Method for constructing animal model of epilepsy syndrome associated with hot infection

Abstract

The invention belongs to the technical field of animal models, and relates to a construction method of a febrile infection associated epileptic syndrome (FIRES) animal model. The method comprises the following steps: firstly, injecting lipopolysaccharide (LPS) into the abdominal cavity of a normal mouse to induce systemic inflammatory reaction, and simulating a fever process in combination with thermal exposure; 4-aminopyridine (4-AP) is injected into the brain of the mouse 24 hours after the injection of lipopolysaccharide (LPS) is finished to induce the occurrence of the epilepsy syndrome related to the hot infection of the normal mouse, and the model of the epilepsy syndrome related to the hot infection of the mouse is constructed. Typical characteristics of FIRES are successfully simulated: precursory fever (2 weeks to 24 hours before attack), an electroencephalogram (EEG) shows beta-delta complex waves and rhythmic sharp waves of extreme delta brushing, and a multi-drug-resistant epileptic state. The invention provides the novel construction method of the mouse fever infection related epileptic syndrome model, the model characteristics are typical, the modeling success rate is high, and the modeling period is short. Wide application prospects are realized.

Description

Technical Field

[0001] This invention relates to the field of research on febrile infection-related epilepsy syndrome or the field of mouse model construction, and more specifically, to a method for constructing an animal model of febrile infection-related epilepsy syndrome. Background Technology

[0002] New-onset refractory status epilepticus (NORSE) is a rare and devastating condition characterized by a new onset of refractory status epilepticus (RSE) without an identifiable acute or active structural, toxic, or metabolic cause. Febrile infection-associated epilepsy syndrome (FIRES) is a subtype of NORSE, requiring a prior febrile infection, with fever starting between 2 weeks and 24 hours prior to the onset of refractory SE, and may or may not be accompanied by fever during the SE seizure. FIRES is a rare and catastrophic epileptic encephalopathy, a severe and complex pediatric neurological disorder that has received widespread attention in recent years. Its pathogenesis remains incompletely understood, posing a significant threat to healthy children. It is characterized by seizures or refractory status epilepticus following an acute febrile illness, subsequently evolving into chronic, multifocal seizures, severely impacting the child's neurological development and quality of life, but without evidence of infectious encephalitis. This condition leads to high mortality, neurological disability, and drug-resistant epilepsy. Furthermore, most FIRES are irreversible, and their sequelae include drug-resistant epilepsy and neuropsychiatric disorders. Therefore, research on febrile infection-related epilepsy syndromes is crucial.

[0003] Currently, there is still a lack of effective drugs and strategies for treating febrile infection-related epilepsy syndrome (FIRES). Animal models are important tools for studying disease pathogenesis and exploring treatment strategies; however, existing mouse models have many shortcomings in simulating the pathological features and pathogenesis of FIRES. Most models cannot accurately reproduce the complex clinical symptoms exhibited by FIRES patients, such as refractory status epilepticus and beta-delta complex waves on EEG resembling extreme delta brushes. These typically begin with prolonged localized rapid activity, followed by the gradual appearance of visible rhythmic spikes or spike-spik complexes. Therefore, establishing a more accurate, stable, successful, and convenient mouse model to simulate FIRES is fundamental and crucial for studying the condition. This has significant practical implications for a deeper understanding of the pathogenesis of FIRES and the development of effective treatments. It will not only contribute to the advancement of basic research but also provide a more reliable theoretical basis and experimental foundation for clinical treatment. Summary of the Invention

[0004] The purpose of this invention is to solve the aforementioned problems in the existing technology and provide a method for constructing an animal model of febrile infection-related epilepsy syndrome. This method can induce febrile infection-related epilepsy syndrome in a short time, is easy to operate, and produces a precise model with a high survival rate.

