Closed loop stimulation system and device for epilepsy

Abstract

The invention provides a closed-loop stimulation system and equipment for epilepsy. A new closed-loop stimulation system is constructed and verified, the system monitors epileptic seizure signals in real time and sends accurate electrical stimulation in time for intervention, the treatment effect is remarkable, and the treatment effect is not only reflected in control over epileptic seizure time, but also comprises improvement of behavior and cognitive functions and effective regulation and control over inflammatory response in the brain.

Description

Technical Field

[0001] This application relates to the field of medical devices, specifically to closed-loop stimulation systems and devices for controlling epileptic seizures and improving anxiety and memory impairment associated with epilepsy. Background Technology

[0002] Epilepsy is a common chronic neurological disorder with a global prevalence of approximately 1%. Its main characteristic is the sudden abnormal discharge of neurons in the brain, which causes transient brain dysfunction. It is characterized by its suddenness and unpredictability, and is usually accompanied by loss of consciousness and inability to control physical activities, which has a significant impact on the patient's work and life.

[0003] Currently, antiepileptic drugs are the primary treatment for epilepsy. However, approximately 30% of patients do not respond to multiple drug treatments and are diagnosed with drug-resistant epilepsy. For these patients, while traditional surgical resection of the lesion is effective in some cases, this method is highly invasive, irreversible, and associated with complications such as cognitive impairment. It is not suitable for all patients, especially those whose epileptogenic lesions are located in important functional areas (such as language, motor, or visual centers) or are multifocal, as such surgery may carry the risk of permanent cognitive impairment. Therefore, new treatment methods need to be explored for patients for whom surgical resection is ineffective or unsuitable.

[0004] Neuromodulation technology offers new hope for patients with drug-resistant epilepsy. It reduces or alleviates seizures by intervening in the nervous system. Early neuromodulation techniques for epilepsy primarily employed open-loop stimulation, such as vagus nerve stimulation, which continuously stimulates the vagus nerve for a predetermined time to reduce seizure frequency. However, while open-loop stimulation can reduce susceptibility to epilepsy and thus decrease the frequency of seizures, it cannot quickly terminate already initiated seizures. Furthermore, patients using open-loop stimulation may experience physical discomfort such as hoarseness, coughing, and headaches, as well as localized pain, memory impairment, and, to some extent, potential side effects such as inducing depression and suicidal tendencies.

[0005] Therefore, in order to overcome the above problems, it is urgent to develop new neuromodulation therapies. Summary of the Invention

[0006] Through repeated exploration, the inventors of this application have developed a new closed-loop stimulation system and device for controlling epileptic seizures and improving anxiety and memory impairment associated with epilepsy.

[0007] On the one hand, this application provides a closed-loop stimulation system for controlling epileptic seizures and improving epilepsy-related anxiety and memory impairment, comprising:

[0008] The signal acquisition module is configured to acquire EEG signals in real time;

[0009] The signal processing module is configured to perform two-stage filtering on the acquired EEG signals;

[0010] The feature value calculation module is configured to calculate the feature values ​​of the processed EEG signals, including line length, amplitude, and slope, using the following formulas:

[0011]

[0012] In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, and n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz.

[0013] A seizure detection module, configured to determine a seizure threshold and dynamically adjust the threshold to adapt to real-time calculation results; and

[0014] An electrical stimulation output module is configured to apply electrical stimulation to terminate a seizure when the calculated EEG signal characteristic value reaches the seizure threshold.

[0015] On the other hand, this application provides a closed-loop stimulation device for controlling epileptic seizures and improving epilepsy-related anxiety and memory impairment, comprising:

[0016] A signal acquisition device configured to acquire electroencephalogram (EEG) signals in real time;

[0017] A signal processing device configured to perform two-stage filtering on the acquired electroencephalogram (EEG) signals;

[0018] A feature value calculation device is configured to calculate the feature values ​​of the processed EEG signal, including line length, amplitude, and slope, using the following formulas:

[0019]

[0020]

[0021] In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, and n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz.

[0022] A seizure detection device configured to determine a seizure threshold and dynamically adjust the threshold to adapt to real-time calculation results; and

[0023] An electrical stimulation output device is configured to apply electrical stimulation to terminate an epileptic seizure when the calculated electroencephalogram (EEG) signal characteristics reach the seizure threshold.

[0024] Therefore, this application provides a closed-loop stimulation therapy for controlling epileptic seizures and improving anxiety and memory impairment associated with epilepsy. This therapy involves real-time monitoring of seizure signals and timely delivery of precise electrical stimulation for intervention. A closed-loop stimulation system and corresponding device for treating epilepsy have been constructed and validated.

[0025] Furthermore, this application also investigates the effects of the provided closed-loop stimulation system on epilepsy-induced brain inflammation and spatial memory impairment from a behavioral and molecular biological perspective, while simultaneously alleviating epileptic seizures. Specifically, in further exploring changes in the brain's inflammatory response, this application found that closed-loop stimulation treatment can downregulate the expression of NLRP3 inflammasome-related genes and their downstream molecules in the hippocampus of epileptic rats, while reducing the activation state or proliferation of microglia and astrocytes, suggesting that closed-loop stimulation may indirectly inhibit the inflammatory response process in the brain by alleviating epileptic seizures.

