Explanation of Preclinical (Animal) Models of Seizure and Epilepsy. General overview of Seizure and Epilepsy and its current Management. Need to develop newer drugs and Newer models. Current models for Acute Seizure. Kindling explained. PPT contains overview and Protocol.

1. Screening of antiepileptic agents and
Demonstration of anticonvulsant action of
Sodium Valproate by PTZ and MES methods
Moderator: Prof. Jagriti Bhatia
Presenter: Dr. Pranav Sopory
Overview & Presentation

2. Contents
1. Overview: Seizure & Epilepsy
2. Current models in use
3. The Maximal Electroshock Seizure (MES) model
4. The Pentylenetetrazol (PTZ) model
5. Acute models
6. Drug resistant models

3. Overview: Definitions
Seizure: Latin - sacire - “to take possession of”
• A paroxysmal event due to abnormal, excessive hypersynchronous discharges
from an aggregate of central nervous system.
Epilepsy: Greek - epilepsia – “taking hold of”
• Characterized by two or more unprovoked seizures
• Which can’t be explained by a medical condition such as fever or substance
withdrawal

4. Overview: Definitions
Convulsion:
An intense paroxysm of involuntary repetitive muscular contractions
Seizure:
1. Convulsive seizure or motor seizure
2. Non convulsive seizure:
Sensory seizure, psychic seizure, autonomic seizure
All seizures are not convulsions.

5. Overview: Classification
Types of Epileptic Seizures
A. Partial (focal) seizures
1. Simple partial (with motor, sensory, autonomic, or psychic signs; consciousness is not
impaired )
2. Complex partial (consciousness in impaired)
3. Partial seizures evolving to secondarily generalized seizures
B. Primarily generalized seizures
1. Absence (petit mal) 4. Tonic
2. Myoclonic 5. Tonic-Clonic (grand mal)
3. Clonic 6. Atonic
C. Unclassified seizures
1. Neonatal seizures
2. Infantile spasms

6. Overview: Classification
Types of Epilepsy Syndromes
A. Juvenile Myoclonic Epilepsy (JME)
• Generalized seizure disorder of unknown cause, appearing in early adolescence chr. by:
1. B/L myoclonic jerks: maybe single or repetitive
2. Progressing to GTCS eventually
3. ~ 30 % : Absence seizure +ve also
B. Lennox-Gustaut Syndrome (LGS)
Severe form of epilepsy beginning at ~ 4 yr. of age
Multiple seizure types: GTCS, atonic, atypical absence and myoclonus
Impaired cognitive functioning seen in most cases
C. Mesial Temporal Lobe Syndrome (MTLS)
M/C epilepsy syndrome
Associated with Complex partial seizures

7. Overview: Etiology
1. Defective synaptic functioning
2. Genetic inheritance
3. Individual susceptibility for threshold
Overview: Pathology
1. Definable brain lesions
2. Epileptogenesis
Overview: Diagnosis
1. EEG
2. Assisted Video with EEG
3. Radiology: CT / MRI / PET Scan

8. Overview: Treatment
Goal:
Prevent further seizures
Aim:
Decrease frequency
Avoid adverse effects of AEDs
Enable patients to lead active lives
Initiate AED after a single episode of seizure: Controversial
Start if identifiable lesions is +ve
Eg. CNS tumour, Trauma, CNS infection, Stroke

9. Overview: AEDs available
Pathology: Overstimulation on Brain
Rx: DEPRESS IT !
1. GABA: ↑
2. Glutamine: ↓
3. Na+: ↓
4. Ca+: ↓

10. I. Drugs ↑ GABA

11. I. Drugs ↑ GABA
1. Drugs + GABA release (from vesicle)
• GABAPENTINE
• PREGABLIN
• Useful in Partial (focal) seizures
• DOC:
• Diabetic Neuropathy
• Post-Herpetic Neuralgia

12. I. Drugs ↑ GABA
2. Drugs # GABA metabolism (# GABA Transaminase)
• VIGABATRIN
• DOC: Infantile spasm in Tuberous
Sclerosis
• Used mainly for Infantile Spasm
• Seen M/C in West Syndrome
• DOC: ACTH
• S/E: Visual field defects

