CN109820845B - Novel method for drug-induced epilepsy model - Google Patents
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Abstract
The invention relates to the technical field of epilepsy model construction, in particular to a novel method for inducing an epilepsy model by using a medicament, namely a novel application method for finding a pharmacological tool medicament, and inducing the epilepsy model in vivo and in vitro. The invention provides a method for establishing an epilepsy inducing animal model and an epilepsy cell model by using KN93 as a pharmacological tool drug, the KN93 can simultaneously induce the epilepsy inducing animal model and the epilepsy cell model, and the existing epilepsy model caused by the drug is one of an animal model (in vivo) or a cell model (in vitro), so that the KN93 can establish the epilepsy model in two layers of the in vivo and the in vitro, further deeply discuss the epilepsy disease mechanism and screen related antiepileptic drugs.
Description
Technical Field
The invention relates to the technical field of epilepsy model construction, in particular to a novel method for inducing an epilepsy model by using a medicament, namely a novel application method for discovering a pharmacological tool medicament, and inducing the epilepsy model in vivo and in vitro.
Background
Epilepsy is a chronic brain disease characterized by transient cerebral dysfunction caused by abnormal firing of cerebral neurons. Studies have shown that the worldwide incidence of epilepsy is 0.5-1%. The total prevalence rate of epilepsy in China is 7.0 per thousand, and the annual incidence rate is 28.8/10 ten thousand. So far, the pathogenesis of epilepsy is still unclear, so that establishing reliable and effective epilepsy animal models and cell models is the technical basis for searching the pathogenesis of epilepsy and screening new targets of drugs.
KN93 is calcium ion/calmodulin dependent kinase II (Ca)2+The inhibitor of calmodulin dependent kinase II, CaMKII) is derivative of methoxybenzenesulphonamide (methoxybenzenesulphonamide), and the molecular formula is as follows: 2- [ N- (2-hydroxyethyl) -N- (4-methoxybenezenesulfonyl)]-N- (4-chlorocinnamyl) -N-methylcinnamylamine. The mechanism of KN93 inhibition is to prevent the binding of CaM to CaMKII by interacting with the CaM binding site, so that CaMKII cannot autophosphorylate without being activated. This inhibition is in competition with calmodulin. The phosphorylation activity of CaMKII can be completely inhibited by KN93 of 0.37 mu mol/L, and a pharmacological tool drug KN93 is applied to the related research of a signal path of CaMKII.
The animal models of epilepsy are various, including a persistent epilepsy animal model, a febrile convulsion epilepsy animal model, a microbial infection model, a nerve injury epilepsy model, a blood brain barrier injury model and the like. Common molding drugs include pimaric acid, pilocarpine, tetanus toxin, and the like. The medicine causes a small number of epileptic cell models, and compared with a classical magnesium-free epileptic cell model, the magnesium ions are removed from normal extracellular fluid, the normal extracellular fluid is recovered after 3 hours of culture, and neurons generate epileptic high-frequency high-amplitude discharge waves. Therefore, the existing drug modeling has the problem that no drug can form an epileptic discharge model on animals and neurons, and the deep discussion of applying two in-vivo and in-vitro epileptic models to epileptic pathogenesis and drug screening is limited to a certain extent. Therefore, the research of a pharmacological tool medicine which can simultaneously induce an epilepsia animal model and an epilepsia cell model is imminent, and has important scientific significance and application value.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a novel method for inducing an epilepsy model by using a medicament, provide a pharmacological tool medicament which can simultaneously induce an epilepsy animal model and an epilepsy cell model, and lay a solid technical foundation for an epilepsy pathogenesis and the screening of a novel medicament target.
In order to achieve the above object, the present invention adopts the following technical solutions.
A novel method for a drug-induced epilepsy model is characterized in that KN93 is used as a pharmacological tool drug to be applied to building an epilepsy-induced animal model and an epilepsy cell model.
A novel method for drug-induced epilepsy modeling is characterized by comprising the following steps.