[0026] The closed-loop stimulation therapy provided in this application has shown significant therapeutic effects in animal models of epilepsy, not only in controlling the duration of epileptic seizures, but also in improving behavioral and cognitive functions, as well as effectively regulating the inflammatory response in the brain, demonstrating important value in the treatment of epilepsy. Attached Figure Description

[0027] Figure 1 The exemplary operating interface (left) and settings interface (right) of the epilepsy closed-loop stimulation program established in this application are shown.

[0028] Figure 2 This invention demonstrates a closed-loop stimulation therapy used to alleviate epileptic seizures in rats, wherein:

[0029] A shows a representative electroencephalogram (EEG) of a non-SE rat that has been injected with saline intracranially.

[0030] B shows a representative electroencephalogram (EEG) of rats during an acute attack following KA injection, revealing high-frequency paroxysmal discharges.

[0031] C shows a magnified spike-wave EEG on the abscissa of a representative rat during an acute attack after KA injection.

[0032] D shows the effect of a single stimulation during an acute epileptic seizure in a rat. The blue area indicates the time point of electrical stimulation. After a single stimulation, the paroxysmal discharge was suppressed.

[0033] E shows the effect of multiple stimulations during acute epileptic seizures in rats. The blue positions indicate the time points of electrical stimulation. After multiple stimulations, paroxysmal discharges are suppressed and transformed into low-frequency spike discharges.

[0034] F shows the statistical rating of epileptic seizures in the KA group and CLS group after KA injection;

[0035] G shows the average hourly paroxysmal discharge (FS) duration in rats from different treatment groups; the KA group had a significantly longer paroxysmal discharge duration than the CON and CLS groups.

[0036] H shows the average number of spike discharges per hour in rats of different treatment groups. The number of spike discharges in the KA group was significantly higher than that in the CON group and the CLS group.

[0037] Figure 3 This invention demonstrates a closed-loop stimulation therapy used to improve motor function in epileptic rats, wherein:

[0038] A shows a representative trajectory of the CON group rats moving in an open field;

[0039] B shows a representative trajectory of rats in the KA group moving in an open field;

[0040] C shows a representative trajectory of the CLS group rats moving in an open field;

[0041] D shows the distance rats traveled in the open field. The distance traveled by rats in the KA group was significantly lower than that in the CON and CLS groups.

[0042] E shows the rats' movement speed in the open field; the KA group rats' movement speed was significantly lower than that of the CON and CLS groups; and

[0043] F shows the time rats spent in the central region; there were no significant differences among the CON, KA, and CLS groups.

[0044] Figure 4 The closed-loop stimulation therapy established in this application is shown to reduce anxiety levels in epileptic rats, wherein:

[0045] A shows a representative trajectory of the CON group rats in the elevated cross maze;

[0046] B shows a representative trajectory of the KA group rats in the elevated cross maze;

[0047] C shows a representative trajectory of the CLS group rats in the elevated cross maze;

[0048] D shows the distance the rats traveled in the elevated cruciate maze; there was no significant difference among the CON, KA, and CLS groups.

[0049] E showed the time rats spent in the open arm; the open arm time of rats in the KA group was significantly lower than that in the CON and CLS groups; and

[0050] F shows the number of times the rat's open arm entered the rat, with no significant difference among the CON, KA, and CLS groups.

[0051] Figure 5This invention demonstrates the use of closed-loop stimulation therapy to improve spatial working memory in epileptic rats, as established in this application, wherein:

[0052] A shows a representative trajectory of the CON group rats in the Y-maze test;

[0053] B shows a representative trajectory of the KA group rats in the Y maze test;

[0054] C shows a representative trajectory diagram of the CLS group rats in the Y maze test;

[0055] D shows the percentage of times rats entered the new arm in the Y maze test; the percentage in the KA group was significantly lower than that in the CON group.

[0056] E shows the percentage of distance traveled by rats in the new arm during the Y-maze test; the KA group rats had significantly lower new arm travel distances than the CON and CLS groups.

[0057] F shows the percentage of time rats spent in the new arm during the Y-maze test. The percentage of time rats spent in the new arm in the KA group was significantly lower than that in the CON and CLS groups.

[0058] Figure 6 This invention demonstrates the reduction of inflammation levels in the hippocampus of acutely epileptic rats using closed-loop stimulation therapy established in this application, wherein:

[0059] A shows the real-time quantitative PCR results of NLPR3 gene in the CON group, KA group and CLS group after 7 days. The NLPR3 gene expression level in the KA group was significantly higher than that in the other two groups.

[0060] B shows the real-time quantitative PCR results of ASC gene in the CON group, KA group and CLS group after 7 days. The ASC gene expression in the KA group and CLS group was significantly higher than that in the CON group, while there was no significant difference between the KA group and CLS group.