13. I. Drugs ↑ GABA
3. Drugs # GABA reuptake (# GAT 1)
• TIAGABINE
• Used in Focal Seizures

14. Overview: GABA Cl- ion channel

15. I. Drugs ↑ GABA
4. Drugs acting on channel:
Barbiturates Benzodiazepines
GABAmimetic: same action
as GABA.
Can work without GABA
GABAfacilitating: helps endogenous GABA
attach to its receptors.
Cannot work without GABA
Increases duration of single
opening
Increases the frequency of opening
Phenobaritone Diazepam, Lorazepam, Clonazepam, Clobazam
etc.
• BZD used to Rx Acute seizures
DOC: Status Epilepticus:
Lorazepam>Diazepam
• DOC: Febrile Seizure: Diazepam (P/R)
• Px: Febrile Seizure: Clobazam
• DOC Catamenial Epilepsy
• Rx: Absence Seizure: Clonazepam
• Rx: Lennox-Gustat Synd: Clobazam

16. Substances acting on GABA Cl- ion channel
1. GABA
• Endogenous agonist
2. Positive Allosteric modulation:
• Benzodiazepines
• Muscle relaxants
• Alcohol
• Propofol
• Etomidate
3. Negative Allosteric Modulation
• Flumazenil
Anxiolytics
Anticonvulsant
Sedation
Amnesia
Muscle relaxation
BZ1 modulation
(Only sedative effects)
• Zolpidem
• Zaleplon
• Zolpielone

17. II. Drugs ↓ Glutamate
Glutamate: Excitatory NTM acting via NMDA
• NMDA #
Drug Use
Felbamate Only NMDA # previously approved for Epilepsy
Now discontinued due to Aplastic anemia
Methadone Opioid deaddiction
Acamprosate Alcohol craving
Memantine Alzheimer’s disease
Riluzole DOC: Amyotropic Lateral Sclerosis
Ketamine Anesthetic agent
LSD Psychotropic usage

18. III. Drugs # Na+ channel
Drugs Uses
Topiramate GTCS, atypical absence, myoclonic seizures
Zonisamide Partial Seizures
Carbamazepine GTCS, partial seizures, trigeminal neuralgia,
bipolar mania
Phenytoin GTCS
Rufinamide Partial seizures, lennox-gustaut syndrome
Lancosamide Partial seizures, lennox-gustaut syndrome

19. IV. Drugs # Ca+ channel
Ethosuximide
Only used in Absence seizures
V. Unconfirmed MOA
Valproic acid
Lamotrigine
GABA: ↑, Glutamine: ↓, Na+: ↓, Ca+: ↓
Use in any seizure

20. VI. Recent advances
1. K+ channel opener
• Ezogapine
2. AMPA #
• Parempanel
• Terampanel
Approved for use in Partial seizures

21. Overview: Treatment
Primary GTCS Partial Seizures Absence Seizures Atypical Absence,
Myoclonic and
Atonic Seizures
First-line agents
Valproic acid
Lamotrigine
Topiramate
Carbamazepine
Phenytoin
Oxcarbazepine
Valproic acid
Valproic acid
Ethosuximide
Valproic acid
Lamotrigine
Topiramate
Alternative agents
Zonisamide*
Phenytoin
Carbamazepine
Oxcarbamazeine
Phenobarbital
Primidone
Felbamate
Levetiracetam*
Topiramate
Tiagabine*
Zonisamide*
Gabapentin
Phenobarbital
Primidone
Felbamate
Eslicarbazepine
Vigabatrin
Lacosamide
Pregablin
Rufinamide
Lamotrigine
Clonazepam
Clonazepam
Felbamate
* Adjunctive treatment

