IN-VIVO SCREENING METHODS FOR NEURODEGENERATIVE DISEASE.pptx

SCREENING MOTHODS FOR
NEURODEGENERATIVE DISEASE
Presented by: Gautamkumar Sosa
Mpharm Pharmacology sem-1
1

Content
PARKINSON
1. INTRODUCTION
2. TYPES
3. ETIOLOGY
4. PATHOPHYSIOLOGY
5. DRUG CLASSIFICATION
6. SCREENING MODELS
ALZHEIMER
1. INTRODUCTION
2. PATHOGENESIS
3. DRUG CLASSIFICATION
4. SCREENING MODELS
5. MONITORING PARAMETER
MULTIPLE SCLEROSIS
1. INTODUCTION
2. TYPES OF MS
3. PATHOPHYSIOLOGY AND CAUSE OF MS
4. MEDICATION
5. SCREENING MODELS
REFERENCES
2

Parkinson Disease
Introduction
• Parkinson’s disease is a slowly progressing neurologic movement disorder that eventually leads
to disability.
• The degenerative or idiopathic form is the most common; there is also a secondary form with a
known or suspected cause. Although the cause of most cases is unknown, research suggests
several causative factors, including genetics, atherosclerosis, excessive accumulation of
oxygen free radicals, viral infections, head trauma, chronic antipsychotic medication use, and
some environmental exposures.
• Parkinsonian symptoms usually first appear in the fifth decade of life; however, cases have been
diagnosed at the age of 30 years. It is the fourth most common neurodegenerative disease.
• Parkinson’s disease affects men more frequently than women and nearly 1% of the population
older than 60 years of age (Gray & Hildebrand, 2000).
Sign and Symptoms
• Involuntary tremors
• Pill rolling movement of fingers and nodding of head
• Bradykinesia (difficulty and slowness of movements)
• Rigidity of muscles
3

Cont...
• Jerk during voluntary movement
• Aphonia (inability to speak)
 eg. Muhammad Ali
 Cause of disease: The cause of selective degeneration of nigrostriatal neurones is not precisely
known, but appears to be multifactorial.
• Ageing, genetic predisposition, oxidative generation of free radicals, N-methyl-4-phenyl
tetrahydropyridine (MPTP)-like environmental toxins and excitotoxic neuronal death due to NMDA-
receptor (excitatory glutamate receptor) mediated Ca2+ overload have all been held responsible.
4

Types
1. Primary or Idiopathic Parkinson’s:
• Cause of the disease is unknown
• Over 60 years of age
• Over the years the dopaminergic neurons degenerate due to hydrogen peroxide and free radicals
and OONO-(Peroxynitrite).
2. Secondary or Drug Induced Parkinson’s
• No degeneration of neurons but decrease ion dopamine is drug induced
7

Photomicrograph of regions of substantia nigra in a Parkinson’s patient showing Lewy bodies and Lewy neurites in various magnifications. Top
panels show a 60x magnification of the alpha-synuclein intraneuronal inclusions aggregated to form Lewy bodies. The bottom panels are 20×
magnification images that show strand-like Lewy neurites and rounded Lewy bodies of various sizes.
9

Pathophysiology of Parkinson
10

1. MPTP
• It is byproduct of the illicit manufacture of synthetic meperidine derivative.
MPTP
(1,2,3,6-methyl-phenyl-
Tertahydopyridine)
• MPPC is taken up by dopaminergic neurons and cause mitochondrial complex I (ERC) defect similar
to that found in PD.
2. Alpha-synuclein
• Abnormal accumulation of this protein in brain cells cause damage by binding with ubiquitin in the
damaged cells.
• This protein accumulation forms proteinaceous cytoplasmic inclusions called lewy bodies.
• Latest research shows that death of dopaminergic neurons by alpha-synuclein is due to defect
in machinery that transports proteins between two major organelles-the endoplasmic reticulum
(ER) and Golgi apparatus.
MAO-B MPPC
(pyridinium ion)
12

3. Oxidative stress:
• Because of oxidative metabolism of dopamine to yield hydrogen peroxide H2O2 and other reactive
oxygen species (ROS).
• Oxidative stress and consequent cell death could develop in SNc under such circumstances in
which there is :
a) increase dopamine turnover resulting in excess peroxide formation.
b) A deficiency in glutathione thereby diminishing brain capacity to clear H2O2.
c) increase in reactive iron which can promote OH- radical formation.
13

