CN103505455A - Method for making epileptic seizure animal model - Google Patents
Method for making epileptic seizure animal model Download PDFInfo
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- CN103505455A CN103505455A CN201210228629.2A CN201210228629A CN103505455A CN 103505455 A CN103505455 A CN 103505455A CN 201210228629 A CN201210228629 A CN 201210228629A CN 103505455 A CN103505455 A CN 103505455A
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Abstract
The invention provides a method for making an epileptic seizure animal model. The method for making the epileptic seizure animal model comprises the step of applying kainic acid to primate animal sea horses by adopting a stereospecific technique. The made epileptic seizure animal model has the advantages of local injection administration, low dosage, little relation between epileptogenic dosage and genders and ages of animals, good repeatability, clear epileptogenic process and good drug safety.
Description
Technical field
The present invention relates to a kind of method of epilepsy animal model and epilepsy animal model that uses the method to prepare prepared.Particularly, the present invention relates to a kind ofly by the Hippocampus micro-injection digenic acid to primate, prepare the method for epilepsy animal model, and the primate epilepsy model of preparing by the method.
Background technology
Epilepsy is that the epilepsy brain function that causes of cerebral neuron paradoxical discharge is abnormal.Outbreak is mostly of short duration and have self limiting, and because the brain domain of paradoxical discharge is different, clinical have a multiple outbreak performance, comprises partial seizures or Generalized seizure, is embodied in motion, paraesthesia, or behavior cognition, vegetative nerve functional disturbance.Generalized seizure and complex partial seizures, often with disturbance of consciousness in various degree.
Epilepsy is the synchronous paradoxical discharge of neuronic transition, and its mechanism is mainly because the unbalance of excitatory amino acid in brain and inhibitory aminoacid causes, the interior irritability mechanism of brain strengthens on the one hand, and inhibition weakens on the one hand.The former is by Excitatory Amino Acids in brain, mainly glutamic acid and aspartic acid, they are combined by the glutamate receptor on neuron, the ion channel of mediation G protein coupling, the person of obtaining and excitatory amino acid receptor (NMDA salt receptor) combination, cause a large amount of calcium ions and sodium ion inflow, potassium ion, magnesium ion outflow, and produce depolarization.The latter is by inhibitory aminoacid γ-aminobutyric acid (GABA) mediation, GABA discharge with its receptors bind after, chloride channel is opened, Chlorion influx makes postsynaptic membrane hyperpolarization.
Current animal epileptic model is divided into according to outbreak performance: acute simple partial seizure, chronic simple partial seizures, recurrence partial seizures, comprehensive grand mal, petit mal, status epilepticus, intractable epilepsy; According to mechanism, be divided into: heritability epilepsy, epilepsy immune model, traumatic epilepsy model, cortical development obstacle animal model; According to preparation method, be divided into: chemicals model, metal is implanted, electricity irritation etc.The cause of disease of intractable epilepsy and mechanism are complicated, and outbreak is without predictability, and attack degree is difficult to control.Loscher (Loscher W.Animal models of intractable epilepsy.Prog Neurobiol, 1997,53:239-258.) propose in experimentation, judge that the whether applicable epileptoid standard of people of animal epileptic model has three: 1. the bio electricity granting of model must be consistent with mankind's epilepsy with mode of onset.If 2., without outbreak symptom, EEG must show bio electricity and extremely provide, and is called epileptiform discharge.3. antuepileptic can not be controlled its satisfied outbreak.Unique approved drug resistant epilepsy model is the rat phenytoin Sodium Amygdaloid-kindled epilepsy model by foundation such as Loscher in the world at present, but the shortcoming of this model is, animal is difficult for raising, the rate of lighting is low, easily dead, and rat and people's species variation, its anatomical structure, physiological function and biochemical metabolism and people's difference are larger, and the biological credibility of molecular biology research is not high.