[0061] C shows the real-time quantitative PCR results of Caspase-1 gene in the CON group, KA group and CLS group after 7 days. The Caspase-1 gene expression level in the KA group was significantly higher than that in the other two groups.

[0062] D shows the real-time quantitative PCR results of the NF-κB gene in the CON group, KA group and CLS group after 7 days, with no significant differences among the three groups;

[0063] D shows the expression of the microglial-specific marker gene AIF in the CON, KA, and CLS groups after 7 days, with no significant difference among the three groups; and

[0064] E showed the expression of the astrocyte-specific marker gene GFAP in the CON, KA, and CLS groups after 7 days. The GFAP expression in the KA group was significantly higher than that in the CON group. Detailed Implementation

[0065] This application first uses SD rats as experimental subjects, implanting electrodes and cannulas in the hippocampus and micro-injecting alginate (KA) to establish a status epilepticus (SE) model. Then, the closed-loop stimulation system for treating epilepsy provided in this application is used to monitor and suppress seizures in the SE rats in real time.

[0066] In a specific implementation plan, the closed-loop stimulation system for treating epilepsy provided in this application includes:

[0067] The signal acquisition module is configured to acquire EEG signals in real time;

[0068] The signal processing module is configured to perform two-stage filtering on the acquired EEG signals;

[0069] The feature value calculation module is configured to calculate the feature values ​​of the processed EEG signals, including line length, amplitude, and slope, using the following formulas:

[0070]

[0071] In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz, x represents the function composed of these 50 points, and i and j represent each of these 50 points.

[0072] A seizure detection module is configured to determine a seizure threshold based on typical dense spike-wave discharge characteristic signals of EEG collected and processed during a seizure, typically 10 times the baseline during a seizure-free period; and

[0073] An electrical stimulation output module is configured to apply electrical stimulation to terminate a seizure when the calculated EEG signal characteristic value reaches the seizure threshold.

[0074] In a specific implementation plan, the closed-loop stimulation system for treating epilepsy provided in this application further includes:

[0075] A recording module is configured to record and save monitored epileptic seizure information; preferably, the epileptic seizure information includes: raw field potential recording, seizure channel, seizure time, and stimulation parameters.

[0076] In a specific implementation plan, the two-stage filtering includes: using a fourth-order bandpass filter to limit the signal frequency to 4-80Hz, and then using a second-order notch filter to eliminate 50Hz interference.

[0077] In a specific implementation scheme, the applied electrical stimulation parameters are as follows: current intensity 50-200μA, preferably 100μA; current frequency 60-130Hz, preferably 130Hz; pulse width 100-200μs, preferably 100μs; interval 5-20 seconds, until the epileptic seizure stops.

[0078] Accordingly, this application also provides a closed-loop stimulation device for treating epilepsy, comprising:

[0079] A signal acquisition device configured to acquire electroencephalogram (EEG) signals in real time;

[0080] A signal processing device configured to perform two-stage filtering on the acquired signal;

[0081] A feature value calculation device is configured to calculate feature values ​​of the electroencephalogram (EEG) signal based on the processed signal, including line length, amplitude, and slope, respectively, using the following formulas:

[0082]

[0083] In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, n represents the 50 points of the EEG signal with a length of 50ms analyzed each time when the sampling rate is 1000Hz, x represents the function composed of these 50 points, and i and j represent each of these 50 points.

[0084] A seizure detection device configured to determine a seizure threshold and dynamically adjust the threshold to adapt to real-time calculation results; and

[0085] An electrical stimulation output device is configured to apply electrical stimulation to terminate an epileptic seizure when the calculated electroencephalogram (EEG) signal characteristics reach the seizure threshold.

[0086] In a specific implementation plan, the closed-loop stimulation device for treating epilepsy provided in this application further includes:

[0087] A recording device configured to record and store monitored epileptic seizure information; preferably, the epileptic seizure information includes: raw field potential recording, seizure channel, seizure time, and stimulation parameters.

[0088] In a specific implementation, the signal processing device includes: a fourth-order bandpass filter for limiting the signal frequency to 4-80Hz, and a second-order notch filter for eliminating 50Hz interference.

[0089] In a specific implementation, the applied electrical stimulation parameters are as follows: current intensity 50μA-200μA, preferably 100μA; current frequency 60-130Hz, preferably 130Hz; pulse width 100-200μs, preferably 100μs, with an interval of 5-20 seconds, until the epileptic seizure stops.

[0090] As can be seen, this application successfully constructed and validated a closed-loop stimulation system. This system effectively inhibits epileptic seizures in rats by real-time monitoring of seizure signals and timely delivery of precise electrical stimulation. Furthermore, the closed-loop stimulation provided in this application improved motor function, reduced anxiety levels, and decreased spatial memory impairment in rats with acute epileptic seizures. Further investigation into changes in intracranial inflammatory responses revealed that closed-loop stimulation treatment downregulated the expression of NLRP3 inflammasome-related genes and their downstream molecules in the hippocampus of epileptic rats, while simultaneously reducing the activation or proliferation of microglia and astrocytes, suggesting that closed-loop stimulation may indirectly inhibit intracranial inflammatory processes by alleviating epileptic seizures.

