A method for constructing an epileptic animal model based on temporal coherent electrical stimulation

Abstract

The present invention relates to the field of epilepsy, and specifically to a method for constructing an epilepsy animal model based on time-coherent electrical stimulation, comprising using time-coherent electrical stimulation technology, applying an alternating current of a first frequency to a first time-coherent electrical stimulation electrode pair, applying an alternating current of a second frequency to a second time-coherent electrical stimulation electrode pair, and stimulating the experimental animal to construct an epilepsy animal model starting from a deep brain region. The present invention can effectively and non-invasively construct an epilepsy animal model with small individual differences, comparable epileptic seizure severity, and more consistent with the epilepsy development of actual epilepsy patients, and is particularly suitable for research on the occurrence and development of epilepsy, pathological mechanisms, and early intervention.

Description

Technical Field

[0001] The present invention relates to the field of epilepsy, and in particular to a method for constructing an epileptic animal model based on temporal coherence electrical stimulation. Background Art

[0002] Epilepsy is a serious chronic neurological disease that affects about 1% of the world's population. Currently, the mechanism of epilepsy is still unclear, and about one-third of epilepsy patients are ineffective against existing anti-epileptic drugs.

[0003] In order to explore the neurobiological mechanisms behind epilepsy, determine the efficacy of drugs in treating specific types of epilepsy and preventing the development of epilepsy, and efficiently develop and screen anti-epileptic drugs, it is crucial to construct a suitable animal model of epilepsy. At present, the methods for constructing animal models of epilepsy mainly include chemical drugs, physical stimulation, gene knockout or overexpression. Among them, the animal model of epilepsy constructed by chemical drugs induces animal epileptic seizures through neuroexcitatory drugs (such as pentylenetetrazol). Although it is relatively simple and non-invasive, the constructed animal model of epilepsy can only imitate human temporal lobe epilepsy and mesial temporal lobe epilepsy, and cannot imitate the clinical characteristics and pathological changes of other types of epilepsy. In addition, the modeling mechanism is complex and it is impossible to rule out the epileptogenic effects of drugs in the whole body or brain. Since gene knockout or overexpression may affect other aspects besides epileptic seizures, the animal model of epilepsy that purposefully knocks out or overexpresses the gene to be studied has the problem of difficult to predict potential phenotypes, is not yet mature and difficult to promote. Physical stimulation methods, including electrical stimulation, require electrodes to be implanted directly into specific areas of the animal brain to induce epileptic-like electrical activity in the brain tissue there. For example, the document "Orientation of Temporal Interference for Non-invasive Deep Brain Stimulation in Epilepsy" uses temporal coherent electrical stimulation technology to invasively implant subdural electrodes into experimental animals to induce epileptiform electrical activity. On the one hand, the method reported in the document is still invasive, and there are still problems such as severe damage to brain tissue, easy loss or loss of function of the implant, infection, bleeding and other complications. On the other hand, the method reported in the document only involves a one-time induction of epileptiform electrical activity in experimental animals, and cannot construct a stable animal model of epilepsy.

[0004] Chinese patent application CN117679672A discloses a method for constructing an epilepsy animal model based on ultrasonic brain stimulation. Although it can construct an epilepsy animal model in a non-invasive way, its potential mechanism is to cause abnormal discharges of neurons by causing damage to the brain area actually stimulated, which is inconsistent with the progressive development of epilepsy in actual epilepsy patients to a certain extent. Summary of the invention

[0005] The present invention provides a method for constructing an epileptic animal model based on temporal coherence electrical stimulation, which is characterized by comprising the following steps:

[0006] S101 Determine the placement of the temporal coherence electrical stimulation electrode on the scalp of the experimental animal according to the deep brain area to be stimulated;

[0007] In some embodiments, the experimental animal comprises a rat or a mouse.

[0008] In some embodiments, the deep brain regions include one or more of the hippocampus, thalamus, and nuclei.

[0009] In some embodiments, the temporally coherent electrical stimulation electrodes include a first temporally coherent electrical stimulation electrode pair and a second temporally coherent electrical stimulation electrode pair.

[0010] S102 Using the time-coherent electrical stimulation technology, apply an alternating current of a first frequency to the first time-coherent electrical stimulation electrode pair, apply an alternating current of a second frequency to the second time-coherent electrical stimulation electrode pair, and perform a first stimulation on the experimental animal.

