aminal models for seizure
TRANSCRIPT
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Dr. Jitendra Agrawal
Second year resident
ANIMAL MODELS FOR ANTIEPILEPTIC DRUGS
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INTRODUCTION
A Seizure (from the Latin sacire, “To take possession of”) is a paroxysmal event due to abnormal, excessive, hyper synchronous discharges from an aggregate of central nervous system (CNS) neurons.
Epilepsy describes a condition in which a person has reccurent seizures due to a chronic, underlying process.
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WHY WE NEED ANIMAL MODEL?
Discovery of new AED Characterization of spectrum of
anticonvulsant activity of new AED Evaluation whether efficacy of new AED
changes during chronic treatment of epilepsy Discovery of antiepileptogenic or disease
modifying agent
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Models for Epilepsy
Induction of Seizure in normal animal Genetic animal Model
Electrically induced Seizure
Chemically induced Seizure Animals with spontaneous Recurrent seizures
Acute induced Seizure
MES PTZ
Chronic induced Seizure
Electrical or Chemical Kindling
Post Epilepticus model with spontaneous recurrent seizures
Electrical SE Induction
(Perforanth path)
Chemical SEInduction
( Pilocarpine)
e.g. Rats or Mice with Spike wave discharge
(lethargic mice,tottering mice)
e.g. DBA/2 MiceGEPRs, Photosensetive
baboons,Gebrils
Animal with reflex seizures
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ELECTROSHOCK SEIZURES IN MICE & RATS
Protection against electroshock induced seizures in mice and
rats is used as an indication for compounds which may prove
effective in Generalized tonic clonic seizures
Electric stimuli evoke tonic hind limb extensions, which are
suppressed by anti-epileptic drugs.
CC50 : current for inducing hind limb extension 50% of animal
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MAXIMAL ELECTROSHOCK SEIZURE
Merritt and Putnam (1938)
Animals are stimulated 2-5 times the threshold current strength
The purpose of this test is to induce the most intense
physiologically possible seizure by a method analogous to human
electroshock therapy.
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METHODOLOGY
ANIMALS: Groups of 6-10 male Swiss mice (20-32g) or Wistar rats (100-150g) are used.
ROUTE OF DRUG ADMINISTRATION:
i. Intraperitoneal
ii. Oral 30 min after i.p. injection and 60 min after oral
administration the animals are subjected to electroshock.
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An electro-convulsiometer with Corneal or Ear electrodes is used to deliver the shock.
Current used: o Rat : 150mAo Mice : 50 mAo 0.2 second duration
METHODOLOGY
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The PHASES of maximal seizure shown by normal mice typically
consists of :
Phase of tonic limb flexion
Full extension of limbs
Clonic interval ( variable )
Death (in some animals)
video
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EVALUATION
Supression of hind limb extenson - measure of eficcacy
Calculation of ED50 for supression of tonic
hind limb extension – anticonvulsant potency Phenytoin, carbmazepine, phenobarbitone –
effective Ethosuximide - ineffective
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DISADVANTAGE
Do not give clue about mechanism of action of drug
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PENTYLENETETRAZOL INDUCED SEIZURES
Pentylenetetrazol (PTZ) produces generalized asynchronized
clonic movements which are superceded by tonic convulsions
characterized by flexion of limbs followed by extension.
Act by antagonizing the inhibitory GABAergic transmission
The test is considered as indicative of anticonvulsant activity
of drugs against Absence seizure
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METHODOLOGY ANIMALS: Groups of 6-10 mice (18-22g) of either sex
ROUTE OF DRUG ADMINISTRATION: Determine S.C. CD97 (convulsive dose in 97% animals) 1% solution of PTZ , 80-100mg/kg S.c. in scruff of neck
There are 3 distinct phases constituted the PTZ seizure sequence i.e.
Myoclonic jerk Clonic seizures Tonic-clonic hind limb extension. Death
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EVALUATION
End point
First episode of clonic jerking last for 5 sec
First clonic seizure with loss of righting reflex
Evaluation
Efficacy: measured by ED50 for suppression of
clonic seizure
Ethosuximide, valproate – effective
Phenytoin, Carbamazepine – not effective
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STRYCHNINE INDUCED SEIZURES The convulsant action of strychnine is due to
interference with post-synaptic inhibition that is mediated by Glycine.
