ANIMAL MODELS FOR

ANTIEPILEPTIC DRUGS

Dr. Jitendra Agrawal

Second year resident

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
Electrically induced
Seizure

Chemically induced
Seizure

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 SE
Induction
( Pilocarpine)

Genetic animal Model

Animals with
spontaneous
Recurrent seizures

Animal with reflex

seizures

e.g. Rats or Mice with

Spike wave discharge
(lethargic
mice,tottering mice)

e.g. DBA/2 Mice

GEPRs,
Photosensetive
baboons,Gebrils

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.

METHODOLOGY

An electro-convulsiometer with Corneal or Ear

electrodes is used to deliver the shock.

Current used:
o Rat :
150mA
o Mice :
50 mA
o 0.2 second duration

The PHASES of maximal seizure shown by normal mice typically

consists of :
Phase
Full

of tonic limb flexion

extension of limbs

Clonic
Death

video

interval ( variable )

(in some animals)

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 :

Route : Intravenous.

The tonic convulsions appear in all treated rats

1 mg/kg

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 nonNMDA 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

(270400 g)

The rats are implanted with an

electrode in the right amygdala

After 1 week electrical

stimulation 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 1 st 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 eletrode
In right angular bundle
Unipolar reccording electrode
In right hippocampal dentate granule
Pathway is stimulated by
2mA 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 rats
Betamethasone 0.4mg/kg i.p. two doses
at 8:oo am and 6:00 pm on gestational day15

Postnatal day 15
Pups
NMDA 15mg/kg ip
Twisting movements of tail, arching for several seconds
Finally 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

Model

for tonic clonic epilepsy

Generelized tonic clonic in older animals

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):359368.

THANK YOU !