[0011] In some embodiments, the first frequency comprises 1040 Hz.

[0012] In some embodiments, the second frequency comprises 1000 Hz.

[0013] In some embodiments, the placement position of the temporally coherent electrical stimulation electrode on the scalp of the experimental animal can be determined by a simulation platform.

[0014] In some embodiments, the simulation platform includes COMSOL software.

[0015] In some embodiments, the first stimulation method includes: using a pulse current, with a first current intensity as the starting current intensity, a first amplification current as the step, and a first time as the single stimulation duration to perform a first stimulation on the experimental animal.

[0016] In some embodiments, the pulse width of the pulse current comprises 4-5 ms.

[0017] In some embodiments, the duty cycle of the pulse current comprises 20-30%.

[0018] In some embodiments, the first current intensity comprises 400 - 800 µA.

[0019] In some embodiments, the first boost current comprises 50-100 µA.

[0020] In some embodiments, the first time comprises 20-30s.

[0021] S103 performing a first screening on the experimental animal during the first stimulation period;

[0022] In some embodiments, the first screening method includes: taking the current intensity when the experimental animal first exhibits epileptic seizure behavior as the second current intensity, and performing a first screening of the experimental animals based on the second current intensity, classifying the experimental animals with a second current intensity less than 1400 µA as first experimental animals, classifying the experimental animals with a second current intensity of 1400-2100 µA as second experimental animals, and classifying the experimental animals with a second current intensity greater than 2100 µA as third experimental animals, and incorporating the first experimental animals and the second experimental animals into the subsequent S104 step.

[0023] In some embodiments, the epileptic seizure behavior is grade IV epileptic seizure behavior on the Racine scale.

[0024] S104 performing a second stimulation on the first experimental animal and the second experimental animal;

[0025] In some embodiments, the second stimulation method includes: using a pulse current, using the second current intensity as the current intensity and using a second time as the stimulation duration to perform a second stimulation on the experimental animal.

[0026] In some embodiments, the pulse width of the pulse current comprises 4-5 ms.

[0027] In some embodiments, the duty cycle of the pulse current comprises 20-30%.

[0028] In some embodiments, the second time comprises 1-5 min.

[0029] S105 Perform a second screening on the experimental animals according to the proportion of epileptic seizure time of the first experimental animal and the second experimental animal during the second stimulation period.

[0030] In some embodiments, the second screening method includes: excluding experimental animals without epileptic seizure behavior, first experimental animals with epileptic seizure time accounting for 5%-40%, and second experimental animals with epileptic seizure time accounting for 5%-20%.

[0031] S106 performing the third stimulation of the first cycle on the experimental animals obtained in S105;

[0032] In some embodiments, the first period comprises at least 6 days.

[0033] In some embodiments, the third stimulation method includes using a pulse current, using the second current intensity as the current intensity and a third time as the stimulation duration to perform the third stimulation on the experimental animal.

[0034] In some embodiments, the third time comprises 5-10 min.

[0035] S107 A second cycle of observation is performed on the experimental animal obtained in S106. When the experimental animal exhibits at least three spontaneous epileptic seizures of grade IV or above during the second cycle, the animal model is considered to be successfully constructed, and the epilepsy is focal epilepsy initiated in deep brain regions.

[0036] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0037] Temporal coherence electrical stimulation technology usually requires invasive electrode implantation to stimulate the desired deep brain region. The invasive electrode implantation position determined based on the simulation platform is not completely suitable for non-invasive placement on the scalp of experimental animals, and it is difficult to ensure the stability and homogeneity of the constructed epilepsy animal model. Compared with the prior art, the present invention adopts a lower carrier frequency (i.e., close to the critical frequency of "high frequency" and "low frequency") and a lower difference frequency, and cooperates with a relatively "diffuse" stimulation method. On the basis of the placement position of the temporal coherence electrical stimulation electrode determined by the conventional simulation platform, it can accurately locate the desired brain region for stimulation and ensure that the neurons in the desired deep brain region produce field potentials and epileptiform electrical activities synchronized with the stimulation frequency.