It acts as a selective competitive antagonist to block the inhibitory effect of glycine at all glycine receptors.
The convulsions has a characteristic Motor pattern. Dose : 2 mg/kg. Route : i.p. Time for onset of tonic extensor convulsions and death of
animals is noted. Strychnine abolishes the flexor latency completely,
leading to almost instantaneous onset of the extensor seizure.
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PICROTOXIN-INDUCED CONVULSIONS Picrotoxin is a GABA-antagonist and it modifies
the function of chloride ion channel of the GABA receptor complex.
Dose : 3.5 mg/kg Route : subcutaneous
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BICUCULINE TESTS IN RATS Bicuculine is a GABA-antagonist.
Dose : 1 mg/kg
Route : Intravenous.
The tonic convulsions appear in all treated
rats within 30 seconds of injection.
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4-AMINOPYRIDINE INDUCED SEIZURES IN MICE
4-Aminopyridine, K+ channel antagonist is a powerful convulsant.
The epileptiform activity is predominantly mediated by non-NMDA type excitatory amino acid receptors.
Dose : 13.3 mg/kg Route : Subcutaneous
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EPILEPSY INDUCED BY FOCAL SEIZURES Topical or intracerebral application of metal and chemical can
lead to simple partial seizures Cortical imlanted metals:
Alumina cream, cobalt, tungstic acid Appliead onto or into the cerebral cortex Injection of iron in brain cortex
Aluminium Hydroxide gel model 4% aluminium hydroxide is injected into surgically exposed monkey
neocortex One or two month after injection spontaneous and recurrent seizures
begins Model for focal epilepsy
Chemical Intrahippocampal – kainic acid, tetanus toxin Topical application – penicillin, picrotoxin, bicuculline
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KINDLED RAT SEIZURE MODEL The kindled seizure model in rats offer a method to study the
anticonvulsant activity on the basis of pathophysiological model.
Kindling results from repetitive sub convulsive electrical stimulation of certain areas of brain .
On continued stimulation electrical activity spreads and generalized convulsions occur.
The animals are given stimulation through an electrode implanted with in right amygdala.
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Adult female Sprague-Dawley rats (270–400 g)
The rats are implanted with an electrode in the right amygdala
After 1 week electricalstimulation of the brain is started
Other brain areas like
Neocortex, hippocampus
in rats
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Duration and amplitude, behavioral seizure duration and seizure stage are recorded
Seizure severity is graded into 5 stages. 1: immobility, eye closure, twitching of vibrissae, sterotyping
sniffing 2: facial clonus and head nodding 3: facial clonus , head nodding and forelimb clonus 4: rearing , often accompanied by bilateral forlimb clonus 5: rearing with loss of balance and falling accompanied by
generalized clonic seizures
Rats are considered to be kindled on the 1st stimulation causing a stage 5 seizure which is followed by at least two consecutive stage 5 seizures
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EVALUATION
Test animals are tested on the day before and after the test compound is given orally or i.p.
Test and control are compared with four different measures of efficacy Seizure latency – time from stimulation to the
first sign of seizure activity Seizure severity Seizure duration After discharge duration
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Drug efficacy can be measured by determining separate ED50 value for total supression of Generalized seizure (stage 4,5) Focal seizure (Stage 1-3) Amygdala after discharges
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ADVANTAGE:
Efficacy of drug : Process of epileptogenesis Fully kindled state
Efficacy against generalized seizures provides model for effective in secondary generalized seizures of partial epilepsy
Efficacy against the focal component of kindled seizures provides a valid model for drugs effective in complex partial seizures
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OTHER METHODS OF KINDLING
Corneal Electroshock kindling Mice: once daily application of 3 mA current 60
Hz for 2 sec Rat : once daily application of 8 mA current 60
Hz for 4 sec Stage 5 seizure is considered as animal is
kindled
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CHEMICAL INDUCED KINDLING
Rat: 3o mg/kg of PTZ i.p. 3 dose/week for 9 weeks Scoring : 0 - no response 1 – ear and facial twitching 2 – one to 20 myoclonic jerck 3 – more than 20 body jerck 4 – clonic forelimb convulsion 5 – generalized convulsions with rearing and falling
down episodes 6 – generelized convulsions with tonic extension
episodes and status epilepticus At the end of the 9th week 90% animals are
kindeled Seizure score more than or equal to 3
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MODELS FOR STATUS EPILEPTICUS
Electrical Stimulation of hippocampal perforant pathway:
Implantation of bipolar stimulating eletrodeIn right angular bundle
Unipolar reccording electrode In right hippocampal dentate granule
Pathway is stimulated by2mA monopolar pulse for 50mcs, 20 Hz, for 2 h
Development of self sustained limbic status epilepticus
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CHEMICAL INDUCED STATUS EPILEPTICUS
Pilocarpine Cholinomimetic Can produce status epilepticus in rats Dose : 380-400 mg/kg Route : ip
Lithium- Pilocarpine; Pretreatment with lithium – 3meq/kg ip Followed by pilocarpine – 30-40 mg/kg ip
Lithium – methomyl Pretreatment with lithium Methomyl – 5.2mg / kg s.c.
