| Epilepsy, one of the most common neurological disorders, is characterized by epileptic seizures. Different types of anti-epileptic drugs (AEDs) are used to control epileptic seizures. But one third of the patients showed tolerance to these drugs, which is called refractory epilepsy. In temporal lobe epilepsy (TLE), one of the most common epilepsy types, the ratio is even higher. Only a part of patients are suitable for epileptic surgery, and even some patients cannot reach seizure free after surgery, and face the problem of cognitive impairment. Therefore, it is urgent to find new treatment approach for refractory TLE. Low frequency stimulation (LFS) turns out to be an alternative strategy for neurological disorders especially epilepsy. Our previous reports showed that LFS at the foci or other regions can inhibit the epileptogenesis and seizures of TLE. But when applying LFS for treating refractory TLE, some difficulties still exists:first, the mechanisms underlying refractory TLE are complex (different from normal TLE), and most previous reports about LFS focused on normal TLE, it’s still urgent to define the possible mechanism underlying refractory TLE and to acess the effect of LFS on it. On the other hand, our previous report shows that only delivering LFS immediately or in a short time after seizure onset can be effective. Delivering LFS after the end of seizures may even aggravate the seizures. Due to that clinical epileptic seizures are sudden and unpredictable. Predicting TLE seizure onset becomes very important for controlling seizures.At first, we try to define the possible mechanisms underlying refractory TLE. Now the mechanisms underlying genesis of refractory TLE still remain unclear. Traditional viewpoint thinks that increased level of drug-transport protein may decrease the drug concentration intraparenchymally. But controversy exists. Now the abnormal effects of drugs on neural activity in some regions are thought to be linked with drug resistant in epilepsy. But the role of temporal lobe regions in this process needs to be further clarified. The subiculum is the major gating region in hippocampus (the most common foci in TLE), and is the origin of epileptic discharge in TLE patient. The subicular excitatory neurons play an important role in the maintenance and spread of epileptic excitatbility. Thus we tried to analyze the relationship between neuron activity in subiculum and drug resistance in a refractory TLE model. And found that in drug resistant animals, AEDs could not inhibit the firing of subicular excitatory neurons as usual. In addition, effects of AEDs could be changed by modulating subicular excitatory neurons with optogenetics. Our findings provide new clew in investigating the mechanism underlying genesis of refractory TLE.Sencondly, due to the fact that optogenetics remains difficulty in clinical translation. Other treatments need to be investigated for treating refractory TLE. LFS is considered as a potential strategy for neurological disorders, and can modulate the neural activity in target region. So combined with the findings in the first part, we further investigated the effect of LFS on subiculum for two refractory TLE animal models. And we found that LFS on subiculum could not only inhibit the seizures, but also reverse the drug-resistant state of refractory TLE. These suggest that LFS at subiculum might be a new strategy for refractory TLE.At last, therapeutic "time window" exists in the treatment of LFS for epilepsy. The optimum stimulating time is correlated with seizure onset time. Due to the fact that clinical epileptic seizures are sudden and unpredictable. Thus we try to find an approach for predicting seizures. So far, predicting seizures are performed by analyzing the preictal EEG retrospectively to find the specific changes preictally. The exist seizure prediction methods based on EEG has usually been performed using a complex mathematical algorithm, or a rule-based method that requires the collection of a large database from a single patient for individual analysis. Lacking reliable biomarkers makes it difficult to predict seizures. Accumulating evidence has shown that high frequency oscillations (HFOs) are closely associated with seizure onset zones, which suggest they might be connected with seizure process. But pre-ictal changes of HFOs are not fully understood and need further investigation. So in the first part, we recorded and analyzed the preictal changes of HFOs in TLE animal models and patients. And found that rate of ripple but not fast ripple decrease from 2 min to 1 min preictally in both mTLE model and patients. These suggest that ripple rate may be used for seizure prediction in TLE.Part ⅠSubicular pyramidal neurons showed abnormal response to