[0091] Example

[0092] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0093] Unless otherwise specified, all reagents used in the examples are commercially available and all technical means used in the examples are conventional means well known to those skilled in the art.

[0094] Example 1: Establishment of a rat model of status epilepticus (SE)

[0095] In this embodiment, SD rats (purchased from Spiford (Beijing) Biotechnology Co., Ltd.) were used as experimental subjects to establish a rat model of status epilepticus (SE) induced by alginate (KA).

[0096] In this embodiment, the surgical equipment and tools were purchased from Shenzhen Ruiwode Life Technology Co., Ltd., the dental cement was purchased from Beijing Laiate Technology Development Co., Ltd., and the KA was purchased from Hebei Pinke Biotechnology Co., Ltd.

[0097] The specific steps are as follows:

[0098] 1. Electrode and cannula implantation surgery

[0099] Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a standard dose of 0.5 mL / 100 g body weight, and then the rats' heads were fixed on a stereotaxic apparatus.

[0100] The rat's head was disinfected three times alternately with alcohol and iodine. The skin and scalp tissue were cut open, and the soft tissues such as the fascia and periosteum on the surface of the skull were separated until the skull and sutures were clearly visible.

[0101] Brain region coordinates were determined according to the Paxinos & Watson Rat Brain Atlas (6th Edition). Using the Bregma point as the zero point, stereotaxic positioning was used to determine the CA3 brain region where the electrode implantation site was located (AP = -3.6 mm, ML = +2.0 mm, DV = -3.0 mm) and the CA3 region where the cannula was implanted (AP = -4.2 mm, ML = +4.0 mm, DV = -3.0 mm), and the sites were marked.

[0102] Subsequently, a window of appropriate size was made at each marked site using a skull drill to open the dura mater. The electrodes and cannulas were then slowly lowered into the target brain region and fixed with denture base resin.

[0103] After the surgery, the rats were placed back in clean cages to rest for at least a week before subsequent experiments were conducted.

[0104] 2. Acute epilepsy modeling

[0105] First, rats were anesthetized with isoflurane gas and fixed to a stereotaxic apparatus. Then, a microsyringe containing KA solution (1 μg / μL) was fixed to the stereotaxic apparatus. The needle was lowered 0.5 mm below the bottom of the drug delivery cannula, and 0.5 μL of KA solution was injected using a microinfusion pump at a rate of 0.1 μL / min. After injection, the injection was left in place for 5 minutes to allow for full absorption before the microsyringe was slowly withdrawn.

[0106] After the rats regained consciousness, they were observed for seizures, and the seizure severity was assessed according to the Racine criteria. Racine criteria: Grade 0: No response; Grade 1: Staring spells; Grade 2: Head nodding and myoclonic jerks; Grade 3: Unilateral forelimb clonus; Grade 4: Bilateral forelimb clonus, standing with falls; Grade 5: Random running and jumping; Grade 6: Tonic-clonic seizures. Rats with seizure severity levels of 4 or 5 were included in subsequent studies.

[0107] Example 2: Construction of a closed-loop stimulation system and treatment of SE rats

[0108] This embodiment constructs a closed-loop stimulation system and uses it to treat SE rats, as detailed below.

[0109] 1. Closed-loop stimulation program development

[0110] The epilepsy closed-loop stimulation system uses a custom Matlab program to continuously read and analyze real-time acquired rat EEG data, perform real-time filtering to remove noise, and calculate EEG characteristic parameters reflecting the epileptic seizure state. When these characteristic values ​​reach the preset epilepsy threshold, the program automatically triggers control commands to send microcurrent pulses to the rat brain via an electrical stimulator, intervening in time and attempting to terminate the epileptic seizure.

[0111] The specific operation steps include: data reading and signal preprocessing, which involves real-time acquisition of EEG signals and filtering through digital signal processing; waveform feature value calculation, which calculates line length, amplitude, and slope parameters based on the typical dense spike discharge characteristics of EEG during epileptic seizures; seizure judgment, which determines the seizure threshold (generally 10 times the baseline when there is no seizure) to adapt to the real-time calculation results; electrical stimulation output, which judges the seizure status based on the feature values ​​and applies electrical stimulation to terminate the seizure; finally, the system records and saves information on each detected seizure, including the seizure channel number, seizure time, and stimulation parameters.

[0112] The implementation method and technical details of this closed-loop stimulation system are described in detail, covering key steps such as data processing, feature calculation, threshold setting, and real-time intervention. This provides a reliable tool and process for effectively monitoring and intervening in epileptic seizures. Figure 1 ).

[0113] The specific operating steps include the following:

[0114] (1) Data reading: Data segments are extracted from the real-time EEG signals of the PlexStim in vivo multichannel electrical stimulation system every 50ms.