[0038] Due to the individual differences of experimental animals, conventional methods for constructing animal models of epilepsy are difficult to construct animal models with the same degree of epilepsy in large quantities. The present invention effectively constructs an animal model of epilepsy that is more similar to actual clinical epilepsy, has the same degree of epilepsy, and has more controllable neuronal discharges through methods such as the first screening and the second screening. It better simulates epilepsy attacks initiated in deep brain areas, while avoiding damage to the brain areas actually stimulated to a certain extent. It achieves the non-invasive construction of an animal model of epilepsy with small individual differences, the same degree of epilepsy, and more matching the epilepsy development of actual epilepsy patients (specifically, a model of focal epilepsy initiated in deep brain areas and focal secondary generalized epilepsy), which is particularly suitable for research in the fields of occurrence and development of epilepsy, pathological mechanisms, and early intervention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0040] Figure 1 This is the result of the EEG activity of the mouse hippocampus before and after stimulation;

[0041] Figure 2 This is the result of EEG activity before and after stimulation of the frontal lobe of mice;

[0042] Figure 3 This is the result graph of mice showing epileptic seizure behaviors above grade IV;

[0043] Figure 4 Schematic diagram of pulse current. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] Herein, suffixes such as "module", "component" or "unit" used to represent elements are only used to facilitate the description of the present invention, and have no specific meanings by themselves. Therefore, "module", "component" or "unit" can be used mixedly.

[0046] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0047] In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0048] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0049] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0050] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0051] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0053] Embodiment 1

[0054] In this example, 6-8 week old C57BL / 6 mice were selected, and anesthesia was induced using 5% isoflurane / 95% oxygen, and anesthesia was maintained using 3% isoflurane / 97% oxygen. The shaved area should extend laterally from the left ear to the right ear, and from behind the eyes to the rear end of the skull, with a shaved area of ​​at least 1×1.5 cm. The shaved area was disinfected three times continuously with povidone iodine, and then the exposed skin around was disinfected with ethanol. A medial incision was made at the top of the skull from between the eyes to the back of the skull to cut open the skull. The skull was cleaned with hydrogen peroxide and sterile saline (0.9% NaCl), and an electric knife was used to stop bleeding in time during the incision.

[0055] In order to better observe whether the mice produce epileptic electrical activity, this embodiment takes the stimulation of the hippocampus as an example, takes the bregma as the coordinate 0 point in the stereotaxic, and adjusts the parameters of the brain stereotaxic instrument to place the recording electrode. Among them, the upper left x = -3mm, y = 3mm is the frontal electrode placement point; the lower left x = -3mm, y = -3mm is the hippocampal electrode placement point; the upper right x = 3mm, y = 3mm is the reference electrode placement point, and the lower right x = 3mm, y = -3mm is the ground electrode placement point. Use a high-speed dental drill with a 0.5 mm spherical burr drill bit to establish each implantation site in the skull. Use a 0.7 mm spherical burr drill bit to chamfer the outer edge of each hole to facilitate screw insertion. Insert the EEG screws into the holes and screw them in about 4-5 turns with a flat-blade manual screwdriver to obtain the desired depth. Use glass ionomer cement to cover the motor screw site and the surrounding skin, and use ultraviolet light to accelerate curing. Solder the ends of the wires coming from the EEG leads to the contacts on the plastic connector, connect the electrode amplifier, trim the excess, and secure the amplifier.

[0056] In this embodiment, the temporal coherent stimulation electrodes (4) are placed on the mouse scalp surface at the following positions: -1.94mm behind the anterior fontanelle, 0.5mm left of the midline, 0.5mm right of the midline, 3.7mm right of the midline, and 4.3mm right of the midline. The placement method can be:

[0057] Cut the ECG electrode patch into a suitable size and stick it on the mouse scalp surface, and connect the electrode to the stimulator using a connecting wire; or

[0058] Use dental cement to stick a polyimide tube of appropriate diameter to the surface of the mouse scalp, fill it with saline, immerse one end of the silver wire in the saline, and connect the other end to the stimulator; or

[0059] Use dental cement to stick a stainless steel screw to the surface of the mouse scalp, wrap one end of the silver wire around it and weld it to the screw, and connect the other end to the stimulator; or

[0060] The single-pin female header was fixed to the mouse skin surface with dental cement, and the female header and the stimulator were connected using a pin header and a jumper wire.