phenytoin in refractory temporal lobe epilepsy.The mechanisms of drug resistant in refractory TLE are complex and unclear. Traditional "abnormal drug transportation" hypothesis remains controversial. Recently, epilepsy is considered as a disease caused by the dysfunction of specific brain regions and their circuits. AEDs can modulate the neural activity directly in these regions to control epileptic seizures. When this function is abnormal, epilepsy terms to be drug resistant. Thus in this part, we built a rat PHT resistant TLE model, and recorded the neuron activity and their response to AEDs in some epileptic brain regions. Surprisingly, PHT could inhibit firing of subicular excitatory neurons in response but not nonresponse group. In addition, this phenomenon was probably subiculum specific, as in hippocampal CA1, CA3, dentate gyrus (DG) and entorhinal cortex (EC), no similar phenomenon was observed. Furthermore, in nonresponse and some variety rats, with the existence of PHT, photoinhibition of subicular excitatory neurons genetically targeted with archaerhodopsin-3 (Arch) can reverse the bad PHT response. In response and some variety rats, with the existence of PHT, photoactivation of subicular excitatory neurons genetically targeted with channelrhodopsin-2 (ChR2) can partly reverse the good PHT response. These results indicate that subicular excitatory neurons’abnormal response to PHT might mediated the genesis of refractory TLE, and subicular pyramidal neuron might be a potential therapeutic target for innervating refractory TLE.Part ⅡLow frequency stimulation can inhibit seizure activity and reverse drug-resistant state in refractory temporal lobe epilepsyIn the first part, we found that subicular excitatory neurons played an important role in the genesis of drug resistance in TLE, and might be a potential therapeutic target. But due to the fact that optogenetics remains difficulty in clinical translation. Other treatments need to be investigated for treating refractory TLE. LFS is considered as a potential strategy for neurological disorders, and can modulate the neural activity in target region. Thus we tried to investigate the effect of LFS subiculum for refractory TLE. In this part, we build rat phenytoin sodium (PHT) resistant and mouse lamotrigine (LTG) resistant refractory TLE models, and effects of LFS on these two models were investigated. We found that LFS could inhibit the incidence of generalized seizures, the average seizure stage and the average generalized seizure duration, and also could extend the average latency in drug-resistant and other groups. Interestingly, we found that after LFS treatment, drug-resistant state in refractory TLE animals could be reversed. These results suggest that LFS on subiculum can inhibit the seizure and reverse the drug-resistant state in refractory TLE model, and indicate that LFS might be a potential treatment for refractory TLE.Part ⅢThe preictal changes of HFOs in refractory temporal lobe epilepsyTherapeutic "time window" exists in the treatment of LFS for epilepsy, which meas only delivering the stimulation immediately or a short period after seizure onset can be effective. Due to the fact that clinical epileptic seizures are sudden and unpredictable. Thus finding an approach for predicting seizures is urgent. So far, predicting seizures are performed by analyzing the preictal EEG retrospectively to find the specific changes preictally. HFOs (80-700 Hz) are promising biomarkers for epileptic focus; however, their characteristic changes during the pre-ictal period remain unclear. Here, the pre-ictal HFOs were recorded and detected by an automated HFOs detection method in the mouse pilocarpine model as well as in patients with mesial temporal lobe epilepsy (mTLE) and neocortical epilepsy. A total of sixteen low-voltage fast (LVF) and fifty-three hypersynchronous-onset (HYP) seizures were recorded in ten mice. The rate of ripple (80-250 Hz) but not fast ripple (250-700 Hz) decreased during 1 min before the onset of LVF and HYP seizures, which was primarily due to the reduction of type Ⅱ (independent of epileptiform discharges) rather than type Ⅰ ripples (superimposed on epileptiform activities). The ripple rate decreased until 30 s before HYP seizure, whereas it increased with a peak at 40 s during the 1 min pre-ictal period of LVF seizures. Furthermore, the "ripple reduction" phenomenon was also observed in all twelve seizures from nine patients with mTLE but not in neocortical epilepsy. Those results indicate that ripples can potentially be used for seizure prediction in mTLE, and the different change of ripples 1 min before LVF and HYP seizures might also be beneficial for the diagnosis of seizure types. |
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