[0115] Data acquisition: Field potential signal data is acquired in real time from the Plexon in vivo multichannel EEG recording system. Every 50ms, a 50ms segment of EEG signal is acquired for filtering and feature calculation in each loop of detection.

[0116] (2) Signal preprocessing: Implement two-stage filtering - first, use a 4th-order bandpass filter to limit the signal frequency to 4-80Hz, and then use a 2nd-order notch filter to eliminate 50Hz power line interference.

[0117] (3) Feature value calculation: Extract EEG features (line length LL, amplitude AMP, slope SLP), and use a cumulative averaging strategy to reduce the risk of misjudgment and improve detection accuracy. The calculation formula is as follows:

[0118]

[0119] Among them, line length (LL) represents the total fluctuation of the extracted EEG signal, which fluctuates violently during an epileptic seizure; amplitude (AMP) represents the sum of the amplitudes of each point of the extracted EEG signal, which increases significantly during an epileptic seizure; and slope (SLP) represents the slope of the line between the maximum and minimum values ​​of the extracted EEG signal, which increases significantly during an epileptic seizure.

[0120] (4) Seizure detection: Personalized thresholds are set through offline analysis to ensure that a seizure is detected within 2 seconds after the onset of the seizure.

[0121] (5) Electrical stimulation output: When the characteristic value is met, electrical stimulation (130Hz, 100μs pulse width, 50μA) is immediately performed through PlexStim SDK. If the first attempt is ineffective, the test is repeated after 5 seconds and a second stimulation is considered until the attack stops.

[0122] 2. EEG signal acquisition and closed-loop stimulation

[0123] Connect the recording cable and stimulation cable to the electrode interface on the rat's head, turn on the signal acquisition system and the electrical stimulator, and simultaneously collect EEG signals and record video of the animal's behavior.

[0124] Three animals served as the blank control group (CON group), during which saline was injected into the CA3 area of ​​the hippocampus through a cannula in their brains to ensure the effectiveness of the control conditions; five animals were used in the acute epilepsy induction group (KA group); and eight animals were used in the acute epilepsy closed-loop stimulation group (CLS group) for the study.

[0125] Subsequently, a closed-loop stimulation procedure will be initiated, and closed-loop stimulation therapy will continue for 8 hours. For example... Figure 2 As shown in Figure A, the control group (CON group) rats did not exhibit any abnormal behavior or physiological response after saline injection. Their EEG characteristics were within the normal range, without epilepsy or other abnormal brain activity, such as spike discharges. In the KA-induced group, 5 rats successfully developed acute epileptic seizures, with high-frequency paroxysmal discharges visible on their EEG (Figure B), and the seizure intensity ranged from 3 to 5. Similarly, in the closed-loop stimulation treatment group (CLS group), 8 rats developed acute epileptic seizures after KA injection, with seizure intensity also ranging from 3 to 5 (Figure C). Half an hour after KA injection, closed-loop stimulation treatment was administered to the CLS group rats that developed acute epileptic seizures. Observation of changes in brain activity during epileptic seizures during closed-loop stimulation revealed that when the system detected epileptic seizures and applied single or multiple electrical stimulation interventions, the originally high-frequency discharges gradually transformed into a low-frequency discharge state with lower frequency and amplitude (Figures D and E).

[0126] Statistical analysis showed no significant difference in seizure severity scores between the KA and CLS groups, as shown in F. This indicates that closed-loop stimulation therapy did not reduce the severity of acute seizures. However, it is noteworthy that after quantifying the duration and number of FS (FS defined as paroxysmal discharges lasting at least 10 seconds with a mean amplitude more than three times the baseline and a frequency greater than 2 Hz) in each rat over 8 hours (as shown in G and H), it was found that the number of spikes and FS duration were significantly reduced in the CLS group compared to the KA group. This indicates that closed-loop stimulation therapy effectively reduced the duration and number of seizures, suggesting that closed-loop stimulation treatment helps control seizures and reduce seizure duration.

[0127] Example 3 Behavioral testing of SE rats after treatment

[0128] To assess the recovery, anxiety levels, and spatial memory impairment in epileptic rats 7 days after acute epileptic modeling, the rats underwent open field, elevated cruciate maze, and Y-maze tests. Specific experimental procedures are described below.

[0129] 1. Open field experiment

[0130] The Open Field Test (OFT) is a widely used animal behavior assessment tool, primarily used to investigate the spontaneous activities, exploratory behaviors, and stress levels of rats and mice in new environments.

[0131] The open field test was used to systematically assess the recovery of motor function and changes in anxiety in rats.

[0132] Open field experiments were conducted on day 7 after the onset of SE in rats. One hour before the experiment, the rats were placed in the open field laboratory to acclimatize. At the start of the experiment, the rats were placed in the open field (a 100cm × 100cm × 39cm square dark box), and the test lasted for 10 minutes. After each rat completed the experiment, it was immediately transferred to a new, independent cage for isolation to prevent contact with other unexperimented rats. Simultaneously, the open field was cleaned, disinfected with alcohol, and dried to prevent odors left by the previous rat from interfering with the experiment of the next rat.

