioral abnormalities. We also elucidated alterations in synaptic architecture and myelination in TLE epileptic model.
Methods:
After focal injection of pilocarpine into the right hippocampus of male SD rats, seizure severity was assessed as spontaneous recurrent seizures (SRS) according to the modified Racine classification at least four weeks post-initial SE. Behavioral testing of the animals using the Open Field Test (OFT), Elevated Plus Maze (EPM), and Forced Swimming Test (FST) were performed. Neural marker (NeuN), allograft inflammatory factor 1 (Iba-1) and glial fibrin protein (GFAP) antibodies were used for immunihistochemistry. In addition, we assessed the synaptic density, neuronal density, size of postsynaptic density (PSD), and myelin sheath thickness versus axon diameter (g-ratio) in hippocampal subregions using transmission electron microscopy (TEM).
Results:
SRS occurred in four to eight weeks after the initial SE. Epileptic rats had abnormalities in OFT, EPM and FST at 8 weeks after the initial SE, in addition to decreased NeuN cells in CA1 and CA3, increased Iba-1 in CA3, and increased GFAP cells in CA1 and dentate gyrus (DG), respectively. Using TEM, excitatory synaptic density per unit area was significantly reduced in the DG and CA1 of epileptic rats compared to controls. Excitatory synaptic density per neuron was decreased in the inner molecular layer, whereas inhibitory synaptic density per neuron was increased in the outer molecular layer of epileptic DG compared to controls. More importantly, PSD size of excitatory synapses was increased in all hippocampal subregions CA3 stratum lucidum. We also observed that myelin sheath thickness was increased (decreased G-ratio) in the CA1 area of epileptic rats compared to controls.
Conclusions:
Pilocarpine-induced seizures led to microstructural alterations in hippocampal neuronal as well as glial cells. In addition to SRS occurrence, increased depressive and anxiety-like behaviors were observed at eight weeks after the initial SE. Our ultrastructural data suggest that, the increased PSD length of excitatory synapses (enhanced synaptic strength) and the thickened myelin sheath may contribute to neuronal hyperexcitability. Additionally, the number of synaptic vesicles, the availability of pre-synaptic mitochondria, and the density of inhibitory synapses in the cell body will be further analyzed to find further clinical applications.
Funding:
Supported by the National Research Foundation of Korea(NRF-2021R1A6A3A13043922, 2021R1I1A1A01047392,2020R1A2C2013216,2019M3C1B8090803,2019M3C1B8090802) and the KBRI Basic Research Program(23-BR-01-03).