1) Animal modeling administration and grouping: normal 12-week-old male and female Wistar and TRM were placed individually in a standard environment with all rats at controlled temperature, in 12 hours of light/dark cycle, with sufficient food and water to randomly divide the animals into 6 groups of 10 animals each; vehicle groups 1, 2: normal Wistar and TRM received artificial cerebrospinal fluid lateral ventricular injection; low dose groups 3, 4: normal Wistar and tremor rats received low doses of KN93 and KN92 (TOCRIS; 9. mu.g/kg body weight) dissolved in artificial cerebrospinal fluid (mixed with DMSO); high dose groups 5, 6: normal Wistar and TRM were given high doses of KN93 and KN92, TOCRIS, respectively; 18 μ g/kg body weight); all lateral ventricles were injected in a volume of 5 μ l using a rat stereotaxic apparatus, 1.5 mm posterior to bregma, 1.5 mm lateral to the left and right of the sagittal suture, and 3.5 mm below the surface of the brain.
2) Electroencephalogram detection of animal brain waves: anesthetizing a rat with 10% of 0.3ml/100g chloral hydrate, and implanting an electroencephalogram electrode; the stereotaxic apparatus guides to implant cortex and hippocampal electrodes into cortex and left hippocampus respectively for a long time; after dosing and electrode implantation, neuroelectrical activity was recorded for 24 h; carrying out electroencephalogram monitoring on the rat for more than 20 minutes in a 40 x 40 cm sound insulation box by using a BL420 type biological signal and data acquisition analyzer, and then carrying out electroencephalogram recording for 30 minutes; analysis of seizure time was performed using BL420 data analysis software.
3) Behavioral grading assessment of epileptic rats: rat epileptic behavior was according to the Racine rating scale, grade 0: no reaction is carried out; stage I: facial clonus including blinking, palpitations, rhythmic chewing, wet dog-like tremor, and the like; II stage: i-level adding rhythmic nodding and/or head swinging; grade III: stage II plus forelimb clonus; stage IV: standing on hind limbs in level III; and V stage: level IV plus bilateral rigidity of the anterior and posterior limbs, rigidity of the dorsiflexion, falling down, continuous standing and falling down, unbalance and four limbs twitching.
4) Rat primary hippocampal neurons were cultured in vitro: taking the brain of a newborn rat, separating bilateral hippocampus under a microscope, and placing the hippocampus in D-Hanks solution; adding 0.125% trypsin, and digesting for 15-30 min in an incubator at 37 ℃; the digestion was stopped by the plating medium (DMEM/F12 + 15% serum) and the cell density was adjusted to 2X 105/cm2Then planted on a 2.0 cm × 2.0 cm cover glass. Changing the feeding culture solution by half 3-4 days (2% B27+ Neurobasal)TM-a-Medium); the hippocampal neurons cultured for 12-14 days form obvious processes under an optical microscope, cell bodies are rich in stereoscopic impression, the neurons can form network connection, and cell electrophysiological experiments are carried out after the neurons are mature.
5) Cell administration and determination of the discharge function of primary hippocampal neurons in rats by patch clamp technique: firstly, detecting the spontaneous discharge condition of neurons by using patch clamp, wherein the neurons are divided into a normal vehicle group, a 24 h treatment group of 5 mu M KN93, a 24 h treatment group of 5 mu M KN92 (an inactive derivative of KN 93) and a magnesium-free epileptic cell model group, and each group is used for testing 6-20 neurons; recording spontaneous discharge by applying an Axomatch 700B patch clamp amplifier and adopting a whole-cell patch clamp technology in a current clamp I-normal mode; normal extracellular fluid (mmol. L)-1）：145NaCl、4 KCl、1.8 CaCl2、1 MgCl 210 Glucose, 10 HEPES, pH 7.35; magnesium-free liquid (mmol. L)-1）：145NaCl、4 KCl、1.8 CaCl 210 Glucose, 10 HEPES, pH 7.35; electrode internal solution (mmol. L)-1）： 140 potassium gluconate、2 KCl、 3 MgCl 210 HEPES, 5 phosphoricetine, 2 potassium ATP and 0.2 sodium GTP, and the pH is adjusted to be 7.4; when the patch clamp technology is used, a cover glass is placed in a bath above a microscope, a microelectrode is formed by drawing through a two-step method, the resistance is 2-5M omega, and the patch clamp current clamp technology is used for applying Clampex10.0 and Clampfit 10.0 data acquisition and analysis; then, detecting the excitability of the neurons under depolarization current, wherein the neurons are divided into a normal vehicle group, a 5 mu M KN93 treatment 24 h group, a 5 mu M KN92 treatment 24 h group and a 5 mu M KN93 treatment 1 h group, each group of experiment has 6-20 neurons, 1 s depolarization current is injected into the neurons to induce a series of action potentials, and the maximum stimulation current is 200pA in a slope mode; the excitability parameters were further analyzed using the claufft 10.0 software.