22. Valproic Acid
Chemically: Carboxylic Acid
Dissociates in GIT: Valproate ion
True MOA:
Unknown
Increases GABA conc. in brain after use
Correlation: possible not absolute
Dosage and administration:
Initial dose: 10-15 mg/kg/day
↑ by 5-10 mg/kg/week to achieve clinical response (CR)
If total dose >250 mg/day: Give in divided doses
If CR not achieved at 60 mg/ day :
↑ R/O adverse effects
If CR not achieved: Measure Plasma Conc.
Therapeutic Range: 50-100 mcg/ml

23. Valproic Acid
Uses apart from Epilepsy
1. Migraine
Prophylaxis
250 mg PO q12h
2. Bipolar Mania
Rx of Manic episodes
750 mg / day PO in divided doses

24. AEDs: Adverse effects
Drugs Principal side effect Serious adverse effect
Valproate thrombocytopenia, GI effects hepatotoxicity, pancreatitis
Clobazam sedation, dizziness, irritability, depression
Felbamate anorexia, GI symptoms, insomnia, nausea,
headache
aplastic anemia, hepatic
failure
Gabapentin weight gain, behavioral changes, somnolence,
ataxia, dizziness
Lamotrigine headache, tremor, vomiting, insomnia allergic toxin reaction (SJS)
Tiagabine confusion, dizziness, GI upset, anorexia,
stupor
weakness
Topiramate cognitive disturbance, renal stones,
paresthesia, glaucoma, tremor, renal calculi
weight loss, language
dysfunction
Vigabatrin depression, psychosis, depression, weight
gain, tremor
irreversible visual field
defects

25. 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
(Perforant pathway)
Chemical SE
Induction
( Pilocarpine)
e.g. Rats or Mice with Spike
wave discharge
(lethargic mice,tottering
mice)
e.g. DBA/2 Mice
Photosensetive
baboons,Gebrils
Animal with reflex
seizures

26. Maximal Electroshock Seizure: Overview
• Analogous to Electro Convulsive Therapy (ECT).
• Considered Gold Standard
• High correlation between ability of a drug to inhibit MES and its effectiveness in GTCS in humans
• Principle:
• Rodents receive an electrical stimulus of sufficient intensity to induce maximal seizure of hind
limbs
• A stimulus about 5-10 times higher than the seizure threshold of the rodent is applied.
• Standardized Parameters:
1. Apply transauricular electrodes
2. Fixed current: 50 mA (mice) or 150 mA (rats)
3. 50 - 60 Hz pulse frequency
4. 0.6 ms pulse width
5. 0.2 s stimulus duration

27. Maximal Electroshock Seizure
Animal recovers to its upright position and starts
moving around apparently recovering its normal
behaviour
Paddling movement of hind limbs and shaking of
the body
Immediate severe Tonic Seizure with maximal
extension of the forelimb and hindlimb
Stimulus given 0.2 s
10-15 s
20 s
Tonic phase
Clonus phase
Phases Time period Significance

28. MES: Seizure Score
4: Post ictal depression
3: THLE: Total hindlimb extension
2: Complete forelimb extension and partial hindlimb extension
1: Forelimb extension without hindlimb extension
0: No Seizure

29. Maximal Electroshock Seizure
• Positive test:
Animals exhibit Tonic extensor seizure with hind limb extension (HLE) at > 90° from
the body sustained for more > 3s
• Measure efficacy of AED:
1. Determined by its ability to abolish THLE
2. Prolongation of latent phase and
3. Decrease in the duration of tonic phase.
MES is used to screen for AEDs active against GTCS

30. Pentylenetetrazol (PTZ) model
PTZ/ metrazol/ pentetrazol
• Binds at Picrotoxin binding site on GABA receptor
• was originally used as a cardiostimulant.
• Dose: (acute model)
• For rats: 60 mg/kg and for mice: 90 mg/kg

31. Pentylenetetrazol (PTZ) model
Death
Tonic extension: tonic contraction of the muscle
as demonstrated by extension of hind limbs
backward and parallel to the surface
Clonus: Forelimb (with/without righting
reflex)
Straub’s tail: tail is held rigidly
perpendicular to the surface
Myoclonic jerks: brief involuntary jerking
of body
Phases
I (5min)
II
III(9 min)
IV
V(18 min)
Seizure score
3: Clonus
2: Straub’s tail
1: Myoclonic jerks
0: No activity