[0005] To achieve the construction of the aforementioned animal model of febrile infection-related epilepsy syndrome, this invention provides the following technical solution: A method for constructing an animal model of febrile infection-related epilepsy syndrome is provided. The method involves first injecting lipopolysaccharide (LPS) into the peritoneum of normal mice to induce a systemic inflammatory response and then simulating the fever process by heat exposure. Timing is then initiated 24 hours after the LPS injection, and 4-aminopyridine (4-AP) is injected into the brain of the mice to induce febrile infection-related epilepsy syndrome, thus constructing a mouse model of febrile infection-related epilepsy syndrome.

[0006] Preferably, the normal mice described in this invention are mice that do not have the physiological characteristics associated with febrile infection-related epilepsy syndrome.

[0007] More preferably, the normal mouse is at least one of C57BL / 6 mouse, MRL / MPJ mouse, or BALB / c nude mouse, more preferably C57BL / 6 mouse; more preferably male, 4-6 weeks old mouse; more preferably the mouse weight is between 15-20g to reduce interference from other factors. All normal mice in the above technical solution can be used to construct a mouse model of febrile infection-related epilepsy syndrome using the method described in this invention, and no other adverse reactions have been found.

[0008] Preferably, the lipopolysaccharide (LPS) is prepared as a solution with a concentration of 0.1 mg / 10 mL for intraperitoneal injection, and more preferably, phosphate-buffered saline (PBS) is used as the solvent. A more preferred injection dose is 0.1 mL / 10 g mouse body weight. The injection is administered once.

[0009] Preferably, the heat exposure process refers to placing the model mouse in a temperature-controlled oven after intraperitoneal injection of lipopolysaccharide (LPS). More preferably, the temperature is controlled at 37-39°C and the humidity is controlled at 50%RH-55%RH. The preferred heat exposure time is 2 hours. During the heat exposure process, the mouse drinks water and eats normally. The rectal temperature of the mouse is measured every half hour during the heat exposure period. The mouse meets the standard if the rectal temperature reaches 38.5°C or above. After the heat exposure, the mouse is raised in its original environment.

[0010] Preferably, mice exposed to heat are housed in a standard environment; more preferably, the animal housing environment is SPF grade, with the ambient temperature controlled at 22±1℃ and the ambient humidity controlled at 50%-55%RH, with automatic 12-hour light-dark cycle switching. Animals are randomly separated into individual cages and have free access to water and food. All animal experiments comply with the national guidelines for the breeding and use of laboratory animals and the animal ethics regulations of the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine, minimizing the number of animals used and their suffering as much as possible during the experiments.

[0011] Preferably, when injecting 4-aminopyridine, mice are first anesthetized, and intracranial screws are implanted in the mice to connect an EEG recorder. The EEG recorder monitors the changes in the mice's brain waves and records the baseline EEG for 10 minutes for use as a control. Then, 4-aminopyridine is injected into the mice's brain at room temperature.

[0012] Preferably, the 4-aminopyridine (4-AP) is prepared as a 40 mmol / L solution for injection, and more preferably, phosphate-buffered saline (PBS) is used as the solvent. A more preferred injection dose is 1.5 μL of 4-aminopyridine solution. By employing intracerebral injection, the abnormal discharges and inflammatory microenvironment of focal epileptogenic networks can be precisely simulated.

[0013] Preferably, the drug injection site is selected in the hippocampus region of the mouse brain. The high excitability of neurons in the hippocampus and the easy activation of glial cells can simultaneously simulate the dual pathological process of epileptiform discharge and neuroinflammation in FIRES. At the same time, the hippocampus is the core affected brain region of FIRES, and its high neuronal density and easy glial activation can accurately simulate the clinical 'fever-epilepsy-inflammation' interaction mechanism.

