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Combination of Matching Responsive Stimulations of Hippocampus and Subiculum for Effective Seizure Suppression in Temporal Lobe Epilepsy

ABSTRACT: Responsive neural stimulation (RNS) is considered a promising neural modulation therapy for refractory epilepsy. Combined stimulation on different targets may hold great promise for improving the efficacy of seizure control since neural activity changed dynamically within associated brain targets in the epileptic network. Three major issues need to be further explored to achieve better efficacy of combined stimulation: (1) which nodes within the epileptogenic network should be chosen as stimulation targets? (2) What stimulus frequency should be delivered to different targets? and (3) Could the efficacy of RNS for seizure control be optimized by combined different stimulation targets together? In our current study, Granger causality (GC) method was applied to analyze epileptogenic networks for finding key targets of RNS. Single target stimulation (100 μA amplitude, 300 μs pulse width, 5s duration, biphasic, charge-balanced) with high frequency (130 Hz, HFS) or low frequency (5 Hz, LFS) was firstly delivered by our lab designed RNS systems to CA3, CA1, subiculum (SUB) of hippocampi, and anterior nucleus of thalamus (ANT). The efficacy of combined stimulation with different groups of frequencies was finally assessed to find out better combined key targets with optimal stimulus frequency. Our results showed that stimulation individually delivered to SUB and CA1 could shorten the average duration of seizures. Different stimulation frequencies impacted the efficacy of seizure control, as HFS delivered to CA1 and LFS delivered to SUB, respectively, were more effective for shortening the average duration of electrographic seizure in Sprague-Dawley rats (n = 3). Moreover, the synchronous stimulation of HFS in CA1 combined with LFS in SUB reduced the duration of discharge significantly in rats (n = 6). The combination of responsive stimulation at different targets may be an inspiration to optimize stimulation therapy for epilepsy.

SUBMITTER: Zhang F

PROVIDER: S-EPMC8426572 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Proteomic analysis Epilepsy was induced using perforant pathway stimulation (PPS) in rats as described (Norwood BA, et al. (2010) Classic hippocampal sclerosis and hippocampal-onset epilepsy produced by a single "cryptic" episode of focal hippocampal excitation in awake rats. J Comp 803 Neurol 518(16):3381-3407). Rats were killed under deep anesthesia (xylazine+ketamine) by transcardial perfusion with ice-cold 0.9 % NaCl solution at the following time points: 1 h, 24 h, 72 h, 10 d and 16 d after PPS (epileptogenesis); within 1 d of first spontaneous seizure (early epilepsy); 1 month after first spontaneous seizure (chronic epilepsy). Control rats were killed on day 17 after surgery (corresponding to day 10 after PPS). Hippocampi were removed and snap frozen at -80°C. Frozen rat hippocampi were resuspended in 200 ul lysis buffer (6M GdmCl, 10mM TCEP, Complete protease inhibitor cocktail, PhosSTOP inhibitor, 100 mM TEAB, benzonase) and dounced 30 times on ice. Samples were boiled for 5 min, sonicated on ice for 2min and centrifuged at 13000 rpm for 3 min. The supernatant was added chloroacetamide to a final concentration of 20 mM followed by incubation in the dark for 30 min at room temperature (RT). Proteins were digested with LysC for 30 min at RT and then diluting 10x— using 100 mM TEAB followed by adding trypsin and incubating overnight at 37°C with shaking. LysC and trypsin were added in a LysC/trypsin:protein ratio of 1:100. Samples were centrifuged at 13000 rpm for 5 min and from the supernatant a volume corresponding to 50 ug peptide was transferred to a new eppendorff tube. Sets of 6 samples were labelled using the TMTsixplex isobaric label reagent. (ThermoFisher Scientific). The labeling reagent was prepared using the manufactures instructions to first equilibrate to RT, then dissolving the labeling reagent in 41 ul anhydrous acetonitrile. The labeling reagent was added to each sample and incubated for 1 hour at RT. The reaction was quenched by incubating with 0.76 M lysine for 15 min. A small amount was taken from each sample, mixed and tested by mass spectrometry for labeling efficiency and mixing ratio. Remaining samples were mixed in a 1:1 ratio. The Stage-tips for the high-pH reverse phase fractionation were prepared by placing first two C18 disks in a p200 pipette tip, then adding a slurry of C18 beads (ReproSil-Pur, 3 um) in methanol and adding one additional C18 disk in the top. The column was activated and equilibrated by washing one time in 150 ul buffer B (50% buffer A, 50% ACN) and then two times in 150 ul buffer A (20mM Ammonium hydroxide, pH 10). The labeled peptide sample was adjusted to pH 10 using buffer A and then loaded onto the activated and equilibrated stage-tip by spinning the sample through the column and collecting 26 the flow-through. The column was washed 2 times with 150 ul buffer 585 A and the washes were collected and combined with the flow-through. Next, peptides were eluted stepwise in 17 fractions by spinning 150 ul of buffer A containing increasing amounts of ACN for each step (3.5%, 4.5%, 6%, 8%, 10%, 10.5%, 11.5%, 13.5%, 15%, 16%, 17.5%, 18.5%, 19.5%, 21%, 27%, 50%, 80%) through the column. After collection, the liquid was evaporated completely in a Concentrator Plus (Eppendorf) and prepared for MS by resuspending in buffer A* (2% ACN, 0.1% TFA). Samples were analyzed using an Easy-nLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source (Proxeon/Thermo Scientific). The peptides were eluted into the mass spectrometer during a chromatographic separation from a fused silica column packed in-house with 3 um C18 beads (Reprosil, Dr. Maisch) using a 120 min gradient of buffer B (80% ACN, 0.5% acetic acid) with a flow rate of 250 nL/min. The Q Exactive HF was operated in positive ion mode with a top 12 data-dependent acquisition method where ions were fragmented by higher-energy collisional dissociation (HCD) using a normalized collisional energy (NCE)/ stepped NCE of 28 and 32. The resolution was set to 60,000 (at 400m/z), with a scan range of 300-1700 m/z and an AGC target of 3e6 for the MS survey. MS/MS was performed at a scan range of 100 - 2000 m/z using a resolution of 30,000 (at 400 m/z), an AGC target of 1e5, an intensity threshold of 1e5 and an isolation window of 1.2 m/z. Further parameters included an exclusion time of 45 sec and a maximum injection time for survey and MS/MS of 15 ms and 45 ms respectively.