So far both at home and abroad researcher with digenic acid as causing epilepsy agent, adopted different route of administration and mode to set up acute epi leptic model, Chronic Kindling model with it at many animals, and be widely used in the research of epilepto genesis, result shows that epilepsy is similar to clinical temporal lobe epilepsy seizure types due to digenic acid, there is process of establishing repeatable strong, dosage is little, lights rate advantages of higher, is a kind of good epilepsy model.
Primate and people's sibship is nearest, both at home and abroad so far still without the relevant report of Rhesus Macacus digenic acid epilepsy model.
Summary of the invention
For solving the problems of the prior art, the object of this invention is to provide a kind of method of preparing epilepsy animal model.
Therefore, aspect first, the invention provides a kind of method of preparing primate epilepsy model, the 2.0 μ g/ μ l digenic acids that it is 6.0-9.0 μ l that the method comprises dosage are applied to the right side Hippocampus of primate and bring out epilepsy.
Aspect second, the invention provides the epilepsy animal model of preparing by method of the present invention.
In the present invention, primate includes but not limited to ，Hou section, pongidae (comprising gorilla, chimpanzee etc.), apes (comprising Gibbon, troglodyte etc.), preferably Rhesus Macacus.
In a preferred embodiment, digenic acid is by stereotaxis method micro-injection administration.
The present invention has selected the Rhesus Macacus nearest with mankind's sibship as study subject, tentatively sets up Rhesus Macacus epilepsy model, and observed behavior and electroencephalogram change.Experimental result shows, 4 Rhesus Macacus of experimental group by horseley-Clarke technique in after Hippocampus micro-injection digenic acid 8-12 minute, all having occurred to comprehensively tetanic-battle array, to distribute work by partial seizures arbituarily is 1-5 level epilepsy outbreak, and by EEG(electroencephalography), arrived the epileptic discharge of different times, confirmed the cause epilepsy effect of digenic acid in primate.This test, Rhesus Macacus has experienced swallowing act and accelerated breathing that companion chews, and there is the autonomic nervous system symptoms such as nausea and vomiting gastric content, develop into gradually independently not twitching of one-sided limbs, then develop into independently not twitching of bilateral limbs, wet Canis familiaris L. sample shake and complete tetanus-clonic spasm etc. are the overall process of the comprehensive tonic clonic seizure of partial seizures secondary, and mode of onset is very similar to the mankind's temporal lobe epilepsy.
EEG(electroencephalography) adopts reference electrode to put A1, left cortical electrode: Fp1, F3, C3, P3,01, F7, left hippocampus depth electrode: T3, left temporal lobe depth electrode: T5 right side cortical electrode: Fp2, F4, C4, P4, O2, F8, right side Hippocampus depth electrode: T4， right side temporal lobe depth electrode: T6.Basis EEG: visible frequency 3-8Hz, wave amplitude is 200-500uV, θ ripple is common, and 10-14Hz, wave amplitude 100-20uV, the by a narrow margin fast rhythm and pace of moving things (Fig. 1).3-5 minute after experimental group injection digenic acid, cortex visible brain wave frequency in right side speeds, and frequency is 16-20Hz, wave amplitude is 100-200uV, and waveform comes to a point (Fig. 2) gradually, and after 7 minutes, wave amplitude increases gradually, reach 200-400uV, waveform is sharper, engenders the similar sharp wave rhythm and pace of moving things (Fig. 3).After 15 minutes, there is distributing spike, spike and ware wave, frequency is 16-18Hz, and wave amplitude is 300-500uV, and now behavioristics is without significant change (Fig. 4).The clonic spasm that occurs single limbs about 30-40 minute, now EEG is that the O2 frequency of occurrences of leading in right side speeds gradually, wave amplitude increases gradually, the fast rhythm and pace of moving things by a narrow margin that waveform comes to a point, lead and occur rhythmicity spike in temporo district in right side subsequently, spike and ware wave, left side C3 leads and occurs the spike rhythm and pace of moving things, expands to entirely subsequently and leads (Fig. 5-7).Now behavioristics shows as extremity flexing grand mal, after 2-3 minute, recovers gradually, and electroencephalogram mainly be take slow wave as main, and entirely leading the frequency of occurrences is 2-4Hz, the high-amplitude slow wave (Fig. 8) that wave amplitude is 200-400uV.