[0014] Preferably, the induction of febrile infection-related epilepsy syndrome in normal mice by intracerebral injection of 4-aminopyridine (4-AP) is performed at room temperature. The specific procedure is as follows: mice are anesthetized by intraperitoneal injection of 1% sodium pentobarbital. After anesthesia, the lateral ventricle is located according to the mouse brain atlas, and the Bergma point of the anterior fontanelle is defined as the origin. The coordinates of the drug injection point relative to the Bergma point are: medial and lateral (X) = -2.5 mm, anterior and posterior (Y) = -2.9 mm, and dorsoventral (Z) = -3.0 mm. 1.5 μL of 40 mmol / L 4-aminopyridine solution is slowly injected into the hippocampus of the mouse brain at a rate of 0.15 μL / min over 10 min, and the needle is left in place for 5 min. The injection is performed once.

[0015] More preferably, the specific procedure for intracerebral injection of 4-aminopyridine (4-AP) is as follows: Mice are anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a concentration of 0.1 mL / 10g of mouse body weight. After anesthesia, the mice are fixed on a stereotaxic apparatus and placed on a rectal thermostat throughout the procedure to ensure that the mouse's body temperature does not fall below 32°C. Based on the brain map of C57BL / 6 mice, the Bergma point of the anterior fontanelle is defined as the origin, with the following coordinates relative to the bregma: Upper frontal cortex coordinates relative to the origin: medial-lateral (X) = -1.5 mm, anterior-posterior (Y) = +2.0 mm; Upper cerebellar cortex coordinates relative to the origin: medial-lateral (X) = 0.0 mm, anterior-posterior (Y) = -4.5 mm; Upper right ventral hippocampus coordinates relative to the origin: medial-lateral (X) = -3.0 mm, anterior-posterior (Y) = -2.9 mm. These three points are filled with screws, and an EEG recording device is connected to begin recording the mouse's EEG changes. The drug injection point coordinates relative to the origin were -2.5 mm lateral (X), -2.9 mm anterior and posterior (Y), and -3.0 mm dorsoventrally (Z). 4-Aminopyridine (4-AP) was dissolved in phosphate-buffered saline (PBS) at 40 mmol / L to prepare a 4-aminopyridine solution (4-AP). 1.5 μL of the 4-aminopyridine (4-AP) solution was slowly injected into the hippocampus region of the mouse brain at a rate of 0.15 μL / min over 10 min, with the needle left in place for 5 min. The injection was repeated once.

[0016] Preferably, the criteria for determining whether the constructed mouse model of febrile infection-related epilepsy syndrome is successful include: (1) Judging by intracranial electroencephalogram (EEG), specifically, the changes in the EEG of mice are recorded by an EEG recorder throughout the process, which serves as the standard for judging whether the model is successful; more preferably, the EEG of mice with febrile infection-related epilepsy syndrome shows that the EEG can be observed as a beta-delta complex wave similar to an extreme delta brush, usually starting from a long period of local rapid activity, followed by the gradual appearance of tangible rhythmic sharp waves or sharp wave-sharp wave complex waves; And / or, (2) by judging whether commonly used antiepileptic drugs can alleviate the epilepsy in mice, more preferably, in mice with febrile infection-associated epilepsy syndrome, at least one of multiple antiepileptic drugs such as diazepam (DZP), sodium valproate (VPA), phenytoin sodium (PHT), levetiracetam (LEV), lamotrigine (LTG) could not alleviate the epilepsy in the mice.

[0017] An application of the above-mentioned method for constructing animal models in the field of animal model construction.

[0018] The application of an animal model constructed using the above-mentioned method in screening drugs for the prevention and / or treatment of febrile infection-related epilepsy syndromes.

[0019] A method for screening candidate drugs for the prevention or treatment of febrile infection-related epilepsy syndrome, the method comprising the following steps: The test candidate drug was administered to the mouse model constructed using the above method; Dynamic monitoring of intracranial-induced heat-related infection-associated epilepsy syndrome in a mouse model using electroencephalography (EEG) recording; Candidate drugs for treating febrile infectious epilepsy syndrome were screened by observing changes in mouse EEG before and after treatment.