[0133] Throughout the experiment, the LABMASE animal behavior trajectory video analysis system was used to monitor and record in detail various behavioral indicators of each rat in real time, such as total movement distance and activity time in the central area, in order to evaluate the rats' motor activity and their potential anxiety level.

[0134] The results are as follows Figure 3As shown, on day 7 after the onset of seizures (SE), compared with the control (CON) group and the closed-loop stimulation (CLS) group, the rats in the KA group showed significantly reduced total distance (D) and speed (E) during the experiment. This indicates that the rats' motor function was significantly impaired and their overall health declined after the onset of SE. The CLS group rats receiving closed-loop stimulation therapy showed significantly improved motor function compared to the KA group, as evidenced by increased distance and speed. This suggests that closed-loop stimulation therapy can effectively reduce seizure activity, help restore motor function impaired by SE, and improve the overall health of rats.

[0135] 2. Elevated Cross Maze Experiment

[0136] The elevated cross maze test is a behavioral experiment designed to systematically assess the anxiety levels of laboratory animals, based on the conflict between their instinct to explore new environments and their natural fear of the open arms. Before the experiment, rats are placed in the elevated cross maze laboratory for one hour to acclimatize. At the start of the experiment, the rat is gently placed in the center of the maze, facing the open arms, and software is simultaneously started to record its exploration activities within the maze. The experiment lasts for 10 minutes. After each rat completes its test, it is immediately transferred to a separate new cage for isolation to prevent interaction with other untested rats. Furthermore, between each experiment, the elevated maze is thoroughly cleaned, disinfected with alcohol, and allowed to dry to eliminate any lingering odors from the previous rat that could affect the behavior of subsequent animals.

[0137] Throughout the experiment, the LABMASE animal behavior trajectory video analysis system was used to capture and record in detail the behavioral characteristics of each rat in real time, such as the total distance traveled, the time spent in the open arm, and the number of times the rat entered the open arm, thereby assessing the rat's level of fear and anxiety.

[0138] The results are as follows Figure 4As shown. Significant changes in anxiety behavior may occur in rats after experiencing seizures (SE). To investigate this phenomenon, on day 7 after SE, after the open field test was completed to ensure behavioral stability, the elevated cruciate maze test was used to assess the rats' anxiety levels. No statistically significant differences were observed in the distance traveled (D) among the CON group, KA group, and rats receiving closed-loop stimulation therapy (CLS group). However, the KA group showed a significantly shorter dwell time in the open arm (E) compared to the CON and CLS groups, indicating a higher level of anxiety. Although there was no significant difference in the number of times rats entered the open arm (F) among the three groups, the KA group rats spent significantly less time in the open arm than the other two groups, revealing that rats exhibited increased anxiety levels after seizures compared to the CON group. Notably, after intervention with closed-loop stimulation therapy, i.e., when the seizure time in the CLS group decreased, the dwell time in the open arm increased compared to the KA group, indicating a certain improvement in anxiety and a reduction in anxiety levels. This indicates that reducing seizure activity through closed-loop stimulation therapy can effectively alleviate anxiety symptoms in rats after SE (seizure-related events).

[0139] 3. Y-maze experiment

[0140] The Y-maze test is a behavioral assessment method widely used in animal spatial working memory research, primarily to evaluate the discriminative learning ability, working memory, and reference memory ability of experimental animals. The maze consists of three identical arms of equal length, forming a Y-shaped layout. The Y-maze test mainly includes two testing protocols: a spontaneous alternation test and a recognition memory test. This study investigated the spatial working memory ability of rats through the recognition memory test. The Y-maze recognition memory test consisted of two phases: an acquisition phase and a recall phase, with a two-hour interval between these phases.

[0141] During the acquisition phase, one of the armways is closed, allowing the animal to freely explore the other two open armways for 3 minutes.

[0142] Two hours later, during the recall phase, all brachial canals were reopened, and the animals were allowed to move freely within each of the three brachial canals for three minutes. During this phase, the exploration time and path length of the animals within each brachial canal were recorded and analyzed. Previous research has shown that individuals with impaired memory typically exhibit significantly reduced activity time and distance in previously unexplored "new arms," ​​a typical manifestation of their memory deficit.

[0143] In this study, the Y-maze was used to test the degree of spatial memory impairment and recovery in rats after SE (Sexually Activated Spatial Memory) and to further explore the potential improvement effects of closed-loop stimulation therapy.

[0144] See results Figure 5Impaired spatial working memory is a prominent feature in rats with temporal lobe epilepsy. To further investigate this phenomenon, a series of behavioral assessments were conducted on day 7 after status epilepticus (SE). After ensuring behavioral stability through open field and elevated cruciate maze tests, a Y-maze test was performed to assess spatial working memory. Compared to the CON group, rats induced by KA showed a significant reduction in the percentage of new arm entry (D), the percentage of new arm movement distance (E), and the percentage of dwell time (F) during new arm exploration, demonstrating a significant impairment of spatial working memory after temporal lobe epilepsy. Notably, rats treated with closed-loop stimulation (CLS) showed a significant improvement in the percentage of movement distance and dwell time during new arm exploration compared to the KA-only treatment group. This result indicates that as CLS therapy effectively reduces the seizure time in rats with status epilepticus, the spatial working memory deficit is significantly improved and restored.