32. Other Models
1. Strychnine induced seizures
2. Picrotoxin induced seizures
3. Isoniazod induced convulsions
4. Bicuculine test in rats
5. 4 – Aminopyridine induced seizures
6. Tetanus toxin as a model

33. Strychnine Induced Seizures
• MOA: Antagonist of Glycine (End result: Excitation)
• It prevents the inhibitory effects of glycine on the postsynaptic
neuron.
• When the inhibitory signals are prevented, the motor neurons are
more easily activated and the animal will have spastic muscle
contractions
• The convulsions has a characteristic Motor pattern.
• Dose: 2 mg/kg
• Route: i.p.

34. Picrotoxin-induced Convulsions
• Picrotoxin inhibits Presynaptic Inhibition mechanism of GABA channel
• # EPSP
• Dose : 3.5 mg/kg
• Route : subcutaneous

35. Isoniazid-induced Convulsions
• INH + GABA Transaminase
• It is known to produce convulsions
in patients with a history of seizure disorders.
• The typical pattern is of tonic-clonic seizures
• Dose : 300 mg/kg
• Route : subcutaneous

36. Bicuculine Tests In Rats
• Bicuculine is a competitive antagonist of GABAA receptors
• Dose : 1 mg/kg
• Route : Intravenous.
• The tonic convulsions appear in all treated rats within 30 seconds of
injection.

37. • 4-Aminopyridine, K+ channel antagonist is a powerful convulsant
• Increased Glutamate release via non-NMDA type excitatory amino acid receptors.
• Dose :13.3 mg/kg
• Route : Subcutaneous
• Commercial use:
• Pesticide use
• Trade name Avitrol as 0.5% or 1%.
• Medical use
• Fampridine has been used clinically in Lambert-Eaton myasthenic syndrome and
multiple sclerosis. It acts by blocking potassium channels, prolonging action potentials
and thereby increasing neurotransmitter release at the neuromuscular junction.
4-Aminopyridine induced seizures in mice

38. Tetanus toxin as a model for Chronic Epilepsy

39. • Tetanus toxin was first used to create chronic epileptiform events in 1962.
• Local application of tetanus toxin to the brains of experimental animals, there
seems to be a relatively short latency period prior to the clinical and
electrographic onset of chronic seizures.
• Onset of spontaneous and recurrent focal seizures in the cat hippocampus, orbital
frontal cortex, and motor cortex within two to three weeks. In addition, tetanus
toxin induces seizure foci which may remain chronically active.
• Observations of the lesions produced in the cat hippocampus confirm that the
lesions are relatively small areas of necrosis and reactive gliosis.
Tetanus toxin as a model for Chronic Epilepsy

40. • Small amounts of radioactively labelled tetanus toxin indicated that the toxin
remains confined to the site of injection.
• The lesion is well confined to the hippocampus, substantia nigra, thalamus,
caudate, cerebral cortex (region unspecified), and motor cortex.
• It induces a chronic epileptogenic focus in a relatively short time. These features
make it an attractive and potentially superior experimental model of focal
epilepsy.
Tetanus toxin as a model for Chronic Epilepsy

41. Kindling

42. Kindling
• Kindling is a chronic model of epilepsy where repetitive and
intermittent administration of sub-convulsant
chemical/electrical stimuli can lead to progressive
amplification of seizures, culminating in generalized seizure
activity (Goddard et al., 1969) (McNamara, 1984)
• Kindling produces enduring changes in the brain:
• Esp. Decrease in population of GABAnergic neurons in epilepsy-
relevant brain regions
• Where:
• Repetitive seizures cause neuronal death in highly susceptible areas,
such as parts of the hippocampus, amygdala, thalamus

43. Kindling
• Different regions of the brain respond differently to
subthreshold stimulation
• Electrical stimulation for kindling:
1. Amygdala: 15 days
2. Hippocampus: 53 days
• PTZ model:
1. Acute: 60-90 mg/kg X single dose
2. Chronic: 20-40 mg/kg X 14 days