[0061] After 7 days of postoperative recovery, the mice were connected to the temporal coherent electrical stimulation generator and stimulated using the following parameters (i.e., the temporal coherent electrical stimulation electrodes emitted alternating currents of T1 and T2, respectively):

[0062] The first group: T1=1430Hz, T2=1300Hz, difference frequency: 130Hz;

[0063] The second group: T1=1040Hz, T2=1000Hz, difference frequency: 40Hz;

[0064] The third group: T1=2001Hz, T2=2000Hz, difference frequency: 1Hz.

[0065] In this embodiment, a pulse current is selected to stimulate the mouse, with a pulse width of 5 ms, a pulse interval of 25 ms, and a duty cycle of 20%. The waveform of the pulse current is a sine wave. The current is set to be driven in reverse for relative isolation. The current intensity is 2000 µA. The stimulation duration is 25 s.

[0066] The mice were video monitored each time they were stimulated to analyze their behavioral responses. The internationally accepted Racine grading standard was used as the standard: ① Grade 0, no response; ② Grade I, facial clonus, including blinking, whisker twitching, rhythmic chewing, etc.; ③ Grade II: Grade I plus rhythmic nodding; ④ Grade III: Grade II plus forelimb myoclonus, but without hindlimb upright position; ⑤ Grade IV: Grade III plus hindlimb upright position; ⑥ Grade V, generalized tonic-clonic seizure.

[0067] The results are as follows Figure 1 and Figure 2 As shown, from the results of recording EEG activity in the hippocampus and frontal lobe of mice before and after stimulation, under the stimulation of the second group of parameters, clustered high-amplitude spikes appeared in the stimulated hippocampus, while the frontal lobe delayed occasional low-amplitude single or multiple spikes. However, the stimulation of the first and third groups of parameters could not induce epileptic-like electrical activity in mice, that is, the EEG activity records of the hippocampus and frontal lobe of mice after stimulation were basically the same as before stimulation. From the observation results of the epileptic behavior of mice, mice stimulated by the parameters of the second group showed grade II-V epileptic seizures. The above results show that the parameters of the second group can induce epileptic-like waveforms in the EEG activity of mice specifically in the brain region, and can be used to construct an animal model of epilepsy, but the degree of epileptic seizures in mice cannot be controlled at the same level.

[0068] Embodiment 2

[0069] This embodiment uses the following parameters (i.e., so that the time-coherent electrical stimulation electrodes emit alternating currents of T1 and T2 respectively) to stimulate mice: T1=1040Hz, T2=1000Hz, difference frequency: 40Hz. The mice are stimulated using pulsed currents with a pulse width of 5ms, a pulse interval of 25ms, and a duty cycle of 20%. The waveform of the pulsed current is a sine wave. The current is set to be reverse driven for relative isolation. The stimulation amplitude increases by 50µA steps, starting from 50µA, and the stimulation increases until the mouse reaches a grade IV epileptic seizure on the Racine scale.

[0070] The results showed that there were certain differences in the current intensity when mice reached grade IV epileptic seizures on the Racine scale, with an average current intensity of 1800µA. The above results show that even if factors such as species and age are controlled at the same level, there are still large individual differences between mice. If the same current intensity is used for stimulation of all mice, this may further lead to different degrees of epileptic seizures in mice. Therefore, this embodiment uses the current intensity when mice reach grade IV epileptic seizures on the Racine scale as the actual stimulation current intensity, and stimulates for 6 consecutive days (plus the stimulation in this embodiment to obtain the actual stimulation current intensity, a total of 7 days), stimulating once a day, and each stimulation lasts for 10 minutes.

[0071] The results are as follows Figure 3 The results show that after one week of temporal coherent electrical stimulation of this embodiment, more than 70% of the mice had spontaneous grade IV and above epileptic seizures, with varying frequencies, and the frequency of epileptic seizures in most mice can be maintained at about 3-5 times per week. During an epileptic seizure, the EEG shows rapid electrical activity with spikes in the hippocampus, followed by synchronous firing of the hippocampus and cortex.

[0072] Embodiment 3

[0073] The above embodiments have proved that the stimulation method based on time-coherent electrical stimulation provided by the present invention can effectively stimulate mice to produce spontaneous epileptic seizures. In order to achieve the construction of an epilepsy animal model with small individual differences, comparable epileptic seizure severity and more consistent with the epilepsy development of actual epilepsy patients without the use of recording electrodes to monitor the brain electrical activity of mice, this embodiment optimizes the stimulation method based on the results of the above embodiments.