[0145] Example 4 Molecular biological studies of SE rats after treatment

[0146] This embodiment uses real-time quantitative PCR to detect the expression level of NLRP3 inflammasome-related signaling molecule mRNA in rat hippocampus. The specific operation is as follows.

[0147] 1. RNA extraction

[0148] 1) Brain removal: After deep anesthetizing the rat, the head was quickly severed and the complete brain tissue was removed.

[0149] 2) Hippocampal dissection: The brain was cut in half along the midline on an ice table, and the hippocampal structures on both sides were bluntly separated with forceps. The separated hippocampus was immediately immersed in a grinding tube pre-filled with 1 mL of TRIzol solution, and the grinding tube was quickly transferred to liquid nitrogen for rapid freezing treatment, and then transferred to a -80°C freezer for storage.

[0150] 3) Low-temperature grinding: Take out the grinding tube containing the hippocampal tissue from the previous step, thaw it, and put it into a pre-cooled cryogenic grinder for grinding.

[0151] 4) Preliminary RNA extraction and precipitation: Transfer the ground sample liquid to a new centrifuge tube free of RNase enzyme contamination, add chloroform at a volume ratio of 5:1 (tissue fluid to chloroform), mix vigorously, let stand for 5 minutes, and then centrifuge at 12000×g for 15 minutes at 4°C.

[0152] 5) Further purification and precipitation: After centrifugation, aspirate the supernatant into another new centrifuge tube free of RNase enzyme contamination, add an equal volume of isopropanol, mix well, let stand for 15 minutes, and then centrifuge again at 12000×g for 10 minutes at 4℃.

[0153] 6) Ethanol washing: After centrifugation, discard the supernatant and retain the precipitate. Then add 500 μL of 75% ethanol to the precipitate for washing. After mixing, centrifuge at 12000×g for 5 minutes at 4℃ to remove the supernatant.

[0154] 7) Drying and storage: Dry the above precipitate at room temperature until it is transparent or semi-transparent, dissolve it in 50 μL of DEPC water, and store it in a -80℃ refrigerator or use it directly for subsequent reverse transcription reactions.

[0155] 2. Reverse transcription

[0156] 1) Obtain the RNA sample prepared above and measure its concentration using a Nanodrop micro spectrophotometer, and record the measured RNA concentration.

[0157] 2) Next, configure the reaction system according to the reverse transcription reaction system.

[0158] 3) Place the prepared reaction solution into a PCR instrument for reverse transcription. Reaction conditions: 37℃ for 15 min, 85℃ for 5 sec, and 4℃ to finish.

[0159] 4) After reverse transcription is complete, the obtained cDNA product is diluted 10-fold and stored in a -20°C freezer for later use.

[0160] 3. Real-time quantitative PCR

[0161] The prepared PCR reaction mixture was added to a 96-well PCR plate and centrifuged at 2000 rpm for 2 minutes to ensure thorough mixing. The 96-well plate was then placed in a real-time quantitative PCR instrument for amplification. The cycling parameters were set as follows: preheating at 95°C for 1 minute, followed by 40 cycles, each consisting of denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 5 seconds, and extension at 72°C for 15 seconds.

[0162] After the amplification reaction is complete, the experimental data are exported from the instrument, and the relative expression level of the target gene is calculated using the ΔΔCt method (see [link to instrument]). Figure 6 ).

[0163] The results showed that after SE (seizure-related syndrome) occurred in rats, the mRNA expression levels of NLRP3 inflammasome and related signaling molecules were significantly upregulated, accompanied by sustained activation of microglia and an increase in the number of astrocytes. To investigate the effect of closed-loop stimulation on inflammation in the rat brain after SE, this study used real-time quantitative PCR to detect changes in the mRNA expression of NLRP3 inflammasome-related molecules in the rat brain after closed-loop stimulation treatment.

[0164] On day 7 after the onset of seizures in rats, compared with the control group, the expression of NLRP3 (A), ASC (B), and Caspase-1 (C) genes was significantly upregulated in the KA-induced group. While the expression of these genes was also increased in rats receiving closed-loop stimulation (CLS group), the expression levels of NLRP3 and Caspase-1 were lower than in the KA-induced group alone, suggesting that closed-loop stimulation may indirectly alleviate the inflammatory response in the brain by reducing seizures. Although the NF-κB gene (D) did not reach statistical significance, it also showed a similar downregulation trend. In addition, the expression of the microglia-specific marker protein AIF1 (E) and the astrocyte marker GFAP (F) was analyzed. There was no significant difference in AIF1 expression among the groups, but the CON and CLS groups showed a trend of lower expression compared to the KA group. GFAP expression was significantly higher in the KA group than in the CON group, while in the CLS group, although no significant difference was observed between the KA and CON groups, its expression level showed a decreasing trend.