[0020] This invention employs a method combining intraperitoneal injection 24 hours in advance with oven-assisted heat exposure and intracerebral injection to induce febrile infection-related epilepsy syndrome. No other accompanying diseases have been observed, allowing for a more focused study of febrile infection-related epilepsy syndrome. Furthermore, the construction method provided by this invention can induce febrile infection-related epilepsy syndrome in mice within a short period (one day), with a short cycle and high success rate.

[0021] Compared with existing technologies, the advantages of this invention are: it provides a novel method for constructing a mouse model of febrile infection-related epilepsy syndrome, with a high success rate, typical model characteristics, and a short modeling cycle. It can effectively induce febrile infection-related epilepsy syndrome in normal mice and has broad application prospects. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the modeling process for experimental group mice and control group mice in Example 1 of the present invention; Figure 2 This is a schematic diagram of the coordinate positioning of the mouse connected to the electroencephalogram (EEG) recorder in Embodiment 1 of the present invention; wherein, ①, ②, ③: fixing points, filled with screws and connected to the EEG recording device; ④: drug injection point; Figure 3 This is the electroencephalogram (EEG) of a mouse model of febrile infection-related epilepsy syndrome after inducing epileptic seizures in Example 1 of this invention; Figure 4 These are the electroencephalogram (EEG) recordings of mice in the control group and experimental group (labeled as the model group in the figure) in Example 1 of this invention. It can be seen that the experimental group mice exhibited beta-delta complexes with extreme delta brushes on their EEG. These were followed by visible rhythmic sharp waves or sharp-wave-sharp-wave complexes. In contrast, the control group mice developed multidrug resistance after treatment with antiepileptic drugs. Figure 5 This is a comparative model diagram showing the number of successful mice in the experimental group of Example 1 of the present invention and Comparative Examples 4 and 5. Detailed Implementation

[0023] To better clarify and understand the objectives, technical solutions, and advantages of the present invention, the technical solutions and implementation manners of the present invention will be further described clearly, completely, and in detail through specific embodiments and in conjunction with the accompanying drawings. It should be noted that the described embodiments are implemented on the premise of the technical solutions of the present invention, and detailed implementation manners and specific operation processes are given. However, they are only a part of the embodiments of the present invention, rather than all embodiments. The described specific implementation manners are only for explaining and interpreting the present invention and do not limit the present invention. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0024] In the embodiments of the present invention, the experimental methods and conditions used are conventional methods and conventional conditions unless otherwise specified. The materials, reagents, or instruments used in the embodiments can be obtained from commercial sources or prepared by conventional methods unless otherwise specified. The reaction conditions reflected in the content of the present invention can all achieve the described reactions and obtain products with expected effects. Due to space limitations, some embodiments are listed below to further illustrate the advantages of the technical solutions of the present invention.

[0025] In the examples of the present invention, the mice are C57BL / 6 mice (male mice, 4 - 6 weeks old, with a body weight between 15 - 20 g), and all are purchased from the Experimental Animal Center of Zhejiang Chinese Medical University (Experimental Animal Use License No.: SYXK(Zhe)2013 - 0184). Example 1

[0026] Preparation process of mice in the experimental group (model group): 1. Lipopolysaccharide (LPS) injection and heat exposure 24 hours in advance Weighing: Select 6 C57BL / 6 mice (male, 4 - 6 weeks old, with a body weight between 15 - 20 g), and weigh the body weight of the mice. [[ID=)17]]

[0027] Drug preparation: Lipopolysaccharide (LPS) solution is dissolved in phosphate - buffered saline (PBS) solution at a concentration of 0.1 mg / 10 ml.

[0028] Injection: The experimental group draws the prepared LPS solution with a concentration of 0.1 mg / 10 ml at a rate of 0.1 ml / 10 g of mouse body weight and administers it by intraperitoneal injection.