44. Animal models of drug-resistant epilepsy
Considering 25-40% of epilepsy patients are refractory to treatment, it is
important to study the effect of drugs in models that are resistant to the
drugs.
Models with limited or poor drug response
6-Hz model
Lamotrigine-resistant kindled rats
Methylazoxymethanol (MAM) model

45. 1. 6-Hz model
The model is based on electrical stimulation of mice via corneal electrodes
with low-frequency pulses (6-Hz) for 3 seconds, which results in
immobility, forelimb clonus, and behavioral automatisms, reflecting seizure
characteristics of human limbic epilepsy.

46. 1. 6-Hz model
Drug responsiveness differs depending on the current intensity:
• At 32 mA, the sensitivity to phenytoin, lamotrigine, carbamazepine, and topira- mate proved to be
reduced with no efficacy at all or ED50 levels close to or even above TD50 levels (Barton et al.,
2001).
• In contrast, phenobarbital, ethosuximide, valproic acid, felbamate, tiagabine, and levetiracetam
displayed dose-dependent protection in a dosing range not associated with severe adverse effects
(Barton et al., 2001).
• At 44 mA only, two antiepileptic drugs, levetiracetam and valproic acid, resulted in complete
protection (Barton et al., 2001).
• Subsequent studies revealed efficacy of lacosamide using the 32 mA stimulation intensity, and
retigabine, brivaracetam, and several test compounds at 32 and 44 mA (Loscher, 2011; Duncan
and Kohn, 2005).
• Shannon and colleagues (Shannon et al., 2005) have also assessed the efficacy of several
antiepileptic drugs in the 6-Hz model.

47. 1. 6-Hz model
• Based on these findings, the 6-Hz seizure model has been implemented in
the early phase of the NIH anti- convulsant drug screening program, such
that drugs failing to demonstrate efficacy in the MES or PTZ test are
tested a second time in the 6-Hz test.
• Considering available pharmacological data, the 6-Hz model is clearly
characterized by a poor response to classic modulators of sodium
channels which primarily target fast inactivation of voltage-gated sodium
channels.
• However, it should be noted that as an acute model, it is unlikely to
reflect chronic network, cellular, and molecular alterations that might
contribute to therapeutic failure in drug- resistant epilepsy.

48. Lamotrigine-resistant kindled rats
• Kindling is based on repeated administration of the convulsant
pentylenetetrazole (Srivastava et al., 2004).
• Using this experimental approach, lamotrigine pretreatment during
kindling development, reduced drug responsiveness was not only reported
for lamotrigine but also for carbamazepine, phenytoin, and topiramate.
• Considering that the concept of reduction in the drug response occurs in
response to sub-chronic drug treatment, this might suggest that the
approach is rather based on mechanisms of tolerance and cross-tolerance
development than those of multi- drug resistance.

49. Methylazoxymethanol (MAM) model
• Epilepsy associated with neuronal migration disorders in paediatric patients is
often characterized by a poor drug response or mere drug refractoriness.
• Therefore, efforts have been made to develop animal models mimicking
cortical dysplasias.
• In rats, treatment with methylazoxymethanol acetate (MAM) on gestational
day 15 produces a neuronal migration disorder with histological features
including cortical laminar disorganisation and ectopic neurons in subcortical
white matter, cortical layer I and CA1 sub- field of the hippocampus
(Germano and Sperber, 1998).
• Resistance to valproate was demonstrated in rats exposed to the convulsant
kainic acid. Whereas val- proate prolonged seizure latency in control animals,
no such effect was observed in MAM-exposed animals.

50. Methylazoxymethanol (MAM) model
• In this model of transplacentally induced neuronal migration disorder, the
efficacy of different antiepilep- tic drugs has been assessed (Smyth et al.,
2002). Resistance to valproate was demonstrated in rats exposed to the
convulsant kainic acid.
• Whereas valproate prolonged seizure latency in control animals, no such
effect was observed in MAM-exposed animals. In addition to in vivo
drug testing, the authors evaluated the response to phenobarbital,
carbamazepine, valproate, ethosuximide, and lamotrigine in an ex vivo
hippocampal slice preparation (Smyth et al., 2002).