[0074] For the first stimulation of mice, this embodiment uses pre-stimulation (first stimulation) to stimulate mice and screens mice based on the pre-stimulation results. The specific method is as follows. The following parameters (i.e., the time-coherent electrical stimulation electrodes emit alternating currents of T1 and T2 respectively) are used to stimulate mice: T1 = 1040 Hz, T2 = 1000 Hz, difference frequency: 40 Hz. Figure 4As shown, the mouse was stimulated with a pulse current with a pulse width of 5ms, a pulse interval of 25ms, and a duty cycle of 20%. The waveform of the pulse current is a sine wave. The current setting is reverse driven for relative isolation. The mouse was stimulated with a starting current intensity of 400µA and a step of 50 µA until the mouse reached a grade IV epileptic seizure on the Racine scale. The stimulation duration was 30s. This stimulation method can determine the individual stimulation current intensity of the mouse and ensure that the mice have the same degree of epileptic seizures to a certain extent, while gradually activating neurons in deep brain areas (such as the hippocampus).

[0075] According to the individual stimulation current intensity of mice, the mice were divided into three categories: the first category, the individual stimulation current intensity was <1400 µA; the second category, the individual stimulation current intensity was 1400-2100 µA; the third category, the individual stimulation current intensity was >2100 µA (including mice without epileptic seizures), and the third category of mice was excluded.

[0076] The screened mice were stimulated for the second time, and the stimulation method was the same as the first stimulation. The difference was that the current intensity was determined according to the individualized stimulation current intensity, and the stimulation duration was 5 minutes, so as to induce the second epileptic seizure in the mice and calculate the proportion of epileptic seizure time of the first type of mice and the second type of mice. According to the proportion of epileptic seizure time of the mice, mice without epileptic seizure behavior, first type of mice with an epileptic seizure time proportion of 5%-40%, and second type of mice with an epileptic seizure time proportion of 5%-20% were excluded for secondary screening. The secondary screening not only helps to exclude mice that are insensitive to the stimulation method of this embodiment, but also helps to screen out a group of mice that are closer to the actual epileptic seizure of epilepsy patients and have small individual differences by only observing the epileptic seizure behavior, and to a certain extent avoid epileptic seizures induced by unexpected means (such as brain damage, non-target brain areas) to ensure the homogeneity of the constructed epilepsy animal model.

[0077] The mice after the secondary screening were stimulated periodically in the following manner. The mice were stimulated using the following parameters (i.e., so that the time-coherent electrical stimulation electrodes emitted alternating currents of T1 and T2 respectively): T1=1040Hz, T2=1000Hz, difference frequency: 40Hz. The mice were stimulated using pulsed current with a pulse width of 5ms, a pulse interval of 25ms, and a duty cycle of 20%. The waveform of the pulsed current was a sine wave. The current was set to reverse drive for relative isolation. The current intensity was determined according to the individualized stimulation current intensity, with stimulation once a day, each stimulation lasting 10 minutes, for 6 days.

[0078] The results showed that after the above stimulation, more than 95% of the mice had spontaneous grade IV and above epileptic seizures and the frequency of seizures was maintained at about 3-5 times / week, and no mice died. The above experimental results show that the construction method provided by the present invention can accurately stimulate the subcortical brain tissue non-invasively, successfully induce focal epileptic seizures initiated in deep brain areas (such as the hippocampus) and focal secondary generalized seizures, while being able to reduce the influence of individual differences in experimental animals on the modeling results to a certain extent, and can stably construct an epilepsy animal model with a comparable degree of epileptic seizures and epileptic seizure conditions that are highly similar to those of epilepsy patients, with a high modeling success rate and saving experimental animal resources.

[0079] Embodiment 4

[0080] Based on the above experimental results, this embodiment provides a method for constructing an epileptic animal model based on temporal coherence electrical stimulation, which is characterized by comprising the following steps:

[0081] S101 Determine the placement of the temporal coherence electrical stimulation electrode on the scalp of the experimental animal according to the deep brain area to be stimulated;

[0082] In some embodiments, the experimental animal comprises a rat or a mouse.

[0083] In some embodiments, the deep brain regions include one or more of the hippocampus, thalamus, and nuclei.

[0084] In some embodiments, the temporally coherent electrical stimulation electrodes include a first temporally coherent electrical stimulation electrode pair and a second temporally coherent electrical stimulation electrode pair.