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MODEL FOR INFANTILE SPASMS Early childhood Insensitive to most of the available antiepileptics Velisek (2007) developed model
Pregnant sprague-dawley ratsBetamethasone – 0.4mg/kg i.p. two doses
at 8:oo am and 6:00 pm on gestational day15
Postnatal day 15Pups
NMDA 15mg/kg ip
Twisting movements of tail, arching for several secondsFinally loss of righting reflex
Flexion spasms with multiple recurrences.
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GENETIC ANIMAL MODEL FOR EPILEPSY
Totterer Mice: Homozygous (tg/tg) strain totterer mice are
prone to spontaneous epileptic seizure Broad based ataxic gate By 3 to 4 weeks of age → develop frequent partial
seizure Spontaneous focal motor seizure occur a few times a
day → unilateral clonic jerk of limbs with secondary generalization
Also exhibit absence seizure with synchronous 6-7 per second spike wave discharges in EEG
Two seizure type in one model
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LETHARGIC MICE
Homozygous (lh/lh) Model for absence seizure Recognized by ataxic gate at the age of 3
weeks Behavioural , EEG, and anticonvulsant profile
is similar to those in absence seizure in human
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DBA/2J MICE
Inbred strain of house mouse (mus musculus) Audiogenic seizure susceptible mice Between age 2-4 weeks these mice exhibit
sound induced seizures Susceptibility gradually declines → at the 8
week totally free of audiogenic seizures Exposed to loud sound (12-16 kHz) Seizure pattern → wild running phase → clonic
convulsion → tonic extension → respiratory arrest/ full recovery
Sensitive gross screening model for anticonvulsant drug
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GEPRS
Genetically epilepsy prone Rats: Seizures can be induced by various stimuli
Sound Hyperthermia Chemcal Electrical
Seizure pattern → wild running phase → clonic jercks → tonic extension → respiratory arrest/ full recovery
Model for tonic-clonic convulsion
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PHOTOSENSITIVE BABOONS
Intermittent light stimulation at frequencies close to 25 flashes/second leads to seizure
Eyelid, face, and body clonus and subsequently tonic spasms or full tonic clonic convulsions
Model for tonic clonic seizure, myoclonic seizure
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MONGOLIAN GEBRILS
Seizure can be provoked by Placing animal in new envioronment Onset of bright light Audiogenic stimulus Vigorous shaking of cage
Seizure can be myoclonic seizures (7 to 10 weeks) Model for petit mal epilepsy
Generelized tonic clonic in older animals Model for tonic clonic epilepsy
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CONCLUSION
Ideal model of epilepsy should show the following characteristics Development of spontaneously occurring
seizures Type of seizure similar to that seen in human
epilepsy EEG correlates of epileptic –like activity Age dependency in the onset of epilepsy as seen
in many epileptic syndromes At present no model follows all criteria Only genetic model come close to call ideal Resemble idiopathic epilepsy in humans
more closely than any other experimental model
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The antiepileptic drug development program primarily based on two seizure model, the MES and the s.c. PTZ
Single method of screening of antiepileptic drugs can not predict the full pharmacological profile of the drug.
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REFERENCES Hans GV. Drug Discovery and Evaluation:Pharmacological
Assays. Springer. 3rd edition. New York :Springer-Verlag Berlin Heidelberg ; 2008.
Gupta SK. Drug Screening Methods (Preclinical Evaluation of New Drugs). 2nd edition.New Delhi:Jaypee Brothers Medical Publishers; 2009.
Wolfgang L. Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs. Seizure. 2011(20):359–368.
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THANK YOU !