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
Dr. Jitendra AgrawalSecond year resident
INTRODUCTION A Seizure (from the Latin sacire, “To take possessionof”) is a paroxysmal event due toabnormal, excessive, hyper synchronous dischargesfrom an aggregate of central nervous system (CNS)neurons. Epilepsy describes a condition in which a person hasreccurent seizures due to a chronic, underlying process.
WHY WE NEED ANIMAL MODEL? Discovery of new AED Characterization of spectrum of anticonvulsantactivity of new AED Evaluation whether efficacy of new AED changesduring chronic treatment of epilepsy Discovery of antiepileptogenic or disease modifyingagent
Models for EpilepsyInduction of Seizure in normal animal Genetic animal ModelElectrically inducedSeizureChemically induced Seizure Animals with spontaneousRecurrent seizuresAcute inducedSeizureMES PTZChronic induced SeizureElectrical or Chemical KindlingPost Epilepticus model with spontaneous recurrent seizuresElectrical SEInduction(Perforanth path)Chemical SEInduction( Pilocarpine)e.g. Rats or Mice with Spikewave discharge(lethargic mice,totteringmice)e.g. DBA/2 MiceGEPRs, Photosensetivebaboons,GebrilsAnimal with reflex seizures
ELECTROSHOCK SEIZURES IN MICE & RATS Protection against electroshock induced seizures in mice andrats is used as an indication for compounds which may proveeffective in Generalized tonic clonic seizures Electric stimuli evoke tonic hind limb extensions, which aresuppressed 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 intensephysiologically possible seizure by a method analogous to humanelectroshock therapy.
METHODOLOGY ANIMALS: Groups of 6-10 male Swiss mice (20-32g) or Wistarrats (100-150g) are used. ROUTE OF DRUG ADMINISTRATION:i. Intraperitonealii. Oral 30 min after i.p. injection and 60 min after oraladministration the animals are subjected to electroshock.
An electro-convulsiometer with Corneal or Earelectrodes is used to deliver the shock. Current used:o Rat : 150mAo Mice : 50 mAo 0.2 second durationMETHODOLOGY
The PHASES of maximal seizure shown by normal mice typicallyconsists 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 ofeficcacy Calculation of ED50 for supression of tonic hind limbextension – 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 asynchronizedclonic movements which are superceded by tonic convulsionscharacterized by flexion of limbs followed by extension. Act by antagonizing the inhibitory GABAergic transmission The test is considered as indicative of anticonvulsant activity ofdrugs 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 seizuresequence 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 clonicseizure Ethosuximide, valproate – effective Phenytoin, Carbamazepine – not effective
STRYCHNINE INDUCED SEIZURES The convulsant action of strychnine is due to interferencewith post-synaptic inhibition that is mediated by Glycine. It acts as a selective competitive antagonist to block theinhibitory 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 ofanimals is noted. Strychnine abolishes the flexor latency completely, leading toalmost instantaneous onset of the extensor seizure.
PICROTOXIN-INDUCED CONVULSIONS Picrotoxin is a and it modifies thefunction of chloride ion channel of the GABA receptorcomplex. Dose : 3.5 mg/kg Route : subcutaneous
BICUCULINE TESTS IN RATS Bicuculine is a Dose : 1 mg/kg Route : Intravenous. The tonic convulsions appear in all treated ratswithin 30 seconds of injection.
4-AMINOPYRIDINE INDUCED SEIZURES INMICE 4-Aminopyridine, is a powerfulconvulsant. 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 canlead 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 monkeyneocortex One or two month after injection spontaneous and recurrent seizuresbegins 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 theanticonvulsant activity on the basis of pathophysiologicalmodel. Kindling results from repetitive sub convulsive electricalstimulation of certain areas of brain . On continued stimulation electrical activity spreads andgeneralized convulsions occur. The animals are given stimulation through an electrodeimplanted with in right amygdala.
Adult female Sprague-Dawleyrats (270–400 g)The rats are implanted with anelectrode in the right amygdalaAfter 1 week electricalstimulation of the brain is startedOther brain areaslikeNeocortex, hippocampus in rats
Duration and amplitude, behavioral seizure duration andseizure stage are recorded Seizure severity is graded into 5 stages. 1: immobility, eye closure, twitching of vibrissae, sterotypingsniffing 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 bygeneralized clonic seizures Rats are considered to be kindled on the 1st stimulationcausing a stage 5 seizure which is followed by at least twoconsecutive stage 5 seizures
EVALUATION Test animals are tested on the day before and afterthe test compound is given orally or i.p. Test and control are compared with four differentmeasures of efficacy Seizure latency – time from stimulation to the first signof seizure activity Seizure severity Seizure duration After discharge duration
Drug efficacy can be measured by determiningseparate 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 providesmodel for effective in secondary generalizedseizures of partial epilepsy Efficacy against the focal component of kindledseizures provides a valid model for drugs effectivein complex partial seizures
OTHER METHODS OF KINDLING Corneal Electroshock kindling Mice: once daily application of 3 mA current 60 Hz for 2sec Rat : once daily application of 8 mA current 60 Hz for 4sec 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 downepisodes 6 – generelized convulsions with tonic extension episodesand 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 bundleUnipolar reccording electrodeIn right hippocampal dentate granulePathway is stimulated by2mA monopolar pulse for 50mcs, 20 Hz, for 2 hDevelopment 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 modelPregnant sprague-dawley ratsBetamethasone – 0.4mg/kg i.p. two dosesat 8:oo am and 6:00 pm on gestational day15Postnatal day 15PupsNMDA 15mg/kg ipTwisting movements of tail, arching for several secondsFinally loss of righting reflexFlexion spasms with multiple recurrences.
GENETIC ANIMAL MODEL FOR EPILEPSY Totterer Mice: Homozygous (tg/tg) strain totterer mice are prone tospontaneous epileptic seizure Broad based ataxic gate By 3 to 4 weeks of age → develop frequent partialseizure Spontaneous focal motor seizure occur a few times aday → unilateral clonic jerk of limbs with secondarygeneralization Also exhibit absence seizure with synchronous 6-7 persecond 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 issimilar 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 soundinduced seizures Susceptibility gradually declines → at the 8 weektotally free of audiogenic seizures Exposed to loud sound (12-16 kHz) Seizure pattern → wild running phase → clonicconvulsion → tonic extension → respiratory arrest/ fullrecovery 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 stimulationat frequencies close to 25flashes/second leads toseizure Eyelid, face, and bodyclonus and subsequentlytonic spasms or full tonicclonic convulsions Model for tonic clonicseizure, myoclonic seizure
MONGOLIAN GEBRILS Seizure can be provoked by Placing animal in newenvioronment 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 followingcharacteristics 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 inmany epileptic syndromes At present no model follows all criteria Only genetic model come close to call ideal Resemble idiopathic epilepsy in humans moreclosely than any other experimental model
The antiepileptic drug development programprimarily based on two seizure model, the MES andthe s.c. PTZ Single method of screening of antiepileptic drugscan not predict the full pharmacological profile ofthe drug.
REFERENCES Hans GV. Drug Discovery and Evaluation:PharmacologicalAssays. Springer. 3rd edition. New York :Springer-Verlag BerlinHeidelberg ; 2008. Gupta SK. Drug Screening Methods (Preclinical Evaluation ofNew Drugs). 2nd edition.New Delhi:Jaypee Brothers MedicalPublishers; 2009. Wolfgang L. Critical review of current animal models of seizuresand epilepsy used in the discovery and development of newantiepileptic drugs. Seizure. 2011(20):359–368.