2. 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.
3. 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
4. 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 SE
Induction
( Pilocarpine)
e.g. Rats or Mice with Spike
wave discharge
(lethargic mice,tottering
mice)
e.g. DBA/2 Mice
GEPRs, Photosensetive
baboons,Gebrils
Animal with reflex seizures
5. 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
6. 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.
7. 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.
8. 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
METHODOLOGY
9. 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
10. 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
12. 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
13. 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
14. 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
15. 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.
16. PICROTOXIN-INDUCED CONVULSIONS
Picrotoxin is a and it modifies the
function of chloride ion channel of the GABA receptor
complex.
Dose : 3.5 mg/kg
Route : subcutaneous
17. BICUCULINE TESTS IN RATS
Bicuculine is a
Dose : 1 mg/kg
Route : Intravenous.
The tonic convulsions appear in all treated rats
within 30 seconds of injection.
18. 4-AMINOPYRIDINE INDUCED SEIZURES IN
MICE
4-Aminopyridine, 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
19. 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
20. 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.
21. Adult female Sprague-Dawley
rats (270–400 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, hippo
campus in rats
22. 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
23. 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
24. 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
25. 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
26. 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
27. 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
28. 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
29. 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.
30. 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.
31. 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
32. 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
33. 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
34. 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
35. 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
36. 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
37.
38. 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
39. 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.
40. 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.
Mutation in myelin protien ZeroA new spontaneous recessive mutation has been discovered and characterized at The Jackson Laboratory. Mice affected by the new totterer mutation have a neuromuscular phenotype causing a shaky gait. PCR and sequence analysis determined that this is a mutation in the myelin protein zero (Mpz) gene.
Lethargic (lh) mice are spontaneous mutant mice presenting a loss of β4 subunit of voltage-gated calcium channels (VGCC) due to a mutation in the gene encoding for β4, Cacnb4. These lh mice are characterized by a complex phenotype with notably severe neurobehavioral defects