[0085] S102 Using the time-coherent electrical stimulation technology, apply an alternating current of a first frequency to the first time-coherent electrical stimulation electrode pair, apply an alternating current of a second frequency to the second time-coherent electrical stimulation electrode pair, and perform a first stimulation on the experimental animal.

[0086] In some embodiments, the first frequency comprises 1040 Hz.

[0087] In some embodiments, the second frequency comprises 1000 Hz.

[0088] In some embodiments, the placement position of the temporally coherent electrical stimulation electrode on the scalp of the experimental animal can be determined by a simulation platform.

[0089] In some embodiments, the simulation platform includes COMSOL software. COMSOL software is a commonly used electric field simulation platform in the field of time-coherent electrical stimulation, which can generate feasible placement position information of time-coherent electrical stimulation electrodes according to the deep brain area desired to be stimulated. However, this embodiment finds that time-coherent electrical stimulation technology usually requires invasive electrode implantation to stimulate the deep brain area desired to be stimulated, and the electrode implantation position that requires invasiveness determined based on the simulation platform is not fully applicable to the non-invasive placement on the scalp of experimental animals in this embodiment. Adjusting the placement position of the time-coherent electrical stimulation electrode based on the placement position information generated by the simulation platform is not only complicated to operate, but also easily causes the problem that the actual stimulated brain area deviates from the deep brain area desired to be stimulated, which is not conducive to the construction of a large number of stable epilepsy animal models.

[0090] Existing technologies (such as the document "Orientation of Temporal Interference for Non-invasive Deep Brain Stimulation in Epilepsy") usually require invasive implantation of temporal coherence electrical stimulation electrodes, and utilize the low-pass filtering characteristics of neurons (i.e., the principle of not responding to high-frequency (>1 kHz) stimulation and only responding to low-frequency signals) to generate a low-frequency envelope stimulation area with a higher carrier frequency and a higher difference frequency to stimulate deep brain areas (such as the hippocampus). This embodiment found that even with the help of a simulation platform, it is difficult for this conventional stimulation method to effectively stimulate the desired deep brain area through temporal coherence electrical stimulation electrodes placed on the surface of the scalp of experimental animals. This embodiment, by adopting a lower carrier frequency (i.e., close to the critical frequency of "high frequency" and "low frequency") and a lower difference frequency, can accurately locate the desired brain area to stimulate neurons therein based on the placement position of the temporal coherence electrical stimulation electrodes determined by the conventional simulation platform.

[0091] In some embodiments, the first stimulation method includes: using a pulse current, with a first current intensity as the starting current intensity, a first amplification current as the step, and a first time as the single stimulation duration to perform a first stimulation on the experimental animal.

[0092] In some embodiments, the pulse width of the pulse current comprises 4-5 ms.

[0093] In some embodiments, the duty cycle of the pulse current comprises 20-30%.

[0094] Furthermore, the present embodiment found that although the settings of the above-mentioned first frequency and the second frequency can effectively locate the deep brain area desired for stimulation, they are not sufficient to stimulate the neurons therein to form a field potential that is synchronized with the stimulation frequency. Although the "intensive" stimulation of neurons in the prior art can induce the generation of epileptic-like electrical activity in a short period of time (e.g., 10s), the degree of stimulation of neurons is too strong, and repeated stimulation can easily lead to the short-term death of experimental animals, and may even affect neurons in other brain areas and cause brain tissue damage. The present embodiment adopts a relatively "diffuse" stimulation method, which can cooperate with the settings of the first frequency and the second frequency to ensure accurate stimulation of neurons in the deep brain area desired for stimulation to generate field potentials and epileptic-like electrical activities synchronized with the stimulation frequency.

[0095] In some embodiments, the first current intensity comprises 400 - 800 µA.

[0096] In some embodiments, the first boost current comprises 50-100 µA.

[0097] In some embodiments, the first time comprises 20-30s.

[0098] S103 performing a first screening on the experimental animal during the first stimulation period;

[0099] In some embodiments, the first screening method includes: taking the current intensity when the experimental animal first exhibits epileptic seizure behavior as the second current intensity, and performing a first screening of the experimental animals based on the second current intensity, classifying the experimental animals with a second current intensity less than 1400 µA as first experimental animals, classifying the experimental animals with a second current intensity of 1400-2100 µA as second experimental animals, and classifying the experimental animals with a second current intensity greater than 2100 µA as third experimental animals, and incorporating the first experimental animals and the second experimental animals into the subsequent S104 step.

