Dr. Jitendra Agrawal

Second year resident

ANIMAL MODELS FOR ANTIEPILEPTIC DRUGS

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.

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

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

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

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.

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.

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

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

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

DISADVANTAGE

Do not give clue about mechanism of action of drug

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

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

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

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.

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

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.

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

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

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.

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

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

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

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

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

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

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

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

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.

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.

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

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

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

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

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

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

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

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.

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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