Compared with the prior art, the invention has the following beneficial effects.
1) KN93 has not been applied to an epilepsy-inducing animal model and an epileptic cell model in previous studies, and the drug has been applied to related studies of a signal path of CaMKII. The invention is therefore a new application of the pharmacological tool drug KN 93.
2) The KN93 can simultaneously induce an epilepsy animal model and an epilepsy cell model, and the current epilepsy model caused by medicines is one of an animal model (in vivo) or a cell model (in vitro). Therefore, KN93 can construct an epilepsy model in two levels in vivo and in vitro, and further deeply discuss the epileptic disease mechanism and screen related antiepileptic drugs.
3) According to the invention, the animal and the cell treated by KN93 can successfully construct an epilepsy model for at least 24 h, 24 h is a necessary condition for successful model construction, and the application time of KN93 in previous researches is not more than 1 h.
Drawings
FIG. 1 shows that KN93 treatment for 24 h induces epileptiform brain waves in normal rats and genetically Tremor rats (Tremor, TRM).
FIG. 2 is a time analysis of KN93 induced seizures in rats.
FIG. 3 is the morphology of rat primary hippocampal neurons cultured in vitro for 12 days.
FIG. 4 shows that the spontaneous discharge of neurons treated for 24 h by KN93 is enhanced by the patch clamp technology, and the modeling effect is similar to that of a classical magnesium-free epilepsy cell model.
FIG. 5 shows the development of hyperexcitability of neurons 24 h after KN93 treatment.
FIG. 6 is a parametric analysis of KN93 resulting in ultrahigh excitability of neurons. Wherein A: the number of action potentials in hippocampal neurons cultured after 5 μ M KN 9324 h application was significantly increased compared to untreated controls. The number of action potentials in the hippocampal neurons treated for 24 h with 5 μ M KN92 was not altered. B: the slope of the ascending branch of the first action potential was significantly increased in hippocampal neurons cultured after 5 μ M KN 9324 h application compared to the untreated group.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments. The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents. The methods of operation in the examples are conventional in the art.
Example KN93 induces epileptic animal models and epileptic cell models.
Animal modeling administration and grouping.
Normal Wistar and TRM (12 weeks old, male and female) were housed individually in standard settings. All rats had sufficient food and water at controlled temperature in a 12 hour light/dark cycle. Animals were randomized into 6 groups of 10 animals each. 1. 2 (vehicle group): normal Wistar and TRM received artificial cerebrospinal fluid lateral ventricular injection; 3. 4 (low dose group): normal Wistar and tremor rats received low doses of KN93 and KN92 (TOCRIS; 9. mu.g/kg body weight) dissolved in artificial cerebrospinal fluid (mixed with DMSO); (5, 6, high dose group): normal Wistar and TRM were given high doses of KN93 and KN92, TOCRIS, respectively; 18 μ g/kg body weight). All lateral ventricle injections were performed in a volume of 5 μ l. A rat stereotaxic apparatus is used, with a 1.5 mm posterior bregma, a 1.5 mm lateral left and right sagittal suture, and a 3.5 mm cerebral subsurface. Injection protocols and procedures were approved by the animal care and use committee of chinese medical university.