[0029] Controlled heat exposure: After intraperitoneal injection, the mice were placed in a controlled-temperature oven for heat exposure at 37-39℃ and 50%-55% RH. During the 2-hour heat exposure, the mice drank and ate normally. After 2 hours, the mice were returned to their original environment, which was SPF grade, with a temperature of 22±1℃ and humidity of 50%-55%, and an automatic 12-hour light-dark cycle. They had free access to water and food.

[0030] 2. Screw implantation surgery and EEG recording: Twenty-four hours after injection of lipopolysaccharide (LPS), mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a concentration of 0.1 ml / 10 g of mouse body weight. After anesthesia, the mice were fixed on a stereotaxic instrument. Throughout the surgery, the mice were placed on a rectal thermometer to ensure that their body temperature did not fall below 32°C. The lateral ventricles were located according to the mouse brain atlas. The Bergma point of the anterior fontanelle was defined as the origin. The coordinates of the upper part of the frontal cortex relative to the origin were: medial and lateral (X) = -1.5 mm, anterior and posterior (Y) = +2.0 mm; the coordinates of the upper part of the cerebellar cortex relative to the origin were: medial and lateral (X) = 0.0 mm, anterior and posterior (Y) = -4.5 mm; the coordinates of the upper part of the right ventral hippocampus relative to the origin were: medial and lateral (X) = -3.0 mm, anterior and posterior (Y) = -2.9 mm. These three points were filled with screws and connected to an EEG recording device to start recording the changes in the mouse's brain electrical activity. The injection point coordinates relative to the origin are: X = -2.5mm (inner / outer), Y = -2.9mm (anterior / posterior), Z = -3.0mm (dorsoventral). This point is designated as the drug injection point. (Refer to...) Figure 1 , Figure 1 This is a schematic diagram of the coordinate positioning of the mouse connected to the electroencephalogram (EEG) recorder in Embodiment 1 of the present invention; wherein, ①, ②, ③: fixing points, filled with screws and connected to the EEG recording device; ④: drug injection point.

[0031] 3,4-AP induction Ten minutes after recording the baseline of the electroencephalogram (EEG) of the model mice, 4-aminopyridine (4-AP) in phosphate-buffered saline (PBS) solution at a concentration of 40 mmol / L was injected into the drug injection site of the mice. The injection volume was 1.5 μL, the injection time was 10 minutes, the injection rate was 0.15 μL / min, and the needle was left in place for 5 minutes after the injection to complete the intracranial injection.

[0032] 4. Observe the changes in the electroencephalogram (EEG) of mice. In this embodiment, EEG changes were observed approximately 15 minutes after intracerebral injection. Compared to the control group, the experimental group mice exhibited beta-delta complexes resembling extreme delta brushes, followed by the gradual appearance of visible rhythmic spikes or spike-spik complexes. This indicates successful establishment of the mouse model. See details... Figure 3 and Figure 4 .

[0033] 5. Routine antiepileptic drug testing Commonly used antiepileptic drugs, including diazepam (DZP), sodium valproate (VPA), phenytoin sodium (PHT), levetiracetam (LEV), and lamotrigine (LTG), were administered to mice at a rate of one drug every 10 minutes. Each drug was injected at a dose of 0.1 ml / 10 g of mouse body weight. The drug concentrations, calculated based on mouse body weight, were as follows: diazepam (DZP) 4 mg / kg, sodium valproate (VPA) 100 mg / kg, phenytoin sodium (PHT) 50 mg / kg, levetiracetam (LEV) 100 mg / kg, and lamotrigine (LTG) 15 mg / kg. Physiological saline was the preferred solvent. There was no specific order for drug testing, but the above-mentioned order was preferred. If mice did not exhibit antiepileptic drug resistance and the EEG did not show epileptic waves, the next antiepileptic drug was discontinued.