SCREENING METHODS
In-vivo models In-vitro models
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SCREENING MODEL
In-vivo Models
1. Tremorine and Oxotremorine antagonism
2. MPTP model in Monkeys
3. Reserpine antagonism
4. Circling behavior in nigrostriatal lesioned rats
5. Elevated body swing test
6. Skilled paw reaching in rats
7. Stepping test in rats
8. Gait analysis
9. Rotenone Induced Parkinsonism
10. Transgenic Animal Models of Parkinson’s Disease
11. Cell Transplantations into Lesioned Animals
12. Transfer of Glial Cell Line-Derived Neurotrophic Factor (GDNF)
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In-vitro Models
1. Experiment using rat strial slices
2. Dopamine stimulated Adenyl cyclase activity
3. Culture of Substantia Nigra
4. Inhibition of Apoptosis in Neuroblastoma SH-SY5Y Cells
5. Radioligand binding studies for D1 and D2 dopamine receptor
6. In vitro neuro protective efficacy
16

1. Tremorine and Oxotremorine antagonism
PURPOSE AND RATIONALE
• The muscarinic agonists tremorine and oxotremorine induce parkinsonism-like signs such as
tremor, ataxia, spasticity, salivation, lacrimation and hypothermia. These signs are antagonized
by anticholinergic drugs.
PROCEDURE:
• Groups of 6–10 male NMRI mice weighing 18–22 g are used.
• They are dosed orally with the test compound or the standard (5 mg/kg benzatropine mesilate) 1
h prior the administration of 0.5 mg/kg oxotremorine s.c.
• Rectal temperature is measured before administration of the compound (basal value) and 1, 2 and
3 h after oxotremorine injection.
• Tremor is scored after oxotremorine dosage in
10 s observation periods every 15 min for 1 h.
Tremor Score
absent 0
slight 1
medium 2
severe 3
18
• Salivation and lacrimation are scored 15 and
30 min after oxotremorine injection.
Salivation Score
absent 0
slight 1
medium 2
severe 3

cont...
19
Drug Groups An
no
.
An
wt
(g)
before drug
administration
after drug administration
Body
temp.
(℃)
Salivati
on &
Lacrim
ation
Tremor (min) Salivation &
Lacrimation
Body temp.
(℃)
15
min
30
min
45
min
15 min 30 min 1h 2h 3h
Saline Normal
benzatropin
e mesilate
(5 mg/kg)
Standard
Test Drug Test1
Test2

Cont...
EVALUATION
• Hypothermia: The differences of body temperature after 1, 2 and 3 h versus basal values are
summarized for each animal in the control group and the test groups. The average values are
compared statistically.
• Tremor: The scores for all animals in each group at the 3 observation periods are summarized.
The numbers in the treated groups are expressed as percentage of the number of the control group.
• Salivation and lacrimation: The scores for both symptoms for all animals in each group are
summarized at the 2 observation periods. The numbers in the treated groups are expressed as
percentage of the number of the control group.
MODIFICATION
1. Matthews and Chiou (1979) developed a method for quantifying resting tremors in a rat model of
limb dyskinesias. The model involved permanent cannulation of the caudate nucleus for the
introduction of carbachol. Tremors were quantified with a small transducer and an electronic
data collecting system. The system allows the construction of dose-response curves for
tremor inhibition by potential antiparkinsonism drugs.
2. Johnson et al. (1986) developed a procedure for quantifying whole-body tremors in mice.
Displacement of a free floating platform by animal movement created a change in resistance across
a strain gauge.
20

