Anti-inflammatory treatment with a soluble epoxide hydrolase inhibitor attenuates seizures and epilepsy-associated depression in the LiCl-pilocarpine post-status epilepticus rat model

Abstract

Purpose: This study aimed to investigate whether 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase inhibitor with anti-inflammatory effects, could alleviate spontaneous recurrent seizures (SRS) and epilepsy-associated depressive behaviours in the lithium chloride (LiCl)-pilocarpine-induced post-status epilepticus (SE) rat model.

Methods: The rats were intraperitoneally (IP) injected with LiCl (127 mg/kg) and pilocarpine (40 mg/kg) to induce SE. A video surveillance system was used to monitor SRS in the post-SE model for 6 weeks (from the onset of the 2nd week to the end of the 7th week after SE induction). TPPU (0.1 mg/kg/d) was intragastrically given for 4 weeks from the 21st day after SE induction in the SRS + 0.1 TPPU group. The SRS + PEG 400 group was given the vehicle (40% polyethylene glycol 400) instead, and the control group was given LiCl and PEG 400 but not pilocarpine. The sucrose preference test (SPT) and forced swim test (FST) were conducted to evaluate the depression-like behaviours of rats. Immunofluorescent staining, enzyme-linked immunosorbent assay, and western blot analysis were performed to measure astrocytic and microglial gliosis, neuronal loss, and levels of soluble epoxide hydrolase (sEH), cytokines [tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6], and cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB).

Results: The frequency of SRS was significantly decreased at 6 weeks and 7 weeks after SE induction in the 0.1TPP U group compared with the SRS + PEG 400 group. The immobility time (IMT) evaluated by FST was significantly decreased, whereas the climbing time (CMT) was increased, and the sucrose preference rate (SPR) evaluated by SPT was in an increasing trend. The levels of sEH, TNF-α, IL-1β, and IL-6 in the hippocampus (Hip) and prefrontal cortex (PFC) were all significantly increased in the SRS + PEG 400 group compared with the control group; neuronal loss, astrogliosis, and microglial activation were also observed. The astrocytic and microglial activation and levels of the pro-inflammatory cytokines in the Hip and PFC were significantly attenuated in the TPPU group compared with the SRS + PEG 400 group; moreover, neuronal loss and the decreased CREB expression were significantly alleviated as well.

Conclusion: TPPU treatment after SE attenuates SRS and epilepsy-associated depressive behaviours in the LiCl-pilocarpine induced post-SE rat model, and it also exerts anti-inflammatory effects in the brain. Our findings suggest a new therapeutic approach for epilepsy and its comorbidities, especially depression.

Fig. 1

Schematic diagram showing the timeline for drug administration and behavioural testing.

Fig. 2

The frequency of SRS shows a reduction after TPPU administration, with a significant decrease at 21d and 28d after TPPU administration (equal to 6 w and 7 w after SE induction) in the SRS + 0.1 TPPU group compared with the SRS + PEG 400 group, n = 9 in every group, *P<0.05, **P<0.001.

Fig. 3

A) The IMT was significantly increased in the SRS + PEG 400 group compared with the Control group, and it was significantly decreased after TPPU treatment (*P<0.05, **P<0.01); B) The CMT was significantly decreased in the SRS + PEG 400 group compared with the Control group, and it was significantly increased after TPPU treatment (*P<0.05); n=13 in every group.

Fig. 4

The fluorescent intensity of GFAP-positive cells was significantly increased in the CA1 (A), CA3 (B), and DG (C) areas of the Hip and the PFC (D) in the SRS + PEG 400 group compared with the Control group. The astrocytic gliosis in the CA1, CA3, and DG areas of Hip and the PFC was all significantly attenuated after TPPU treatment (n = 4 in every group, *P<0.05, **P<0.01).

Fig. 5

The fluorescent intensity of Iba-1-positive cells was significantly increased in the CA1 (A), CA3 (B), and DG (C) areas of the Hip and the PFC (D) of the SRS + PEG 400 group compared with the Control group. TPPU treatment significantly attenuated microglial gliosis in the CA3 (B) and DG (C) areas of the Hip (n = 4 in every group, *P<0.05, **P<0.01).

Fig. 6

The expression of CD11b in the PFC (A) and Hip (B) was significantly increased in the SRS + PEG 400 group compared with the Control group, and it was significantly decreased in the PFC (A) after TPPU treatment, n = 4 in every group, *P<0.05, **P<0.01.

Fig. 7

The levels of the cytokines IL-1β, IL-6, and TNF-α in the Hip (A-C) and PFC (D-F) were significantly increased in the SRS + PEG 400 group compared with the Control group, and they were significantly decreased after TPPU treatment, n = 4 in every group, *P<0.05, **P<0.01.

Fig. 8

The level of sEH in the PFC (A) and Hip (B) was significantly greater in the SRS + PEG 400 group than in the Control group, and it was significantly decreased in the PFC after TPPU treatment, n = 4 in every group, *P<0.05.

Fig. 9

The number of NeuN-positive cells in the CA1 (A), CA3 (B), and DG (C) areas of the Hip, the PFC (D), and the layer V of PFC (E) was all significantly decreased in the SRS + PEG 400 group compared with the Control group, *P<0.05, **P<0.01. TPPU treatment significantly attenuated the damage of NeuN-positive cells in the DG (C) area of the Hip and the PFC (D and E), n = 4 in every group, *P<0.05, **P<0.01. The co-expression of NeuN and p-CREB was increased after TPPU treatment as shown in the layer V of PFC (the yellow arrow shows that p-CREB is triple-labelled with NeuN and DAPI). 20× magnification micrographs are shown in the figures of CA1, CA3, DG, and PFC; 40× magnification micrographs are shown in the figure of layer V.

Fig. 10

The ratio of p-CREB/CREB in the PFC (A) was significantly decreased in the SRS + PEG 400 group compared with the Control group, and it was significantly increased in both of the PFC (A) and Hip (B) after TPPU treatment, n = 4 in every group, *P<0.05.