[0165] The application has been described in detail above with general descriptions and specific implementation schemes. Any modifications or improvements made based on this application without departing from its spirit shall fall within the scope of protection claimed in this application.

Claims

1. A closed-loop stimulation device for controlling epileptic seizures and improving epilepsy-related anxiety and memory impairment, comprising: A signal acquisition device configured to acquire electroencephalogram (EEG) signals in real time; A signal processing device configured to perform two-stage filtering on the acquired electroencephalogram (EEG) signals; A feature value calculation device is configured to calculate feature values ​​of the processed electroencephalogram (EEG) signal, including line length, amplitude, and slope, which are calculated using the following formulas: In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, and n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz. A seizure detection device is configured to determine a seizure threshold and dynamically adjust the threshold to adapt to real-time calculation results. as well as An electrical stimulation output device is configured to apply electrical stimulation to terminate an epileptic seizure when the calculated electroencephalogram (EEG) signal characteristics reach the seizure threshold.

2. The device as claimed in claim 1, further comprising: A recording device configured to record information about monitored epileptic seizures; Preferably, the epileptic seizure information includes: original field potential recording, seizure channel, seizure time, and stimulation parameters.

3. The device as claimed in claim 1 or 2, wherein the EEG signal processing device comprises: A fourth-order bandpass filter is used to limit the frequency of EEG signals to 4-80Hz, and a second-order notch filter is used to eliminate 50Hz interference.

4. The device according to any one of claims 1 to 3, wherein the applied electrical stimulation parameters are as follows: current intensity 50-200μA, preferably 100μA; current frequency 60-130Hz, preferably 130Hz; pulse width 100-200μs, preferably 100μs; interval 5-20 seconds, until the epileptic seizure stops.

5. A computer program product for controlling epileptic seizures and improving epilepsy-related anxiety and memory impairment, comprising a computer program that, when executed by a processor, performs the following methods: Real-time acquisition of electroencephalogram (EEG) signals; The acquired EEG signals were filtered in two stages. Based on the processed EEG signals, EEG signal feature values ​​are calculated, including line length, amplitude, and slope, which are calculated using the following formulas: In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, and n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz. When the calculated EEG signal characteristic value reaches the epileptic seizure threshold, electrical stimulation is applied to terminate the seizure; preferably, the parameters of the applied electrical stimulation are as follows: current intensity 50-200 μA, preferably 100 μA; current frequency 60-130 Hz, preferably 130 Hz; pulse width 100-200 μs, preferably 100 μs; interval 5-20 seconds, until the epileptic seizure stops; and optionally, Record monitored epileptic seizure information; preferably, the epileptic seizure information includes: Recordings of the original field potential, the attack pathway, the attack time, and the stimulation parameters.

6. A computer-readable storage medium for controlling epileptic seizures and improving epilepsy-related anxiety and memory impairment, having a computer program stored thereon, the computer program being executed by a processor to perform the following methods: Real-time acquisition of electroencephalogram (EEG) signals; The acquired EEG signals were filtered in two stages. Based on the processed EEG signals, EEG signal feature values ​​are calculated, including line length, amplitude, and slope, which are calculated using the following formulas: In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, and n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz. When the calculated EEG signal characteristic value reaches the epileptic seizure threshold, electrical stimulation is applied to terminate the seizure. Preferably, the parameters of the applied electrical stimulation are as follows: current intensity 50-200 μA, preferably 100 μA; current frequency 60-130 Hz, preferably 130 Hz; pulse width 100-200 μs, preferably 100 μs; interval 5-20 seconds, until the epileptic seizure stops; and optionally, Record monitored epileptic seizure information; preferably, the epileptic seizure information includes: Recordings of the original field potential, the attack pathway, the attack time, and the stimulation parameters.

7. A computer system for controlling epileptic seizures and improving epilepsy-related anxiety and memory impairment, comprising a memory, a processor, and a computer program stored in the memory, the processor executing the computer program to perform the following methods: Real-time acquisition of electroencephalogram (EEG) signals; The acquired EEG signals were filtered in two stages. Based on the processed EEG signals, EEG signal feature values ​​are calculated, including line length, amplitude, and slope, which are calculated using the following formulas: In the formula, LL represents the line length, AMP represents the amplitude, SLP represents the slope, and n represents 50 points of EEG signal with a length of 50ms each time when the sampling rate is 1000Hz. When the calculated EEG signal characteristic value reaches the epileptic seizure threshold, electrical stimulation is applied to terminate the seizure. Preferably, the parameters of the applied electrical stimulation are as follows: current intensity 50-200 μA, preferably 100 μA; current frequency 60-130 Hz, preferably 130 Hz; pulse width 100-200 μs, preferably 100 μs; interval 5-20 seconds, until the epileptic seizure stops; and optionally, Record monitored epileptic seizure information; preferably, the epileptic seizure information includes: Recordings of the original field potential, the attack pathway, the attack time, and the stimulation parameters.

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