• Administration of oxotremorine, 2.5 mg/kg, i.p, produced numerous high frequency, high-
intensity peaks within 5 min.
3. Clement and Dyck (1989) constructed and tested a tremor monitor that quantitates soman- and
oxotremorine-induced tremors. The device consisted of a force transducer, from which a plastic
beaker was suspended containing a mouse. The signal from the force transducer was fed into
a tremor monitor and quantitated using the Applecounter from Columbus Instruments.
4. Coward et al. (1977) recommended N-carbamoyl-2-(2,6-dichlorophenyl)acetamidine
hydrochloride (LON-954), a tremorigenic agent, as alternative to oxotremorine for the detection
of anti-Parkinson drugs.
5. Rats treated with 3-acetylpyridine show a selective degeneration of neurons in the inferior olive
nucleus and the olivo-cerebellar tract with characteristic motor incoordination and ataxia (Denk et
al. 1968), Watanabe et al. 1997), Kinoshita et al. 1998). Similar motor dysfunction is seen in
patients with olivo-ponto-cerebellar atrophy. To measure the effect of 3-acetylpyridine and the
ameliorating effect of TRH agonists in rats the maximal height of vertical jump after stimulation
by a foot shock was measured.
6. Stanford an Fowler (1997) used a special technique for measuring forelimb tremor in rats induced
by low doses of physostigmine. The rats pressed a force-sensing operandum while a computer
measured force output and performed Fourier analyses on resulting force time waveforms. 21

2. MPTP model in Monkeys
PURPOSE AND RATIONALE:
• N-MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) has been shown to cause symptoms of
Parkinson’s disease in exposed individuals. When administered to primates this compound
causes a partial destruction of basal ganglia and a syndrome that resembles Parkinson’s
disease.
• It is byproduct of the illicit manufacture of synthetic meperidine derivative.
MPTP
(1,2,3,6-methyl-phenyl-
Tertahydopyridine)
• MPPC is taken up by dopaminergic neurons and cause mitochondrial complex I (ERC) defect similar
to that found in PD.
MAO-B
MPPC
(pyridinium ion)
22

Cont...
PROCEDURE:
Take 8 rhesus monkey (5-8 kg) of either sex.
Administered the N-MPTP I.V. with cumulative dose up to 10-18 mg/kg for 5-8 days.
Produces PD like symptoms
Administered test drug
Symptoms are Evaluated
EVALUATION:
• The severity of parkinsonian symptoms is rated by trained observers using a scale of 0 (normal) to 17
(maximum severity) that assesses movement (0: normal; 1: reduced; 2: sleepy),
• checking movements (0: present; 1: reduced; 2: absent),
• attention and blinking (0: normal; 1: abnormal),
• posture (0: normal; 1: abnormal trunk; 2: abnormal trunk and tail; 3: abnormal trunk, tail, and limbs; 4: flexed
posture),
• balance and coordination (0: normal; 1: impaired; 2: unstable; 4: falls),
• reactions (0: normal; 1: reduced; 2: slow; 3: absent) and vocalizations (0: normal; 1: reduced; 2: absent).
24

25
Drug Groups An
no
.
An
wt
(g)
before drug administration after drug administration
ass
ess
es
ch
ec
kin
g
at
te
nti
o
n
&
bli
nk
p
o
s
t
u
r
e
balan
ce &
coor
re
act
ion
s
vo
ca
liz
ati
on
asses
ses
check
ing
attentio
n &
blink
p
o
s
t
u
r
e
balanc
e &
coor
re
act
ion
s
vo
cal
iza
tio
n
Saline Normal
benzatropin
e mesilate
(5 mg/kg)
Standard
Test Drug Test1
Test2

3. Reserpine antagonism
• Reserpine induces depletion of central catecholamine stores.
• The sedative effect can be observed in mice shortly after injection,followed by signs of eyelid
ptosis, hypokinesia, rigidity, catatonia, and immobility. These phenomena can be antagonized
by dopamine.
REQUIREMENTS:
• Animal: male NMRI mice
• Drug - Chloral hydrate (350mg/kg I.P.), Reserpine (5mg/kg I.P.)
• Apparatus- photocell
PROCEDURE:
Male NMRI mice of either sex are taken 20-25g.
reserpine (5mg/kg i.p.) injected to mice and tested 24hrs later
30min. Prior to observation test compound is injected
26

Animals are placed singly on to floor of Perspex container (30×26×20 cm high.) which situated on
panlab proximally sensor unit.
Horizontal movements are recorded for 10min.
Rearing & grooming episodes are registered.
EVALUATION:
• Locomotors activity & grooming scores of test group is compared with control group.
27