[0034] Preparation process of control group mice: 1. Phosphate-buffered saline (PBS) injection and heat exposure 24 hours in advance. (1) Weighing: Six C57BL / 6 mice (male, 4-6 weeks old, weighing between 15-20g) were selected and their weight was measured.

[0035] (2) Drug injection: In the control group, phosphate-buffered saline (PBS) solution was drawn at a rate of 0.1 ml / 10 g mouse body weight and administered via intraperitoneal injection.

[0036] (3) Controlled heat exposure: The procedure was the same as that for the experimental group mice. Specifically, after the intraperitoneal injection, the mice were placed in a controlled-temperature oven for heat exposure. The oven temperature was 37-39℃, and the humidity was controlled at 50%-55%RH for 2 hours. During this period, the mice drank water and ate normally. After 2 hours, the mice were returned to their original environment, which was SPF grade, with the ambient temperature controlled at 22±1℃ and the ambient humidity controlled at 50%-55%RH. The environment had an automatic 12-hour light-dark cycle. The mice had free access to water and food.

[0037] 2. Screw filling surgery and EEG recording; 4-aminopyridine (4-AP) induction was performed in the same manner as in the experimental group mice.

[0038] 3. Observe the changes in the electroencephalogram (EEG) of mice. Electroencephalography (EEG) was performed on mice in the experimental group as a reference.

[0039] 4. Routine antiepileptic drug testing The mice in the experimental group were tested using routine antiepileptic drugs.

[0040] The results showed that the control group mice exhibited generalized spike-and-slow-wave complexes (3Hz), polyspike-and-slow-wave complexes, sharp-wave rhythms, or focal discharges on EEG. Sequential administration of one or two antiepileptic drugs significantly reduced these abnormal discharges, restoring the normal rhythm of background EEG activity, manifesting as non-drug-resistant status epilepticus. In contrast, the experimental group mice showed beta-delta complexes with extreme delta brushes on EEG, followed by visible rhythmic sharp waves or sharp-wave-sharp-wave complexes. Even after treatment with multiple antiepileptic drugs, the EEG still showed persistent epileptiform discharges, indicating that the drugs failed to effectively inhibit neuronal overexcitation, manifesting as FIRES-like multidrug-resistant status epilepticus.

[0041] Figure 1 This is a schematic diagram of the modeling process for the febrile infection-related epilepsy syndrome mouse model in Example 1 of the present invention. Control refers to the control group in Example 1 of the present invention. FIRES refers to the febrile infection-related epilepsy syndrome mouse model in Example 1 of the present invention.

[0042] Figure 2 This is a schematic diagram of the coordinate positioning of two groups of mice connected to the electroencephalogram (EEG) recorder in Embodiment 1 of the present invention; wherein, ①, ②, ③: fixing points, filled with screws and connected to the EEG recording device; ④: drug injection point.

[0043] Figure 3 This is an electroencephalogram (EEG) of mice in the febrile infection-related epilepsy syndrome model (experimental group mice) after inducing epileptic seizures, as shown in Example 1 of this invention. The spectrogram shows delta waves at 0-10 Hz and beta waves at 12-30 Hz after epilepsy induction. The EEG recording shows beta-delta complex waves with extreme delta brushes. Subsequently, visible rhythmic sharp waves or sharp-sharp wave complexes appear.

[0044] Figure 4 These are the electroencephalogram (EEG) recordings of mice in the control group and experimental group (labeled as the model group in the figure) in Example 1 of this invention. It can be seen that the EEG of the experimental group mice shows beta-delta complex waves with extreme delta brushes. Subsequently, visible rhythmic sharp waves or sharp wave-sharp wave complexes appear.

[0045] Comparative Example 1, based on Example 1, after injecting lipopolysaccharide (LPS) into normal mice, the mice were not placed in an oven for heat exposure, or during the 2-hour heat exposure period, their rectal temperature did not reach 38.5°C or higher. The observed EEG of the experimental group mice did not show extreme delta brush beta-delta complex waves, and subsequently, no visible rhythmic spikes or spike-spik complex waves appeared. This approach cannot well simulate the EEG characteristics of the disease in this model.