2. Electroencephalograms detect animal brain waves.
Rats were anesthetized with 10% chloral hydrate (i.p., 0.3ml/100 g) and electroencephalogram electrodes were implanted. Stereotaxic instruments guide the long-term implantation of cortical and hippocampal electrodes into the cortex and left hippocampus, respectively. Neuroelectrical activity was recorded 24 h after drug administration and electrode implantation. The rats were subjected to electroencephalographic monitoring for 20 minutes or more in a sound-proof box (40 × 40 × 40 cm) using a BL420 type biosignal and data collection analyzer (gendtai alliance), and then subjected to electroencephalographic recording for 30 minutes. As shown in figure 1, it is clear from EEG recordings that both Wistar and TRM rats exhibited epileptiform seizure electrical activity following administration of low doses of KN 93. After 24 h of high dose KN93 administration, TRM rats developed high amplitude, high frequency brain waves that were more severe than seizures. The brain wave severity of epileptic seizures in normal rats and TRM rats was KN93 concentration dependent.
Further, analysis of seizure time using BL420 data analysis software, high dose KN93 treatment for 24 h increased the total duration of Wistar and TRM seizures in rats as shown in figure 2 (p <0.01, n = 6) compared to the non-dosed control group animals. Thus, it was found by EEG detection that lateral ventricle administration of KN93 for 24 h induced brain waves in normal and TRM rats with epileptiform seizures and more severe epileptiform seizures, respectively.
3. Behavioral grading assessment of epileptic rats.
The rat epileptic behavior was according to the Racine rating scale. Level 0: no reaction is carried out; stage I: facial clonus including blinking, palpitations, rhythmic chewing, wet dog-like tremor, and the like; II stage: i-level adding rhythmic nodding and/or head swinging; grade III: stage II plus forelimb clonus; stage IV: standing on hind limbs in level III; and V stage: level IV plus bilateral rigidity of the anterior and posterior limbs, rigidity of the dorsiflexion, falling down, continuous standing and falling down, unbalance and four limbs twitching. It is found that normal rats have grade I-III seizures induced by treating the lateral ventricle with high KN93 dose for 24 h, and TRM rats have grade IV seizures induced by treating the lateral ventricle with high KN93 dose for 24 h.
4. Rat primary hippocampal neurons were cultured in vitro.
Taking the brain of a newborn rat, separating bilateral hippocampus under a microscope, and placing the hippocampus in D-Hanks solution; adding 0.125% trypsin, and digesting for 15-30 min in an incubator at 37 ℃. The digestion was stopped by the plating medium (DMEM/F12 + 15% serum) and the cell density was adjusted to 2X 105/cm2Then planted on a 2.0 cm × 2.0 cm cover glass. Changing the feeding culture solution by half 3-4 days (2% B27+ Neurobasal)TM-a-Medium). As shown in figure 3, the hippocampal neurons cultured for 12-14 days form obvious processes under an optical microscope, the cell bodies are rich in stereoscopic impression, and the neurons can form network connection. Cell electrophysiology experiments were performed after neuronal maturation.
5. Cell administration and measurement of the discharge function of primary hippocampal neurons in rats were performed using the patch clamp technique.
First, the spontaneous discharge of neurons is detected by using patch clamp. The neurons were divided into a normal vehicle group, a 24 h group treated with 5 μ M KN93, a 24 h group treated with 5 μ M KN92 (inactive derivative of KN 93), and a magnesium-free epileptic cell model group, with 6-20 neurons per group tested. Spontaneous electrical discharge recordings were performed in current clamp I-normal mode using the whole-cell patch clamp technique using an Axopatch 700B patch clamp amplifier. Normal extracellular fluid (mmol. L)-1）：145NaCl、4 KCl、1.8 CaCl2、1 MgCl 210 Glucose, 10 HEPES, pH 7.35. Magnesium-free liquid (mmol. L)-1）：145NaCl、4 KCl、1.8 CaCl210 Glucose, 10 HEPES, pH 7.35. Electrode internal solution (mmol. L)-1）： 140 potassium gluconate、2 KCl、 3 MgCl 210 HEPES, 5 phosphoripatine, 2 potassium ATP, 0.2 sodium GTP, and the pH was adjusted to 7.4. In the patch clamp technique, a cover slip is placed in a bath above the microscope. The microelectrode is formed by drawing with a two-step method, and the resistance is 2-5M omega. Data acquisition and analysis were performed using the patch-clamp current-clamp technique using clautext 10.0 and clautft 10.0. As shown in FIG. 4, after 12 days of culture, 5 μ M KN 9324 h treatment is given to neurons, and similar to a magnesium-free epileptic cell model, spontaneous discharge with continuous strong direct high-frequency outbreak appears, the discharge frequency is 6-20 Hz, and the voltage amplitude is 45-80 mV. Neurons exhibited normal episodic firing in either the control vehicle group or given KN 92.