4. Circling Behavior in Nigrostriatal Lesioned
• Unilateral lesion of the dopaminergic nigrostriatal pathway in the rat by the neurotoxin 6-
hydroxydopamine (6-OHDA) induces hypersensitivity of the postsynaptic dopaminergic
receptors in the striatum of the lesioned side.
• The rats rotate in a direction towards the lesioned side (ipsilateral) when an indirect acting
compound such as amphetamine is administered, but to the opposite direction (contralateral)
when a directly acting dopamine agonist,e.g.,apomorphine, dopamineprecursor L-dopa is given.
28

• Therefore, this test can be used for the study of central dopamine function and the evaluation of
dopamine antagonists and agonists, particularly the activity of novel antiparkinsonian drugs.
• An imbalance of dopaminergic activity within the basal ganglia is associated with markedly
asymmetric circling behavior (Rotation turning) which measured by Rota meter.
• This model is used in drug induced rotating behavior and understanding of extra pyramidal disorder
& of their treatment by dopaminergic agents.
REQUIREMENTS:
• Animal - Male Wistar rats(200-250gm)
• Dose - 6-OHDA neurotoxin 6-hydroxydopamine(8µg in 4µL of 0.2mg/ml ascorbic acid in saline ) &
test drug
• Apparatus - Rotameter
29

PROCEDURE:
Male Wistar rats rats are taken
Rats are anesthetized with Pentobarbital (60mg/kg)
Head is placed in stereotaxic device a sagittal cut is made in the skin of the skull, a 2-mm-wide hole is
drilled with an electrical trepan drill.
A 30-gauge stainless-steel cannula connected to a Hamilton syringe is aimed at the anterior zona
compacta of the substantia nigra.
A total of 8µg of 6-OHDA in 4µL of saline is injected at a rate of 1µL/4min
After the intracranial injection the wound is closed. The animal is allowed several weeks for recovery
and for development of the lesion.
30

31
stereotaxic device for rat and mouse

Rats are divided into groups:
• Control groups for base value of ipsilateral rotation -2.5mg/kg of d-amphetamine injected i.p. to
rats.
• Control groups for base value of contralateral rotation- 1mg/kg of Apomorphine injected i.p. to
rats.
• Test compounds are given ip or s.c. and the animals placed into the circling chambers. Circling is
recorded over a 1hr period.
• Further studied test group as compared to control group.
• No. of full turns( either ipsilateral Or contralateral turning to lesion) are recorded an automatic print
out counter every 15 min. for one or two hr session.
OBSERVATION:
• For ipsilateral turning : - administer 2.5 mg/kg Amphetamine & placed in circling chamber for 2 hour
• For contralateral turning: - administer 1 mg/kg apomorpine & placed in circling chamber for hour
• Test compound are given i.p. or s.c. & record reading with 15 min. interval for 1 or 2 hr.
EVALUATION :% change of drug turns from control turns is recorded. 32

5. Elevation body swing test
• EBST measures asymmetrical motor behavior of hemiparkinsonian animals in a drug free state
& drug induced state.
• Drug induced motor behavior widely used as-behavioral index of hemiparkinsonian animals.
• High positive co-relations between swing & Apomorphine induced rotational behavior.
REQUIREMENTS:
• Animal - Sprague Dawley rats
• Drug - 6-OHDA (8 µg in 4µL 0.9% saline containing 0.02% ascorbic acid).
• Test drug.
PROCEDURE:
40 rats are taken as test group(8 weeks old sprague dawley rats)
Anesthetised with sod. Pentobarbital(60mg/kg i.p.) mounted in stereotaxic device
stereotaxically lesioned in left substantia nigra
34

6-OHDA solution are injected over 4min. & needle left in place for an additional 5 min. before retraction
7 days after lesion, behavioral testing is performed.
Remaining 24 animal served as-control group.
The animals are placed into a plexiglass box (40x40×35.5 cm.)
OBSERVATION:
• A swing is recorded whenever the animal moves its head out of vertical axis. swing are counted for
60 sec. with interval of 15sec. 35

EVALUATION:
%LEFT SWING= Σno. Of swing towards left side
Σ L+R
%RIGHT SWING = Σno. Of swing toward right side
Σ L+R
Sr.No. 15 sec 30 sec 45 sec 60 sec
LS RS LS RS LS RS LS RS
36