[0100] In some embodiments, the epileptic seizure behavior is grade IV epileptic seizure behavior on the Racine scale.

[0101] In fact, inducing epileptiform electrical activity in experimental animals once does not mean that a stable animal model can be constructed. On the one hand, as mentioned above, although some stimulation methods can induce the generation of epileptiform electrical activity once, repeated stimulation can easily lead to short-term death of experimental animals and even damage to brain tissue, which is inconsistent with the actual situation of epilepsy patients. On the other hand, due to the individual differences of experimental animals, it is difficult for conventional epilepsy animal model construction methods to construct animal models with the same degree of epilepsy in large quantities. The present invention stimulates experimental animals by gradually increasing the current intensity within a set range, and based on the current intensity corresponding to the preset epilepsy behavior evaluation standard (such as Racine scale IV epilepsy), the minimum current intensity (i.e., the second current intensity) that can cause neuronal response is obtained. While accurately regulating the degree of epilepsy in experimental animals, personalized current intensity can be applied to experimental animals to achieve standardized stimulation effects and avoid excessive stimulation of experimental animals, with higher safety and stability.

[0102] Since this embodiment adopts a non-invasive placement of the time-coherent electrical stimulation electrodes on the scalp of the experimental animal, the first screening step can quickly eliminate the experimental animals that are not suitable for the stimulation method of this embodiment and will not cause substantial trauma to them, thereby avoiding the waste of experimental animal resources and ensuring the stability and homogeneity of the animal models constructed in large quantities by the present invention.

[0103] S104 performing a second stimulation on the first experimental animal and the second experimental animal;

[0104] In some embodiments, the second stimulation method includes: using a pulse current, using the second current intensity as the current intensity and using a second time as the stimulation duration to perform a second stimulation on the experimental animal.

[0105] In some embodiments, the pulse width of the pulse current comprises 4-5 ms.

[0106] In some embodiments, the duty cycle of the pulse current comprises 20-30%.

[0107] In some embodiments, the second time comprises 1-5 min.

[0108] S105 Perform a second screening on the experimental animals according to the proportion of epileptic seizure time of the first experimental animal and the second experimental animal during the second stimulation period.

[0109] As used herein, the proportion of epileptic seizure time refers to the ratio of the time when the experimental animal first reaches the preset epileptic behavior evaluation standard (such as grade IV epileptic seizure on the Racine scale) during the second stimulation period to the stimulation duration of the second stimulation (i.e., the second time).

[0110] In some embodiments, the second screening method includes: excluding experimental animals without epileptic seizure behavior, first experimental animals with epileptic seizure time accounting for 5%-40%, and second experimental animals with epileptic seizure time accounting for 5%-20%.

[0111] It should be noted that in order to achieve the construction of an epilepsy animal model with small individual differences, equivalent epileptic seizure severity and more consistent with the development of epilepsy in actual epilepsy patients without the aid of recording electrodes to monitor the EEG activity of mice, this embodiment creatively proposes "percentage of epileptic seizure time" as a screening indicator based on the second screening step, so that a group of mice that are closer to the actual epileptic seizures of epilepsy patients and have small individual differences can be screened out by only observing the epileptic seizure behavior, and to a certain extent, epileptic seizures induced by unexpected means (such as brain damage, non-target brain areas) are avoided to ensure the homogeneity of the constructed epilepsy animal model. Based on this, this embodiment can effectively construct an epilepsy animal model that is more similar to actual clinical epileptic seizures, has equivalent epileptic seizure severity, and has more controllable neuronal discharges, so as to better simulate epileptic seizures initiated in deep brain areas.

[0112] S106 performing the third stimulation of the first cycle on the experimental animals obtained in S105;

[0113] In some embodiments, the first period comprises at least 6 days.

[0114] In some embodiments, the third stimulation method includes using a pulse current, using the second current intensity as the current intensity and a third time as the stimulation duration to perform the third stimulation on the experimental animal.

[0115] In some embodiments, the third time comprises 5-10 min.