The excitability of the neurons under depolarization current was then examined, and the neurons were divided into a normal vehicle group, a 5 μ M KN 93-treated 24 h group, a 5 μ M KN 92-treated 24 h group, and a 5 μ M KN 93-treated 1 h group, each of which was tested for 6-20 neurons. Injection of a depolarizing current of 1 s into the neuron elicits a cascade of action potentials with a maximum stimulation current of 200pA in the ramp mode. As shown in fig. 5, injection of a depolarizing current of 1 s into the neuron elicits a series of action potentials with a maximum stimulation current of 200pA in the ramp mode. After 5 mu M KN93 treatment for 24 h, the cultured hippocampal neurons show ultrahigh excitability by injecting stimulating current and generate a series of action potentials; whereas the cultured hippocampal neurons treated with 5 μ M KN92 for 24 h or 5 μ M KN93 for 1 h did not show hyperexcitability.
The excitability parameters were further analyzed using the campfit 10.0 software, as shown in fig. 5, and the number of action potentials in cultured hippocampal neurons increased significantly after 24 h treatment with 5 μ M KN93, as shown in fig. 6A, compared to untreated controls (p <0.01, n = 6). The evoked number of neuronal action potentials after 24 h treatment with 5 μ M KN92 was unchanged compared to the untreated group of neurons, as shown in figure 6A (p > 0.05, n = 6). Furthermore, the first action potential rising branch slope was significantly increased in cultured hippocampal neurons after 24 h treatment with 5 μ M KN93, as shown in fig. 6B (p <0.05, n = 6), compared to the untreated group.
In conclusion, the KN93 treatment neuron 24 h successfully constructs the epileptic cell model, and the modeling effect is similar to that of the classical magnesium-free induced epileptic cell model.
Claims (1)
1. A method for inducing an epilepsy model by using a medicament is characterized in that KN93 is used as a pharmacological tool medicament to be applied to establishing an epilepsy inducing animal model and an epilepsy cell model;
the method specifically comprises the following steps:
1) animal modeling administration and grouping: normal 12-week-old male and female Wistar and TRM were placed individually in a standard environment with all rats at controlled temperature, in 12 hours of light/dark cycle, with sufficient food and water to randomly divide the animals into 6 groups of 10 animals each; vehicle groups 1, 2: normal Wistar and TRM received artificial cerebrospinal fluid lateral ventricular injection; low dose groups 3, 4: normal Wistar and tremor rats received KN93 and KN92 at a dose of 9 μ g/kg; high dose groups 5, 6: normal Wistar and tremor rats received KN93 and KN92 at a dose of 18 μ g/kg; all lateral ventricles were injected in a volume of 5 μ l using a rat stereotaxic apparatus, 1.5 mm posterior to bregma, 1.5 mm lateral to the left and right of the sagittal suture, 3.5 mm below the brain surface;
2) electroencephalogram detection of animal brain waves: anesthetizing a rat with 10% of 0.3ml/100g chloral hydrate, and implanting an electroencephalogram electrode; the stereotaxic apparatus guides to implant cortex and hippocampal electrodes into cortex and left hippocampus respectively for a long time; after dosing and electrode implantation, neuroelectrical activity was recorded for 24 h; carrying out electroencephalogram monitoring on the rat for more than 20 minutes in a 40 x 40 cm sound insulation box by using a BL420 type biological signal and data acquisition analyzer, and then carrying out electroencephalogram recording for 30 minutes; analyzing the seizure time by using BL420 data analysis software;