6. Skilled paw reaching test
• This method used to evaluate symptoms and treatment in rat by skilled reaching with fore paw for
food.
• Unilateral DA depletion reduces success by abnormalities in movements including changing in
posture.
REQUIREMENTS:
• Animal - long Evans rats (250-310gm)
• Dose - Desmethyl Imipramine (25 mg/kg i.p.), 6-OHDA (2ul of 4mg/ml in 0.95% saline with 0.02%
ascorbic acid)
• Equipment- Single pellet boxes (25×35×30cm) Food tray
• boxes(10x18x10cm)
PROCEDURE:
20 rats are taken
30minute before surgery, desmethyl imipramine administered (25mg/kg i.p.)
37

Rats anesthetized with pentabarbital(60mg/kg i.p.)
12 rats received 6-OHDA lesions but 8 rats not received
38

• The apparatus consists of a clear Perspex chamber with a hinged lid.
• A narrower compartment with a central platform running along its length, creating a trough on
either side, is connected to the chamber
• The narrowness of the side compartment prevents rats from turning around, so they can use only
their left paw for reaching into the left trough and their right paw for reaching into the right trough.
• A removable double staircase is inserted into the end of the box,sliding into the troughs on either
side of the central platform.
• Each of the steps of the staircase contains a small well, and two 45mg saccharin-flavored pellets
are placed in each well.
Learning procedure
• The week before the start of the training period, the rats are deprived of food and their body
weight is stabilized at 85% of the weight of non-deprived rats.
• At the same time, they are gently manipulated and familiarized with the appetitive saccharin-
flavored pellets.
• The animals then begin to learn the paw reaching task. For 4 weeks they are placed in the test
boxes once per day for 10-15min.
39

• The number of pellets eaten during the test period indicates the rat's success in grasping and
retrieving the pellets; the number of steps from which pellets have been removed provides an
index of the attempts to reach the food and how far the rat can reach the number of missed
pellets remaining at the end of the test on the floor of the side compartment indicates a lack of
sensorimotor coordination in grasping and retrieving the pellets.
Lesions
• The mesotelencephalic system is lesioned by a stereotaxic unilateral injection of 6-OHDA into
the medial forebrain bundle under equithesin anesthesia.
• 6OHDA is injected in a volume of 1.5µl and at a concentration of 4µg/µl of 0.9% saline and 0.01%
ascorbic acid twice over 3min via 30-gaugestainless steel cannula.
Drug Treatment
• The animals are injected i.p. with the test drug or saline 30min before the unilateral 6-OHDA
lesion and 24h thereafter.
40

Scoring reaching success:
• Reaching performance are scored by counting misses and successful reaches for each limb.
1. Scored as "reach".
2. Scored as "hit".
Success% = no. of reaches × 100
no. of hit
Reaching posture
• Two point scale
• Scored as 0
• Scored as 1
EVALUATION
• Test sessions are performed 4, 5, 7 and 8 weeks after 6-OHDA lesion. The parameters success,
attempts and sensorimotor coordination are subjected to a two-way ANOVA with group as the
independent measure and weeks as the dependent measure.
41

MODIFICATIONS OF THE METHOD
• Fricker et al. (1996) investigated the effect of unilat_x0002_eral ibotenic acid lesions in the dorsal striatum, placed at either
anterior, posterior, medial, or lateral loci, in the staircase test of skilled forelimb use.
• Nakao et al. (1996) studied paw-reaching ability in rats with unilateral quinolinic acid lesions of the striatum as an animal
model for Huntington’s disease.
• Barneoud et al. (1996) evaluated the neuroprotective effects of riluzole using impaired skilled forelimb use, circling
behavior, and altered dopaminergic metabolism of the mesotelencephalic system in unilaterally 6-hydroxydopamine-
lesioned rats.
• Fricker et al. (1997) studied the correlation between positron emission tomography, using ligands to the D1 and D2
receptors, and reaching behavior in rats with ibotenic acid lesions and embryonic striatal grafts.
• Grabowski et al. (1993), Marston et al. (1995) Shar_x0002_key et al. (1996) tested drug effects on skilled motor deficits
produced by middle cerebral artery occlusion in rats using the paw reaching test.
• Meyer et al. (1997) described a revolving food pellet test for measuring sensorimotor performance in rats. An animal model
of Huntington’s disease was recommended by Borlongan et al. (1997). Systemic administration of 3-nitropropionic acid, an
inhibitor of the mitochondrial citric acid cycle, produces a very selective striatal degeneration and results in a
progressive locomotor deterioration resembling that of Huntington’s disease.
• Intrastriatal injection of quinolate, a NMDA receptor agonist, replicates many neurochemical, histological, and behavioral
features of Huntington’s disease (Beal et al. 1986; DiFigla 1990; Pérez-Navarro 2000).
42