[0046] Comparative Example 2, based on Example 1, did not place the mice in the experimental group on a rectal temperature monitoring and dynamic temperature control device throughout the experiment, and did not ensure that the mice's body temperature was not lower than 32°C throughout the experiment. After administering the same dose of 4-aminopyridine (4-AP), the EEG waves observed in the experimental group mice showed that epilepsy could not be induced.

[0047] In Comparative Example 3, based on Example 1, the drug inducing epilepsy in mice was changed from 4-aminopyridine (4-AP) to pentylenetetrazol (PTZ). The observed EEG waves in the mice were predominantly spikes or sharp waves, without the appearance of extreme delta brushes or beta-delta complexes. Subsequently, no tangible rhythmic spikes or spike-sharp complexes gradually appeared. This did not effectively simulate the EEG characteristics of the disease in the model.

[0048] In Comparative Example 4, based on Example 1, the injection volume of 4-aminopyridine (4-AP) into the mouse brain was changed to 1 μL. It was observed that in some mice, the electroencephalogram (EEG) failed to successfully induce a beta-delta complex wave with an extreme delta brush, and subsequently, no visible rhythmic spikes or spike-spik complex waves gradually appeared. Figure 5 A. Statistical analysis revealed that the success rate of model simulation decreased after the injection dose was changed to 1 μL.

[0049] In Comparative Example 5, based on Example 1, the injection rate and time of 4-aminopyridine (4-AP) intracerebral injection in mice were modified. The injection rate was changed to 0.3 μL / min and the injection time to 5 minutes. Drug spillage was observed at the injection sites in some mice. The excessively rapid injection rate prevented complete drug absorption, resulting in the failure to successfully induce the beta-delta complex wave of the extreme delta brush, and subsequently, no visible rhythmic spike wave or spike-spik complex wave appeared. Figure 5 B. Statistical analysis revealed that the success rate of the model simulation decreased after the injection time was changed to 5 minutes.

[0050] Current methods in the field involve inducing inflammatory responses through intravenous or intraperitoneal injection of lipopolysaccharide (LPS). Intravenous LPS directly enters the systemic circulation, bypassing the intestinal and peritoneal immune systems, thus failing to simulate the early local immune response of infection. Furthermore, direct intravenous LPS injection into the bloodstream can easily trigger a fulminant cytokine storm (e.g., a sharp increase in TNF-α and IL-6), potentially leading to acute death in animals (e.g., septic shock) and interfering with long-term observation. Therefore, this invention employs intraperitoneal injection of LPS combined with heat exposure, resulting in an EEG pattern closer to the FIRES model, allowing for more accurate simulation. Simultaneously, intracerebral injection of 4-aminopyridine (4-AP) precisely simulates the abnormal discharges and inflammatory microenvironment of focal epileptogenic networks. Meanwhile, the hippocampus is the core affected brain region of FIRES. Its high neuronal density and easily activated glial tissue can simultaneously simulate the dual pathological processes of epileptiform discharge and neuroinflammation in FIRES, accurately simulating the clinical 'fever-epilepsy-inflammation' interaction mechanism. Therefore, this invention successfully constructed an animal model of febrile infection-related epilepsy syndrome by using intraperitoneal injection 24 hours in advance combined with oven-stabilized heat exposure and intracerebral injection.