With respect to other temporal lobe epilepsy models, epilepsy animal model of the present invention has the following advantages: 1, adopt local injection administration, dosage is little; 2, causing epilepsy dosage and Animal Sex, age first closes not quite; 3, favorable repeatability; 4, cause epilepsy process clear; 5, drug safety is good.
Accompanying drawing explanation
Fig. 1 is the basic electroencephalogram of Rhesus Macacus.
Fig. 2 is latter 5 minutes electroencephalograms of injection: behavioristics is without significant change.
Fig. 3 is latter 7 minutes electroencephalograms of injection: behavioristics is without significant change.
Fig. 4 is latter 30 minutes electroencephalograms of injection: single limbs clonic spasm.
Fig. 5 is the rear 31-32 minute electroencephalogram of injection: extremity clonic spasm.
Fig. 6 is the rear 31-32 minute electroencephalogram of injection: orthocolosis, tic.
Fig. 7 is the rear 31-32 minute electroencephalogram of injection: orthocolosis, tic.
Fig. 8 is electroencephalogram after epilepsy.
The specific embodiment
The foundation of Rhesus Macacus epilepsy model of the present invention
1, experiment material
1.1 laboratory animal
5 of healthy Rhesus Macacus, are purchased from animal institute of the Chinese Academy of Medical Sciences, 3 years old age, body weight is respectively 7.9kg, 7.8kg, 8.0kg, 8.1kg, 8.2kg, all raises separately in 90cm * 100cm * 110cm monkey cage, raise every day 3 times, be aided with vegetable, fruit, average indoor temperature (20 ± 12.4) ℃, relative humidity 87.5 ± 12.5, before experiment, all there is the adaptive time of 4 weeks, all carry out neurologic check and EEG (electrocardiogram) examination to get rid of abnormal person.
1.2 Experimental agents
Digenic acid: be purchased from Sigma company.
Anesthetics: the specification 0.1g/2ml of ketalar Hengrui Medicine Co., Ltd., Jiangsu Prov.
Speed is slept new
Atropine
2, key instrument, apparatus
Middle section newly opens up NT9200 series portable quantitative electroencephalogram instrument
20 amplifiers that lead
EEG logging software N9200
Self-control monkey fixed mount
3, animal feeding management
Rhesus Macacus is raised in Beijing Inst. of Neurosurgery's conventional animal room, and before experiment, animal conforms and carries out anthelmintic treatment, selects Healthy female Rhesus Macacus, rustless steel monkey cage sub-cage rearing, tap water is bottled freely to be drunk, every day feed fruit.Animal is hung on neck board and cage and labels as animal identification marking.
4 experimental techniques
4.1 epilepsy models are made
5 Rhesus Macacus are divided into 2 groups: 4 of experimental grouies, 1 of matched group.
Classification is observed by 4.2 behavioristicss
The digenic acid of injecting 6.0 μ l, 7.0 μ l, 8.0 μ l, 9.0 μ l to 4 monkeys of experimental group respectively, to matched group injection same unit dosage normal saline.In monkey cage, observe the outbreak form of expression of Rhesus Macacus epilepsy model, observe the incubation period of epilepsy, the form of expression of outbreak, outbreak rank, performance after persistent period and outbreak rush hour and outbreak dwell time, outbreak, outbreak classification, with reference to the standard of Racine, is divided into 5 grades according to convulsions degree by behavior, 1 grade: Head And Face is twitched, and chews; 2 grades: rhythmicity is nodded or whole body startles; 3 grades: a side forelimb clonic spasm; 4 grades: there is the whole body clonism that hind leg stands and often accompany bilateral forelimb clonic spasm; 5 grades: there is the companion that stands and fall or general tonic-clonic seizure.