7. Stepping Test in Rats
• This model is clinically relevant to unilateral model for parkinsonism akinesia.
• The 6-OHDA lesion induced marked and longlasting impairments in the initiation of stepping
movements with the contralateral paw which can be ameliorated by application of drug.
REQUIREMENTS:
• Animal: Sprague Drawley rats ,
• Dose: 6-hydroxydopamine (3.6µg/µl in 0.2 µg/ml Ascorbate saline),
• test drug
PROCEDURE:
• 6-OHDA Lesion Surgery Female Sprague Dawley rats receive two stereotaxic injections of 6-
OHDA (3.6µg/µl in 0.2µg/ml ascorbate-saline) into the right ascending mesostriatal dopamine
pathway using a 10-µl Hamilton syringe.
• The cannula is left in place for an additional 5min before slowly retracted.
• The tests monitoring initiation time, stepping time and step length are performed using a wooden
ramp with a length of 1m connected to the rat's home cage.
• A smooth-surfaced table is used for measuring adjusted steps. 43

• During the first 3 days the rats are handled by the experimenter to familiarize them with the
experimenter’s grip.
• During the subsequent 1-2days the rats are trained to run spontaneously up the ramp to the
home cage.
1) the time to initiation of a movement of each forelimb, the step length, and the time required for
the rat to cover a set distance along the ramp with each forelimb.
2) the initiation of adjusting steps by each forelimb when the animal was moved side ways along
the bench surface.
The stepping test comprises two parts:
• Each test consists of two tests per day for three consecutive days and the mean of six subtests is
calculated.
EVALUATION PARAMETER:
• Initiation time, Stepping Time, Step length.
• Step length = Length of ramp / no. of steps
• Sequence of testing in right paw & followed by left paw testing, repeated twice.
44

• Stepping test comprises of 2 parts :
1. Each test consists of 2 tests per day for 3 consecutive days
2. The mean of six subtests is calculated.
45

8. Gait analysis
• Gait analysis is useful in objective assessment of walking ability and identify cause for walking
abnormalities in parkinsonism disease.
• The result of gait analysis is useful in determining best course of treatment.
• Catwalk method is mostly used to analyze gait in lab.
46

9. Rotenone induced parkinsonism
Principle :
• Chronic systemic complex 1st inhibition caused by Rotenone exposure induces of parkinsonism in
rats inducing selective nigrostriatal dopaminergic degeneration and formation of ubiquitin & α-
synuclein inclusion.
47

10. Transgenic animal models
Purpose and Rationale :
• The most prominent models are related to α-synuclein.
• The first transgenic mice that express human α-synuclein were generated by Masliah et al.
(2000).
• These mice displayed a progressive accumulation of α-synuclein and ubiquitin-immune reactive
inclusions in neurons of the neocortex, hippocampus, and substantia nigra.
• These derivatives were associated with a loss of dopaminergic terminals in the basal ganglia and
with motor impairment.
48

1. EXPERIMENT USING RAT STAITAL SLICES
• Striatum in brain is primarily affected in parkinsonism. The release of the neurotransmitter like
dopamine and acetylcholine in response to test agent serve as a good in-vitro marker of its
activity.
Male spargue dawley rats(150-250g)are decapitated,the skull is opened.
Right and left striata are removed & placed in ice-cold krebs solution.
The striata is cut into 0.4mm thick slices using a tissue chopper.
The slices are kept floating for 30 min in krebs solution & gassed with 95% O2 & 5% CO2 at room
temperature
The slices are labeled by incubating for 30 min at 37℃ with [3H]dopamine (5µg/ml) &[14c] choline
(2 µg/ml)in the presence of 0.15 mM pargyline chloride & 0.1mM ascorbic acid.
50