Project description:PurposeTo examine the effects of current shunt on rats with temporal lobe epilepsy and neocortex epilepsy.Experimental designA kainic acid (KA)-induced model of temporal lobe seizure and a penicillin-induced model of neocortical partial seizure were used in this study. Rats of each model were randomly allocated into two groups: control and model groups. The model group was further divided into the KA or penicillin group, sham conduction group and conduction group. The current shunt was realized through the implantation of a customized conduction electrode. After surgery, electroencephalogram (EEG) was recorded for two hours for each rat under anesthesia. Subsequently, the rats were video monitored for 72 h to detect the occurrence of behavioral seizures upon awakening. The average number and duration of seizures on EEG and the number of behavioral seizures were measured.ResultsIn KA model, the number of total EEG seizures in conduction group (9.57±2.46) was significantly less than that in sham conduction group (15.13±3.45) (p<0.01). The duration of EEG seizures in conduction group (26.13±7.81 s) was significantly shorter than that in sham conduction group (34.17±7.25 s) (p?=?0.001). A significant reduction of behavioral seizures was observed in the conduction group compared with KA (p?=?0.000) and sham conduction groups (p?=?0.000). In penicillin model, there was a 61% reduction in total EEG seizures in conduction group compared with sham conduction group (p<0.01), and the duration of EEG seizures in conduction group (6.29±2.64 s) was significantly shorter than that in the sham conduction group (12.07±3.81 s) (p?=?0.002). A significant reduction of behavioral seizures was observed in conduction group compared with penicillin (p<0.01) and sham conduction groups (p<0.01).ConclusionCurrent shunt effectively reduces the onset and severity of seizures. Current shunt therapy could be an effective alternative minimally invasive approach for temporal lobe epilepsy and neocortex epilepsy.

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Combination of Matching Responsive Stimulations of Hippocampus and Subiculum for Effective Seizure Suppression in Temporal Lobe Epilepsy

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