Low-frequency rTMS Plays a Neuroprotective role in Pilocarpine-induced Status Epilepticus Rat Models Through the AMPAR GluA1-STIM-Ca2+ Pathway

Abstract

Low-frequency repetitive transcranial magnetic stimulation (rTMS) refers to the stimulation of the brain using repetitive magnetic field pulses at a low frequency (≤ 1 Hz) to reduce seizures. Currently, the mechanism is not well understood. Male Sprague-Dawley rats underwent pilocarpine-induced status epilepticus (SE) and were then stimulated with low-frequency rTMS. An epilepsy cell model was then established by incubating rat hippocampal neurons with Mg²⁺-free extracellular fluids. The effects of the low-frequency rTMS on epileptogenesis and hippocampal neuron injury were evaluated using a video electroencephalogram (vEEG) and Nissl staining, and the expression of AMPAR GluA1 and STIM in the hippocampus and hippocampal neurons was assessed using western blot and immunofluorescence. Additionally, the intracellular Ca²⁺ concentration and reactive oxygen species (ROS) were measured using flow cytometry. Low-frequency rTMS attenuated spontaneous recurrent seizures in rats with epilepsy, with the SE group exhibiting a higher incidence (100%) and frequency (3.00 ± 0.18 times/day) than the SE + 0.3 (50% incidence, 0.06 ± 0.03 times/day), SE + 0.5 (0.20 ± 0.02 times/day) and SE + 1 Hz (1.02 ± 0.05 times/day) groups. Additionally, rTMS reduced the damage and apoptosis of hippocampal pyramidal neurons, increasing their numbers in the CA1 and CA3 regions. Furthermore, AMPAR GluA1 and STIM expression were upregulated in the hippocampus when using rTMS, reversing the downregulation caused by seizures. Immunofluorescence verified the increased fluorescence intensity of AMPAR GluA1 and STIM. Moreover, rTMS inhibited Ca²⁺ overload and ROS in epileptic neuron models. Low-frequency rTMS may exert neuroprotective effects through the AMPAR GluA1-STIM-Ca²⁺ pathway.

Keywords: Epilepsy cell Model; Low-frequency Repetitive Transcranial Magnetic Stimulation; Neuroprotection; Temporal lobe Epilepsy.

Fig. 1

Characteristics of EEG during SRSs in rat models of lithium-pilocarpine-induced status epilepticus, with representative EEG traces from the cortex at interictal periods. (A) The seizure frequency of SRSs in epileptic rats. (B) Latency to the first SRS, indicating that rTMS prolonged the time to SRS onset. (C) The characteristics of interictal EEG in the cortex of rats. Data were expressed as the mean ± SEM. (*** represents p < 0.001, n = 6)

Fig. 2

Effects of the low-frequency rTMS on hippocampal neuronal damage and apoptosis in epileptic rats. (A) The Nissl staining of the whole hippocampus (scale bar = 200 μm, magnification 40x) and its subregions (CA1 and CA3) (scale bar = 50 μm, magnification 200x) in each group. (B) Quantitative analysis of surviving pyramidal neurons in CA1 and CA3 regions of the hippocampus. The data were expressed as the means ± SEM ***p < 0.001, n = 3)

Fig. 3

Effects of low-frequency rTMS on the expression of AMPAR GluA1 and STIM. (A) Representative immunoblots of AMPAR GluA1 and STIM in the hippocampus. (B) The relative intensity of AMPAR GluA1 and STIM in the hippocampus. (C) Representative immunoblots of AMPAR GluA1 and STIM in the hippocampal neurons. (D) The relative intensity of AMPAR GluA1 and STIM in the hippocampal neurons. All values were normalised to the control group and presented as mean ± SEM. (ns, *, **, *** represent p>0.05, p<0.05, p<0.01, p<0.001 respectively, n = 3)

Fig. 4

Effects of low-frequency rTMS on the mean fluorescence intensity of AMPAR GluA1 and STIM. (A) Representative images of mean fluorescence intensity of AMPAR GluA1 and STIM in the hippocampus (scale bar = 50 μm). (B) Quantitative analysis of mean fluorescence intensity of AMPAR GluA1 and STIM in the hippocampus. (C) Quantitative analysis of mean fluorescence intensity of AMPAR GluA1 and STIM in the hippocampal neurons. (D and E) Representative images of mean fluorescence intensity of AMPAR GluA1 and STIM in the hippocampal neurons (scale bar = 100 μm). All results were expressed as the mean ± SEM. (ns, *, **, *** represent p>0.05, p<0.05, p<0.01, p<0.001 respectively, magnification 400x, n = 3)

Fig. 5

Effects of the rTMS in 0.3 Hz on the concentration of Ca²⁺ and ROS in the neuron, model of epilepsy. (A、B) The quantitative analysis of the concentration of Ca²⁺ and ROS in each group. (C) The quantitative analysis of the concentration of Ca²⁺ was represented as the relative fluorescence intensity of Fluo-3 compared with the Control group. (D) The quantitative analysis of ROS was represented as the relative fluorescence intensity of DCF compared with the control group. All values were presented as mean ± SEM (ns, **,*** represent p>0.05, p<0.01, p<0.001 respectively, n = 3)