4.3 electrode arrangement
With ketaject injection, press intramuscular injection 0.2ml/kg, the new II0.15ml/kg of speed dormancy, atropine 0.5mg, 5 monkeys of difference anaesthesia experiment group and matched group, monkey is fixed on to tridimensional orienting headstock, slough head hair, after defat, open cranium and place cortical electrode, left cortical electrode: Fp1, F3, C3, P3,01, F7, left hippocampus depth electrode: T3, left temporal lobe depth electrode: T5 right side cortical electrode: Fp2, F4, C4, P4, O2, F8, right side Hippocampus depth electrode: T4， right side temporal lobe depth electrode: T6, take A1 as reference electrode.
The observation of 4.4 brain electrical acti
Rhesus Macacus is fixed on to tridimensional orienting headstock, places electrode, basic EEG: visible frequency 3-8Hz, wave amplitude is 200-500uV, θ ripple is common, and 10-14Hz, wave amplitude 100-20uV, the by a narrow margin fast rhythm and pace of moving things.3-5 minute after experimental group injection digenic acid, cortex visible brain wave frequency in right side speeds, and frequency is 16-20Hz, and wave amplitude is 100-200uV, and waveform comes to a point gradually, and after 7 minutes, wave amplitude increases gradually, reaches 200-400uV, and waveform is sharper, engenders the similar sharp wave rhythm and pace of moving things.After 15 minutes, there is distributing spike, spike and ware wave, frequency is 16-18Hz, and wave amplitude is 300-500uV, and now behavioristics is without significant change.The clonic spasm that occurs single limbs about 30-40 minute, now EEG is that the O2 frequency of occurrences of leading in right side speeds gradually, wave amplitude increases gradually, the fast rhythm and pace of moving things by a narrow margin that waveform comes to a point, lead and occur rhythmicity spike in temporo district in right side subsequently, spike and ware wave, left side C3 leads and occurs the spike rhythm and pace of moving things, expands to entirely and leads subsequently.Now behavioristics shows as extremity flexing grand mal, after 2-3 minute, recovers gradually, and electroencephalogram mainly be take slow wave as main, and entirely leading the frequency of occurrences is 2-4Hz, the high-amplitude slow wave that wave amplitude is 200-400uV.
5, result of the test
The performance of 5.1 behavioristicss
All there are 5 grades of epilepsy in experimental group Rhesus Macacus, behavioristics shows as, in Hippocampus, inject after digenic acid 3-5 minute, first occur that electroencephalogram changes, cortex visible brain wave frequency in right side speeds, about 10 minutes, there is swallowing act and the accelerated breathing that companion chews, and there is the autonomic nervous system symptoms such as nausea and vomiting gastric content, after 30 minutes, there is independently not twitching of single limbs, then develop into independently not twitching of bilateral limbs, wet Canis familiaris L. sample shake and complete tetanus-clonic spasm etc. are the comprehensive tonic clonic seizure of partial seizures secondary.Contrast monkey has no epilepsy outbreak.
5.2 EEG
Basis EEG: visible frequency 3-8Hz, wave amplitude is 200-500uV, θ ripple is common, and 10-14Hz, wave amplitude 100-20uV, the by a narrow margin fast rhythm and pace of moving things.3-5 minute after experimental group injection digenic acid, cortex visible brain wave frequency in right side speeds, and frequency is 16-20Hz, and wave amplitude is 100-200uV, and waveform comes to a point gradually, and after 7 minutes, wave amplitude increases gradually, reaches 200-400uV, and waveform is sharper, engenders the similar sharp wave rhythm and pace of moving things.After 15 minutes, there is distributing spike, spike and ware wave, frequency is 16-18Hz, and wave amplitude is 300-500uV, and now behavioristics is without significant change (Fig. 3).The clonic spasm that occurs single limbs about 30-40 minute, now EEG is that the O2 frequency of occurrences of leading in right side speeds gradually, wave amplitude increases gradually, the fast rhythm and pace of moving things by a narrow margin that waveform comes to a point, lead and occur rhythmicity spike in temporo district in right side subsequently, spike and ware wave, left side C3 leads and occurs the spike rhythm and pace of moving things, expands to entirely and leads subsequently.Now behavioristics shows as extremity flexing grand mal, after 2-3 minute, recovers gradually, and electroencephalogram mainly be take slow wave as main, and entirely leading the frequency of occurrences is 2-4Hz, the high-amplitude slow wave that wave amplitude is 200-400uV.(seeing Fig. 1-8).