3) behavioral grading assessment of epileptic rats: rat epileptic behavior was according to the Racine rating scale, grade 0: no reaction is carried out; stage I: facial clonus including blinking, palpitations, rhythmic chewing, wet dog-like tremor, and the like; II stage: i-level adding rhythmic nodding and/or head swinging; grade III: stage II plus forelimb clonus; stage IV: standing on hind limbs in level III; and V stage: level IV plus bilateral anterior and posterior limb rigidity, body dorsiflexion rigidity, falling, continuous standing and falling, unbalance and four limbs twitching;
4) rat primary hippocampal neurons were cultured in vitro: taking the brain of a newborn rat, separating bilateral hippocampus under a microscope, and placing the hippocampus in D-Hanks solution; adding 0.125% trypsin, and digesting for 15-30 min in an incubator at 37 ℃; the digestion was stopped by planting the culture solution DMEM/F12+ 15% serum, and the cell density was adjusted to 2X 105/cm2Then planting on a cover glass with the thickness of 2.0 cm multiplied by 2.0 cm; changing the feeding culture solution by half 2% B27+ Neurobasal for 3-4 daysTM-a-Medium; the hippocampal neurons cultured for 12-14 days form obvious processes under an optical microscope, cell bodies are rich in stereoscopic impression, the neurons can form network connection, and cell electrophysiology experiments are carried out after the neurons are mature;
5) cell administration and determination of the discharge function of primary hippocampal neurons in rats by patch clamp technique: first of all, a film is appliedDetecting the spontaneous discharge condition of neurons by using a patch clamp, wherein the neurons are divided into a normal vehicle group, a group treated by 5 mu M KN93 for 24 h, a group treated by 5 mu M inactive derivative KN92 of KN93 for 24 h and a magnesium-free epileptic cell model group, and 6-20 neurons are tested in each group; recording spontaneous discharge by applying an Axomatch 700B patch clamp amplifier and adopting a whole-cell patch clamp technology in a current clamp I-normal mode; normal extracellular fluid: 145 mmol. L-1NaCl、4 mmol·L-1 KCl、1.8 mmol·L-1CaCl2、1 mmol·L-1MgCl2、10 mmol·L-1 Glucose、10 mmol·L-1HEPES, pH 7.35; no magnesium liquid: 145 mmol. L-1NaCl、4 mmol·L-1 KCl、1.8 mmol·L-1 CaCl2、10 mmol·L-1 Glucose、10 mmol·L-1HEPES, pH 7.35; electrode internal liquid: 140 mmol. L-1 potassium gluconate、2 mmol·L-1 KCl、 3 mmol·L-1 MgCl2、10 mmol·L-1 HEPES、5 mmol·L-1 phosphocreatine、2 mmol·L-1 potassium ATP、0.2 mmol·L-1sodium GTP, the pH is adjusted to 7.4; when the patch clamp technology is used, a cover glass is placed in a bath above a microscope, a microelectrode is formed by drawing through a two-step method, the resistance is 2-5M omega, and data acquisition and analysis are carried out by applying Clampex10.0 and Clampfit 10.0 through the patch clamp current clamp technology; then, detecting the excitability of the neurons under depolarization current, wherein the neurons are divided into a normal vehicle group, a 5 mu M KN93 treatment 24 h group, a 5 mu M KN92 treatment 24 h group and a 5 mu M KN93 treatment 1 h group, each group of experiment has 6-20 neurons, 1 s depolarization current is injected into the neurons to induce a series of action potentials, and the maximum stimulation current is 200pA in a slope mode; the excitability parameters were further analyzed using the claufft 10.0 software.
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| Inhibition of calciumycalmodulin kinase II alpha subunit expression results in epileptiform activity in cultured hippocampal neurons;Severn B. Churn et al.;《PNAS》;20050509;第97卷(第10期);第5604-5609页 * |
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