| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 106 === Soluble epoxide hydroxylase (sEH) is a dual activity enzyme with the C-terminal epoxide hydrolase domain and an N-terminal lipid phosphatase domain and is expressed in mammalian brains, especially hippocampus. Inhibition of sEH hydrolase activity has been proven to be protective against ischemic brain injury, but it was also reported to facilitate synaptic plasticity. Synaptic plasticity in the hippocampus is an important role to improve memory function. Also, previous studies demonstrated that inhibition of GABAergic interneuron activity impaired memory function, indicating GABAergic interneuron involved in regulating memory function. However, whether the sEH inhibitor treatment can benefit the recovery of memory function by affecting GABAergic interneurons after excitotoxic insult was unknown. In this study, we examined the post-treatment effect of intranasal delivery (i.n.) of sEH hydrolase inhibitor TPPU (0.5mg/kg once a day for 7 days) on hippocampal damage, with special focus on GABAergic interneurons, and memory impairment 7 days after the intraperitoneal kainic acid (KA) injection, and in comparison with the effect of genetic ablation of Ephx2 (Ephx2-KO). Our results show that the KA-triggered seizure activity 2h after the injection was lower in TPPU-i.n than the vehicle-i.n mice, and also lower in Ephx2-KO than WT mice. Immunohistochemistry staining showed that both Ephx2-KO and TPPU-i.n attenuated KA-induced NeuN+ neuronal loss in hippocampal CA1, CA3 and dentate gyrus (DG). Hippocampus-related pattern separation behavior assessed by the novel object recognition test was impaired by KA, which was ameliorated by TPPU-i.n and also in Ephx2-KO. Another hippocampus-related behavior is the spatial learning memory assessed by the Barnes maze test. The data showed that
TPPU-i.n treatment, not Ephx2 deletion, attenuated KA-induced spatial memory 3 days post-injection. However, this memory-improving effect of TPPU-i.n treatment did not sustain to 7-day post-injection. Notably, the Ephx2-KO mice learn faster than the WT mice to find the target hole during the 4-day training period prior to the drug injection; whereas vehicle-injected Ephx2-KO mice spent more time to find the target hole than the vehicle-injected WT mice 7 days after the training, implying that sEH might be involved in both memory acquisition and consolidation.
Immunohistochemistry study of the GABAergic interneurons as labeled by the GABA synthesizing enzyme glutamic acid decarboxylase 65 (GAD65) show that GAD65 were reduced by KA and rescued by both TPPUi.n and Ephx2-KO in hippocampal subregions. Next, we examined a subset of GABAergic interneurons in the DG hilus region, which expressed a calcium-buffering protein calretinin (CR) to maintain proper neuronal excitability, found that both TPPU and Ephx2-KO could rescue CR+ interneurons in supragranular layer (SG) and hilus of DG after KA insult. Another subtype of GABAergic interneurons, parvalbumin (PV) expressing neurons in the hippocampus, could not be rescued by TPPU treatment after KA-ip insult significantly. Then we tried to confirm this phenomenon in vitro by using primary glia-neuron mix culture treated with glutamate receptor agonist NMDA to induce excitotoxic neuronal death. The results indicated that TPPU pretreatment could ameliorate NMDA-induced loss of NeuN+ neurons and MAP2+ neurons. Next, we examined GABAergic neurons labeled cell cytosol by GAD67, and we found that TPPU pretreatment could not alter NMDA-induced loss of GAD67 protein level or expression. Further examined one type of GABAergic neuron labeled by calretinin, and we found that TPPU pretreatment can significantly ameliorate NMDA-induced reduction of CR+ neurons but not CR protein level. Also, we examined another type of GABAergic neurons labeled by parvalbumin, and the data indicated that TPPU pretreatment cannot alter NMDA-induced the loss of PV+ neurons.
In conclusion, these results suggest that intranasal delivery of sEH inhibitor TPPU and Ephx2 deletion both show moderate protective effects against excitotoxin-induced hippocampal damage and pattern separation deficit accompanied with preservation of CR-expressing GABAergic interneurons in the dentate gyrus. The obtained information may provide clues for the modulation of excitation-inhibition balance in hippocampal circuits.
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