Study In The Antiepileptic Effect And Mechanisms Of Low-Frequency Repetitive Transcranial Magnetic Stimulation

Background:Epilepsy is the second largest disease in neurological disorders. Its prevalence rate is up to 5‰. Of these patients, about 20-40% are unresponsive to currently used drugs, which is termed as refractory epilepsy. Currently, the therapeutic strategies for refractory epilepsy include combinations of antiepileptic drugs, vagus nerve stimulation, deep brain stimulation, responsive cortical stimulation and surgery.However, several drawbacks such as poor efficacy, side effects, invasive procedures , demanding medical equipment and high cost exist in these strategies and hinder their wide use in clinical practice. Therefore, epileptologists never stop to seek for a non-drug, non-invasive method to treat refractory epilepsy. If this comes true, it will open up a promising road for therepay and insight into the epileptogenesis.As a new electrophysiological technique, transcranial magnetic stimulation ( TMS ) has many advantages , such as painless, non-invasive, easy to operate. This merits have made TMS be widely applied in neurology and psychiatry. The good therapeutic effects especifically in patients with depression and parkinsonism have already presaged a brilliant future for TMS. As far as epilepsy is concerned, several preliminary studies experimentally and clinically have shown encouraging results:TMS has antiepileptic effect. However, these results are not totally consistent. The disparity probably manily resulted from different parameters adminstered during rTMS. In addition, the underlying mechanism of TMS on epilepsy has not been established. With regard to this status quo, we assume that:1,rTMS is effective in epilepsy and there are optimal parameters (e.g. frequency and intensity). 2,The mechanism of antiepileptic effect of rTMS may involve in several key links close to epileptogenesis, such as imbalance of cortical excitability and inhibition.Objective:①To observe the effects of different frequency and intensity rTMS pretreatment on the epileptogenity of lithium- pilocarpine in rats, and try to find the optimal frequency and intensity which have the best antilepileptic effects.②To investigate the dynamic expressions of Kca1.1,Na_v1.6,NMDAR1,GAD65in the CA3 region of hippocampus whin 6 weeks after the end of 2-week daily rTMS pretreatment, which delivered with the optimal frequency and intensity, and explore the possible mechanisms of rTMS on epilepsy.③To evaluate the effect and safety of rTMS on patients with refractory focal epilepsy, whose epileptogenic zones are well demarcated, and to explore the effects of rTMS on cortical excitability and cerebral blood perfusion.Materials and methods:①Except for frequency, all other stimulus parameters were established. The SD rats were treated with rTMS trains at one of five frequencies: 0, 0.3, 0.5, 0.8 or 1.0Hz for 14 consecutive days , followed by an intraperitoneal injection of lithium chloride and pilocarpine to make acute epilepsy mode. Subsequently, the latencies and severity of seizures were determined and compared within different frequency groups, hoping to evaluate the effect of rTMS frequency on seizures and find the optimum stimulus frequency.②Except for stimulus intensity, all other stimulus parameters were established. The SD rats were treated with rTMS trains at one of five stimulator maximum outputs: 0%, 10%, 20%, 30% or 40% for 14 consecutive days , followed by an intraperitoneal injection of lithium chloride and pilocarpine to establish acute epilepsy mode. Subsequently, the latencies and severity of seizures were observed and compared within different intensity groups, hoping to evaluate the effect of rTMS stimulus intensity on seizures and select the optimum stimulus intensity.③The SD rats were administered with already determined optimum frequency and intensity rTMS for 14 consecutive days. After finished the rTMS protocol, the animals were killed at 6h, 24h, 1w, 3w and 6w respectively, and the expressions of Kca1.1,Na_v1.6,NMDAR1,GAD65 in the CA3 region of hippocampus were examined by immunohistochemistry.