Labeled slices are transferred to superfusion chambers & perfused with krebs solutions at 37℃ at
flow rate of 0.5ml/min
After washing & stabilization 5min fraction of superfusate are collected.
The perfusion buffer contains 1 µM nomifensine to inhibit dopamine reuptake & 10 µM
hemicholinium to inhibit choline uptake.
The slices are subjected to field strength to the current strength of 10-15 mA/cm2 & pulse duration
of 2 m/sec at stimulation frequency of 3Hz for 5min.
Drugs to be tested are present in the superfusion fluid.
The radioactivity in the superfusate samples & in the tissue is determined by liquid scintillation
counting. 51

• The radiolabelled choline method makes it possible to study Ach release in-vitro without
inhibiting cholinestrase, thus minimizing auto inhibition of transmitter release caused by
accumulation of unhydrolised of Ach.
52

2. DOPOMINE STUMILATED ADENYLY CYCLASE ACTIVITY
Male sprague-dawley Rats(150-250g) are decapitated & right & left striata are removed.
Striatal tissue is homogenized by teflon homogenizer in chilled buffer containing 10mM imidazole,
2mM EDTA & 10% sucrose pH 7.3.
Homogenate is centrifuged at 1000 g for 10min & supernatant is recentifuged at 27000g for 20min.
The pellet obtained is washed twice & suspended in 10mM imidazole, pH 7.3. Membrane protein is
determined by Bradford’s method using bovine serum.
Adenylyl cyclase activity is measured by calculating the conversion rate of (32p) ATP to (32p)
cAMP.
The assay is perform in 250µl solution containing imidazole, MgCl2, papaverine dithiothreitol,
ATP,GTP, phosphocreatine, creatine phosphokinase.
53

The reaction mixture is preincubated at 30℃ for 5min, the reaction is initiated by adding membrane
proteins and incubated for 10min.
The reaction is terminated by adding stopping solution(ATP,SDS). Formed (32p) cAMP is separated
from (32p) ATP by chromatography.
54

Alzheimer
Introduction:
• Alzheimer's disease (AD), also known as the most common form of dementia, is a progressive,
neurodegenerative disorder in adults, afflicting more than 35 million individuals worldwide.
• Extensive extracellular deposits of β-amyloid (Aβ) plaques, intracellular neurofibrillary tangles
(NFTs), as well as subsequent neuronal and synaptic loss, which often begin several years prior to
memory loss and the damage is alread irreversible at the time of diagnosis.
• Study of presymptomatic stages in human is difficult so there is Animal models by which we can
study presymptomatic stage and screen the anti alzheimer drugs using In-vivo and In-vitro medols.
55

• In pathological terms, the AD-affected brain is characterized by widespread senile plaques of
extracellular Aβ peptides, intracellular NFTs, as well as neuronal and synaptic loss.
• Senile plaque is composed of a central core formed by Congo red-positive Aβ proteins and
degenerating nerve endings surrounding the Aβ core. NFTs are neuronal inclusions of the
microtubule-associated protein (MAP) tau and consist of aggregated, hyperphosphorylated
tau. In AD pathology, senile plaques and NFTs appear in the hippocampus, the entorhinal and
polymodal association cortices, and the basal forebrain.
• These brain regions are also severely affected by neuronal and synaptic loss. In addition to the
neuropathological changes, there are also a number of pathophysiological disturbances occurring in
the AD-affected brain, including inflammatory reactions, such as angiogenesis and gliogenesis.
• APP, an ubiquitously expressed transmembrane protein, is involved in the regulation of neuronal
proliferation, migration and differentiation under normal conditions. In AD, overexpressed APP is
hydrolyzed by β-secretase and then γ-secretase into Aβ peptides. Aβ peptides are toxic to
neurons and induce neuronal and synaptic damage when assembled into amyloid fibrils.
• It has also been reported that acetylcholinesterase (AChE), an enzyme responsible for the
hydrolysis of the neurotransmitter acetylcholine (ACh), may promote Aβ assembly and form stable
complexes with Aβ fibrils.
57

