Levetiracetam Protects Against Cognitive Impairment of Subthreshold Convulsant Discharge Model Rats by Activating Protein Kinase C (PKC)-Growth-Associated Protein 43 (GAP-43)-Calmodulin-Dependent Protein Kinase (CaMK) Signal Transduction Pathway

Abstract

<strong>BACKGROUND</strong> Subclinical epileptiform discharges (SEDs) are defined as epileptiform electroencephalographic (EEG) discharges without clinical signs of seizure in patients. The subthreshold convulsant discharge (SCD) is a frequently used model for SEDs. This study aimed to investigate the effect of levetiracetam (LEV), an anti-convulsant drug, on cognitive impairment of SCD model rats and to assess the associated mechanisms. <strong>MATERIAL AND METHODS</strong> A SCD rat model was established. Rats were divided into an SCD group, an SCD+ sodium valproate (VPA) group, and an SCD+ levetiracetam (LEV) group. The Morris water maze was used to evaluate the capacity of positioning navigation and space exploration. The field excitatory post-synaptic potentials (fEPSPs) were evaluated using a bipolar stimulation electrode. NCAM, GAP43, PS95, and CaMK II levels were detected using Western blot and RT-PCR, respectively. PKC activity was examined by a non-radioactive method. <strong>RESULTS</strong> LEV shortens the latency of platform seeking in SCD rats in positioning navigation. fEPSP slopes were significantly lower in the SCD group, and LEV treatment significantly enhanced the fEPSP slopes compared to the SCD group (P<0.05). The NCAM and GAP-43 levels were increased and PSD-95 levels were increased in SCD rats (P<0.05), which were improved by LEV treatment. The PKC activity and CaMK II levels were decreased in SCD rats and LEV treatment significantly enhanced PKC activity and increased CaMK II levels. <strong>CONCLUSIONS</strong> Cognitive impairment in of SCD model rats may be caused by decreased PKC activity, low expression of CaMK II, and inhibition of LTP formation. LEV can improve cognitive function by activating the PKC-GAP-43-CaMK signal transduction pathway.

Figure 1

Latency of platform seeking in positioning navigation and spatial probe test data at 14 days or 28 days after intragastric administration (n=64). (A, B) Latency of platform seeking in positioning navigation at 14 and 28 days after intragastric administration. (C, D) Spatial probe test data at 14 days or 28 days post intragastric administration. * P<0.05, ** P<0.01 vs. Control group, ^# P<0.05 vs. SCD group. Dose of VPA: 150 mg/kg/d, dose of LEV: 150 mg/kg/d.

Figure 2

fEPSP slope at 14 days (A, B) and 28 days (C, D) after intragastric administration, both 1 min after HFS and 30 min after HFS (n=64). * P<0.05, ** P<0.01 vs. Control group, ^# P<0.05, ^## P<0.05 vs. SCD group. Dose of VPA: 150 mg/kg/d, dose of LEV: 150 mg/kg/d.

Figure 3

Evaluation of the expression of NCAM, PSD-95, and GAP-43 by Western blot assay (n=32). (A) Western blot bands for NCAM, PSD-95, and GAP-43 expression. (B) Statistical analysis of NCAM protein expression. (C) Statistical analysis of PSD-95 protein. (D) Statistical analysis of GAP-43 protein. * P<0.05, ** P<0.01 vs. Control group, ^# P<0.05, ^## P<0.05 vs. SCD group. 14N (28N), 14S (28S), and 14L (28L) represent the Control group, SCD group, and SCD+LEV group, respectively, at 14 days (28 days) after intragastric administration. Dose of VPA: 150 mg/kg/d, dose of LEV: 150 mg/kg/d.

Figure 4

Observation for mRNA expression of NCAM, PSD-95, and GAP-43 by using RT-PCR assay (n=32). (A) Statistical analysis for NCAM mRNA expression. (B) Statistical analysis for PSD-95 mRNA expression. (C) Statistical analysis for GAP-43 mRNA expression. * P<0.05, ** P<0.01 vs. Control group, ^# P<0.05, ^## P<0.05 vs. SCD group. 14N (28N), 14S (28S), and 14L (28L) represent the Control group, SCD group, and SCD+LEV group at 14 days (28 days) after intragastric administration, respectively. Dose of VPA: 150 mg/kg.d, dose of LEV: 150 mg/kg.d.

Figure 5

Examination for the p-PKC activity using a non-radioactive method (n=32). (A) The immunoblotting image. (B) Statistical analysis for p-PKC activity. ** P<0.01 vs. Control group, ^## P<0.05 vs. SCD group. 14N (28N), 14S (28S), and 14L (28L) represent the Control group, SCD group, and SCD+LEV group at 14 days (28 days) after intragastric administration, respectively. Dose of VPA: 150 mg/kg/d.

Figure 6

CaMK II expression was evaluated using both Western blot assay and RT-PCR assay (n=32). (A) Western blot assay and statistical analysis for CaMK II expression. (B) Statistical analysis for CaMK II mRNA expression. * P<0.05 vs. Control group, ^# P<0.05 vs. SCD group. 14N (28N), 14S (28S), and 14L (28L) represent the Control group, SCD group, and SCD+LEV group at 14 day (28 days) after intragastric administration, respectively. Dose of VPA: 150 mg/kg/d.

Figure 7

The signal pathway of PKC-GAP43-CaMK.