The above results explanation, the digenic acid epilepsy model of primate-Rhesus Macacus has successfully been set up in this experiment first, and subordinate act and electric physiology two aspects are evaluated, this model shows as part secondary Generalized seizure, well the mode of onset of simulating human temporal lobe epilepsy; And the method has local injection, dosage is little; Repeatable strong; Causing epilepsy dosage and Animal Sex, age first closes not quite; Favorable repeatability; Cause epilepsy process clear; Drug safety is good; Cause the advantages such as the epilepsy time is short.
Claims (4)
1. a method of preparing epilepsy animal model, the method comprises the Hippocampus that the digenic acid of the 2.0 μ g/ μ l of 6.0-9.0 μ l is applied to primate.
2. method according to claim 1, is characterized in that described primate is selected from monkey section, and pongidae, comprises gorilla, chimpanzee, or apes, comprises Gibbon, troglodyte; Preferred Rhesus Macacus.
3. method according to claim 1, is characterized in that digenic acid passes through stereotaxis method micro-injection administration.
4. the epilepsy animal model of preparing by the method described in any one in claim 1-3.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107049552A (en) * | 2017-04-20 | 2017-08-18 | 贵州省人民医院 | A kind of experimental provision for being used to manufacture Chronic Epilepsy animal model |
| CN110269046A (en) * | 2019-07-31 | 2019-09-24 | 大连医科大学附属第一医院 | The method for building up of temporal epilepsy animal model on the basis of brain paralysis |
| CN114099174A (en) * | 2020-06-09 | 2022-03-01 | 首都医科大学宣武医院 | Monitoring system and method for epileptic infant |
| CN114731986A (en) * | 2022-04-14 | 2022-07-12 | 广州医科大学附属第二医院 | Method for determining epileptic pathogenic gene by using drosophila |
| WO2022236749A1 (en) * | 2021-05-12 | 2022-11-17 | 北京太阳电子科技有限公司 | Method and apparatus for detecting abnormal discharge of electroencephalogram, and medium and device |
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2012
- 2012-06-24 CN CN201210228629.2A patent/CN103505455A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107049552A (en) * | 2017-04-20 | 2017-08-18 | 贵州省人民医院 | A kind of experimental provision for being used to manufacture Chronic Epilepsy animal model |
| CN107049552B (en) * | 2017-04-20 | 2018-09-07 | 贵州省人民医院 | A kind of experimental provision for manufacturing Chronic Epilepsy animal model |
| CN110269046A (en) * | 2019-07-31 | 2019-09-24 | 大连医科大学附属第一医院 | The method for building up of temporal epilepsy animal model on the basis of brain paralysis |
| CN110269046B (en) * | 2019-07-31 | 2021-03-26 | 大连医科大学附属第一医院 | Method for establishing temporal lobe epilepsy animal model based on cerebral palsy |
| CN114099174A (en) * | 2020-06-09 | 2022-03-01 | 首都医科大学宣武医院 | Monitoring system and method for epileptic infant |
| WO2022236749A1 (en) * | 2021-05-12 | 2022-11-17 | 北京太阳电子科技有限公司 | Method and apparatus for detecting abnormal discharge of electroencephalogram, and medium and device |
| CN114731986A (en) * | 2022-04-14 | 2022-07-12 | 广州医科大学附属第二医院 | Method for determining epileptic pathogenic gene by using drosophila |
| CN114731986B (en) * | 2022-04-14 | 2023-10-13 | 广州医科大学附属第二医院 | Method for determining epileptic pathogenic genes by using drosophila melanogaster |
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Application publication date: 20140115 |