④4 patients with intractable focal epilepsy, whose epileptogenic zones are well demarcated, were enrolled. All patients were adminstered with rTMS treatment for 10 consecutive days. The seizure frequency, the number of interictal spikes, the cerebral blood flow, and the cortical excitability were determined before and after rTMS by keeping diary, EEG, SPECT and RMT , respectively.Result:①0.3-1.0Hz rTMS dramatically prolonged seizure latencies and also reduced seizure severity to some extent. Compared with the sham group (0Hz), all other group(0.3-1.0Hz) had significant prolongation in seizure latencies(P<0.05 or P<0.01), but for seizure severity, only 0.5Hz and 0.8Hz group had much lighter seizures, with seizure severity scores significantly lower than those in the other groups (P<0.05). Compared among frequency groups, the prologation of seizure latencies and the reduction of seizure severity in the 0.5Hz and 0.8Hz group were more pronounced than in the 0.3Hz and 1. 0 Hz group , and the differences reached statistically significant level (P<0.05).②Compared with the sham group (0%), only groups of 30% and 40% stimulator maximum output had significant prolongation in seizure latencies(p<0.01), and only group of 40% stimulator maximum output had significantly reduced seizure severity(P<0.01).③The expressions of Kca1.1,Na_v1.6,NMDAR1,GAD65 protein could be observed in all regions of hippocampus in all groups of rats. In CA3, positive neurons were densely distributed in the stratum pyramid, but scattered in the molecular layer. Positive cells characterized as brown in the cytoplasm and membrane, but no staining in the nucleus.---The Kca1.1-positive neuron density was dramatically increased in the stratum pyramid of CA3 at 6h after rTMS, reached the peak at 24h, then took on a decline tendency. Compared with the sham group, the Kca1.1- positive neuron densities were significantly increased for at least 3w after rTMS(P<0.01). The nucleus/cytoplasm ratio of the Kca1.1-positive neuron was decreased significantly at 6h after rTMS, reached the lowest at 1w, then demonstrated a tendency to increase. Compared with the sham group, The decreased nucleus/cytoplasm ratios had significant differences in the whole period of 6w after rTMS(P<0.01). --- The Na_v1.6 -positive neuron density in the hippocampal CA3 pyramid layer,compared with the sham group, had no significant difference at 6h,24h,1w,3w,6w following rTMS protocol. The nucleus/cytoplasm ratio of the Na_v1.6 -positive neuron , compared with the sham group, was transiently increased only at 6h point after rTMS(P<0.05),no significant changes at the other time points--- NMDAR1-positive neuron density in the hippocampal CA3 pyramid layer, compared with the sham group, was transiently decreased at 6h after rTMS protocol(P<0.01), but this change was not obvious at other time points. The nucleus/cytoplasm ratios had no significant difference at all time points of the whole 6-week observation compared with the sham group(P>0.05).--- The GAD65- positive neuron density in the stratum pyramid of hippocampus CA3, Compared with the sham group, was obviously elevated at 6h following rTMS(P<0.05), went up to the peak at 24h and maintained at this level for1w(P<0.01), then declined gradually. Nevertheless, the increased GAD65- positive neuron density kept at a statistically different level even after the end of rTMS protocol for 3w . The nucleus/cytoplasm ratio demonstrated a tendency to decrease at 6h and reached the significant level at 1w(P<0.05)after rTMS protocol, then rose gradually.④The seizure frequency in 4 patients with refractory focal epilepsy decreased from 19 (3-month basal period) to 1 (3-month follow-up period after rTMS) after 10 consecutive days'rTMS treatment, that is, 3 patients were seizure free , 1 patient had one seizure in the 3-month follow-up period after rTMS. EEG analysis displayed one patient had less interictal spikes post- rTMS than pre-rTMS: it decreased from 16 to 12, a 20% reduction. The other 3 patients had no spikes on EEG before and after rTMS. Sngle photon emission computed tomography (SPECT) semi-quantitative analysis showed the ROI values further decrease in all of the 4 patients after rTMS. The RMT was clearly elevated in all patients after rTMS treatment.Conclusion:①rTMS has definite antiepileptic effect on both experimental rats and epileptic patients, and this effect is stimulus frequency and intensity dependent, with the best choice of 0.5 Hz and 40% stimulator maximum output , respectively(under our experimental conditions).②Low-frequency rTMS can elevate RMT, further decrease the focal cerebral blood flow, and affect the expressions of Kca1.1,Na_v1.6,NMDAR1,GAD65 proteins in the hippocampal CA3 of rats. It is therefore reasonably infered that the antiepileptic mechanism underlying low frequency rTMS is complex: it probably involves several links related to epileptogenesis.③Low-frequency rTMS is hopeful to be a new tool in the treatment of epilepsy, especially refractory epilepsy, and the study of epileptogenic mechanisms.