[0051] The method for constructing a mouse model of febrile infection-associated epilepsy syndrome (FAES) of the present invention focuses more precisely on FAES itself. It achieves successful construction of a mouse FAES model within one day with a short induction time, boasting a 100% success rate and 100% survival rate. The model is highly stable, exhibits typical characteristics, has a short induction period, high survival rate, and good consistency. It effectively induces the development of corresponding symptoms of FAES in normal mice, without exhibiting other non-febrile infection-associated epilepsy syndrome symptoms. Furthermore, the model construction method of the present invention does not require complex technical steps and relies on minimal individual differences. Therefore, this method can be repeatedly implemented in laboratories, production workshops, etc., using mice and other animals as a basis, making it industrially applicable and meeting practical requirements.

[0052] Furthermore, the mouse febrile infection-associated epilepsy syndrome model successfully constructed by this invention can be effectively used to screen drugs for the prevention or treatment of febrile infection-associated epilepsy syndrome, to determine the preventive or therapeutic effects of drugs on specific symptoms of febrile infection-associated epilepsy syndrome in mice, and to avoid interference from other factors. More preferably, the method for screening candidate drugs for the prevention or treatment of febrile infection-associated epilepsy syndrome can be carried out through the following steps: The test candidate drug was administered to the mouse model constructed using the above method; Candidate antiepileptic drugs were screened based on changes in mouse electroencephalogram (EEG).

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A method for constructing an animal model of febrile infection-related epilepsy syndrome, characterized in that, The method involves first injecting lipopolysaccharide into the peritoneal cavity of normal mice in conjunction with a heat exposure process; then, starting the timing from the end of the lipopolysaccharide injection, injecting 4-aminopyridine into the brain of the mice 24 hours later to construct a mouse model of febrile infection-related epilepsy syndrome.

2. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 1, characterized in that, The normal mice are at least one of C57BL / 6 mice, MRL / MPJ mice, or BALB / c nude mice; And / or, the normal mice are male mice aged 4-6 weeks; And / or, the normal mouse weight is between 15-20g.

3. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 1, characterized in that, The lipopolysaccharide was prepared into a solution with a concentration of 0.1 mg / 10 mL and injected intraperitoneally. The injection dose was 0.1 mL / 10 g based on the mouse's body weight, and the injection was performed once.

4. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 1, characterized in that, The heat exposure process refers to placing mice in a temperature-controlled oven after intraperitoneal injection of lipopolysaccharide, with the temperature controlled at 37-39°C, the humidity controlled at 50%-55%RH, and / or the heat exposure time being 2 hours.

5. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 1, characterized in that, The 4-aminopyridine was prepared as a 40 mmol / L solution for injection, with an injection volume of 1.5 μL. And / or, the injection rate is 0.15 μL / min; And / or, the injection site is selected in the hippocampus region of the mouse brain.

6. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 5, characterized in that, Intracerebral injection of 4-aminopyridine was performed at room temperature. The specific procedure was as follows: Mice were anesthetized, and the lateral ventricles were located according to the mouse brain atlas. The Bergma point of the anterior fontanelle was defined as the origin. The coordinates of the drug injection point relative to Bergma were: medial and lateral (X) = -2.5 mm, anterior and posterior (Y) = -2.9 mm, and dorsoventral (Z) = -3.0 mm. 1.5 μL of 40 mmol / L 4-aminopyridine solution was slowly injected into the hippocampus of the mouse brain at a rate of 0.15 μL / min over 10 min. The needle was left in place for 5 min. The injection was performed once.

7. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 6, characterized in that, Throughout the process, ensure that the mice's body temperature does not fall below 32°C.

8. The method for constructing an animal model of febrile infection-related epilepsy syndrome according to claim 1, characterized in that, The criteria for determining the success of a mouse model of febrile infection-related epilepsy syndrome include: (1) Judgment by intracranial electroencephalogram (EEG); And / or, (2) by judging whether the use of commonly used antiepileptic drugs can alleviate the epileptic condition of mice.

9. The application of the construction method according to any one of claims 1-8 in the field of animal model construction.

10. The use of an animal model constructed by the method according to any one of claims 1-8 in screening drugs for the prevention and / or treatment of febrile infection-related epilepsy syndromes.

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