51. Anticonvulsant activity of sodium valporate by
Pentylenetetrazole (PTZ) method
Ethical clearance
Male Wistar rats and mice were procured from Central Animal
Facility, All India Institute of Medical Sciences, New Delhi. The
protocol was approved by the Institutional Animal Ethics
Committee (125/ IAEC/2005)

52. Requirements
•Animal: Wistar Rats
•Sex/Body weight: Male (200-250g)
•Drugs: Pentylenetetrazole (60 mg/kg i.p.) and Sodium valproate
(300 mg/kg i.p.), both drugs are obtained from Sigma Aldrich and
dissloved in norrmal saline
•Syringe: 1ml
•Equipments: Weighing balance and Stop watch

53. Procedure
• Weigh the animals and mark properly
• Divide animals into two groups
• Group1 (Control group); rats will be given Normal saline as
per body weight
• Group 2 (Experimental group); rats will be given Sodium
valproate (300mg/kg, i.p.)

54. •After 30 minutes, all the rats will be given PTZ (60 mg/kg, i.p.)
•Following parameters will be observed:
 Latency to Myoclonic jerk
Latency to GTCS
Duration of GTCS

55. Observations
Animal Marking Body
Wt.
(g)
Dose Treatment Latency to
Myoclonic
jerk
Latency to
GTCS
Duration
of GTCS
1
2
3
4
5
6

56. Anticonvulsant activity of sodium valporate by
maximum electro-shock (MES) method
Requirements
Animal Mice
Sex/Body weight Male (20-30 g)
Drug Sodium valproate (600 mg/kg)
Syringe 1mL
Equipment Electro convulsiometer
Ear electrode
Stop watch
Weighing balance

57. Procedure
• Weigh the animals and mark properly
• Divide animals into two groups
• Group1 (Control group); Mice will be given Normal saline as
per body weight
• Group2 (Experimental group); mice will be given Sodium
valproate (600 mg/kg, i.p.)
• After 30 minutes mice will be given electroshock at the
intensity of 50 mA, for 0.2 sec

58. Procedure cont’d.
• Electric stimulation applied via ear electrode
• Electrodes will be moistened with normal saline before
application
• Note different stages of convulsion
 Tonic flexion
 Clonic convulsion
 Tonic hind limb extension
 Recovery or death
• Tonic hind limb extension is taken as end point
• Record the latency and duration of tonic hind limb extension

59. Observations
Animal Marking Body
Wt. (g)
Treatment Dose THLE
Seen
(Y/N)
Latency
to
THLE
Duration of
THLE
1
2
3
4
5
6

60. Summary of models
Model Induction Manifestations Human
relevance
Use Limitations
Acute
chemical
models
Systemic
PTZ
induced
injection
Nonconvulsive
absence or
generalized
tonic-clonic
seizures,
depending on
the drug and
dose
Acute and
repetitive
seizures
Rapid screening
of AED, effect of
repetitive
seizures
Lack of
spontaneous
recurrent
seizures and
of neuronal
loss
Electroshock
-induced
seizures
Corneal or
auricular
stimulatio
n
Generalized
tonic-clonic
seizures
Tonic-
clonic
seizures
AED screening Low
predictive
validity for
some AEDs

61. Precautions
• Lab should be dim lighted and noise free
• Animals should be marked properly, to avoid mixing in two
groups
• Handle the animal with care (minimize the stress and pain to
animal)
• After PTZ injection, animals should be kept under wired cages
to observe seizure activity and to restrict their movement.
• Female are more resistant to seizure , hence should be avoided

62. Need for new models of preclinical screening
1. Old models identify only drugs that share characteristics with
existing drugs, and are unlikely to have an effect on
refractory epilepsies
2. The efficacy against drug-resistant seizures are not detected
by seizure screening models.

63. Thank You
?