[0116] S107 A second cycle of observation is performed on the experimental animal obtained in S106. When the experimental animal exhibits at least three spontaneous epileptic seizures of grade IV or above during the second cycle, the animal model is considered to be successfully constructed, and the epilepsy is focal epilepsy initiated in deep brain regions.

[0117] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above-mentioned embodiment methods can be implemented by means of software plus a necessary general hardware platform, and of course by hardware, but in many cases the former is a better implementation method. Based on such an understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product, which is stored in a storage medium (such as ROM / RAM, a magnetic disk, or an optical disk), and includes a number of instructions for a computer terminal (which can be a mobile phone, a computer, a server, or a network device, etc.) to execute the methods described in each embodiment of the present invention.

[0118] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A method for constructing an epileptic animal model based on temporal coherent electrical stimulation, characterized in that: The following steps are involved: S101, according to the deep brain area to be stimulated, determining the placement position of the temporal coherence electrical stimulation electrodes on the scalp of the experimental animal; wherein the temporal coherence electrical stimulation electrodes include a first temporal coherence electrical stimulation electrode pair and a second temporal coherence electrical stimulation electrode pair; S102 Using a time-coherent electrical stimulation technique, applying an alternating current of a first frequency to the first time-coherent electrical stimulation electrode pair, and applying an alternating current of a second frequency to the second time-coherent electrical stimulation electrode pair, to perform a first stimulation on the experimental animal; wherein the first frequency comprises 1040 Hz, and the second frequency comprises 1000 Hz; the first stimulation method comprises: using a pulse current, using a first current intensity as a starting current intensity, using a first amplification current as a step, and using a first time as a single stimulation duration to perform a first stimulation on the experimental animal, wherein the pulse width of the pulse current comprises 4-5 ms, and the duty cycle comprises 20-30%; S103 During the first stimulation, a first screening is performed on the experimental animals; the first screening method includes: taking the current intensity when the experimental animals first exhibit epileptic seizure behavior as the second current intensity, and performing a first screening on the experimental animals according to the second current intensity, classifying the experimental animals with a second current intensity less than 1400 µA as first experimental animals, classifying the experimental animals with a second current intensity of 1400-2100 µA as second experimental animals, and classifying the experimental animals with a second current intensity greater than 2100 µA as third experimental animals, and including the first experimental animals and the second experimental animals in the subsequent S104; the epileptic seizure behavior in S103 is a grade IV epileptic seizure behavior of the Racine scale; S104 performing a second stimulation on the first experimental animal and the second experimental animal, wherein the second stimulation method comprises using a pulse current, using the second current intensity as the current intensity and using a second time as the stimulation duration to perform the second stimulation on the experimental animals, wherein the pulse width of the pulse current comprises 4-5 ms, and the duty cycle comprises 20-30%; S105, performing a second screening on the experimental animals according to the proportion of epileptic seizure time of the first experimental animal and the second experimental animal during the second stimulation period; the second screening method comprises: excluding experimental animals without epileptic seizure behavior, first experimental animals with epileptic seizure time accounting for 5%-40%, and second experimental animals with epileptic seizure time accounting for 5%-20%; S106 performing the third stimulation of the first cycle on the experimental animals obtained in S105; S107 A second cycle of observation is performed on the experimental animal obtained in S106. When the experimental animal exhibits at least three spontaneous epileptic seizures of grade IV or above during the second cycle, the animal model is considered to be successfully constructed, and the epilepsy is focal epilepsy initiated in deep brain regions.

2. The method according to claim 1, characterized in that The first current intensity includes 400-800 µA, and the first amplification current includes 50-100 µA.

3. The method according to claim 1, characterized in that The first cycle comprises at least 6 days.

4. The method according to claim 1, characterized in that The second cycle comprises at least 7 days.

5. The method according to claim 1, characterized in that The deep brain regions include one or more of the hippocampus, thalamus and nuclei.

6. The method according to claim 1, wherein: The second time period includes 1-5 min.

7. The method according to claim 1, characterized in that The third stimulation method includes using a pulse current, using the second current intensity as the current intensity and a third time as the stimulation duration to perform the third stimulation on the experimental animal, wherein the third time includes 5-10 minutes.

8. The method according to claim 1, characterized in that The first time comprises 20-30s.

9. The method according to claim 1, characterized in that The placement position of the temporally coherent electrical stimulation electrode on the scalp of the experimental animal is determined by a simulation platform, which includes COMSOL software.

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