• The Aβ/AChE complex has proven to be more toxic to cultured neurons than Aβ fibrils alone.
• This may explain why cholinergic neurons are the major cell types destroyed in patients with AD.
• In addition, presenilin (PS)1 and PS2, as well as apolipoprotein E (ApoE) bind to soluble Aβ in-
vitro, and promote the formation of Aβ fibrils in an isoform-specific manner. Aβ fibrils can also
induce tau hyperphosphorylation by activating several protein kinases, including glycogen
synthase kinase-3β (GSK-3β), MAP kinase and microtubule affinity- regulating kinase.
Hyperphosphorylated tau, combined with unassembled microtubules, accumulates to form
NFTs in neurons and dendrites, leading to neuronal degeneration and ultimately, AD.
58

Drug Classification
• Cholinesterase inhibitor- Tacrine, Rivastigmine
• Nootropic Agent- Piracetam
• NMDA receptor antagonist- Memantine
• MAO inhibitor- Selegiline
• Other- Piribedil
• NSAID
60

Classification of models used in Alzheimer's disease :-
• Animal models for AD can be classified into :-
• In-vivo models:-
1. Spontaneous models
2. Transgenic models
3. Chemical induced models
4. Miscellaneous models
• Spontaneous animal model is advantageous in identifying the mechanism of aged related defects in
learning and cognition.
• Transgenic model utilizes the genetic mutation associated with familial AD but is expensive to
develop.
• Chemically induced AD model includes disease development after administering a suitable chemical
compound such as streptozotocin, colchicine, Aβ protein, alcohol, scopolamine, etc.
61
• In-vitro models:-
Tissue models Cultured tissues
1. Brain slices
2. Cell models iPSC
3. Neuroblastoma cell
Molecular simulation model
4. Antibubble
5. bio-machinery

Spontaneous models
• Few species, including dogs, cats, (polar) bears, goats and sheep,as well as several non human primate
species spontaneously develop plaque pathology and some species even exhibit tauopathies.
• Unfortunately, the use of these species for experimental research is limited by availability, economical
(based on long lifespan) and/or ethical reasons. Nevertheless, the dog has been pointed out as an
especially appropriate model for the study of human brain ageing and neurodegenerative diseases in
general, and AD in particular, based on its phylogenetic proximity to humans, the in-depth knowledge of canine
(behavioural) neurology, and the histopathological and molecular similarities between clinical AD and the canine
variant.
• Dogs are ideally suited for longitudinal studies, and have therefore been mainly used to study the beneficial
effects of an antioxidant diet, behavioural enrichment and Aβ immunotherapy.
• Ageing rodents do not spontaneously develop AD-like histopathological hallmarks, and are therefore of no
use to the development of drugs targeting these pathological hallmarks.
• Their contribution to the AD-related drug discovery pipeline is based on the occurrence of senescence-
related cognitive decline and behavioural alterations linked to neurochemical and morphological
alterations including age-associated cholinergic hypofunction.
• In addition, they aid in uncovering the boundary between normal and pathological ageing, allowing in-
depth investigation of basic neural mechanisms underlying brain ageing.
63

• Natural age-associated deterioration has culminated in the senescence accelerated mouse (SAM),
a model which was established through phenotypic selection from a genetic pool of AKR/J mice
in the early 1980s. The SAM model includes nine major SAM-prone (SAMP) substrains and
three major SAM-resistant substrains.
• SAM strains have been extensively used as models for various age-related disorders. SAMP
mice undergo accelerated ageing while SAM-resistant mice disorders.
• Interestingly, genes and proteins that undergo significant alterations in SAMP8 brains are related
to the following functional categories: neuroprotection, signal transduction, immune response,
energy metabolism, mitochondrion, protein folding and degradation, reactive oxygen species
production, cytoskeleton and transport, lipid abnormalities and cholinergic dysfunction.
• The SAMP8 strain has proven to be a relevant model for AD and several treatment strategies
have been studied in these mice, including antioxidants, antisense oligonucleotides, directed
at the Aβ region of the amyloid precursor protein (APP) gene, consistent with the notion that
SAMP8 cognitive changes are associated with Aβ-associated oxidative stress.
• Besides pharmacological interventions, dietary restriction as a way to increase lifespan and
improve health, and its effect on various functional categories that are affected in SAMP8 with
ageing.
64

Transgenic models
• Animal models used in preclinical studies can be distinguished in:
I. Tg models of AD, consisting in single or multi Tg animals overexpressing APP, PS and/or Tau
mutations
II. Non-Tg models obtained by toxins injection in the brain, including direct injection of Aβ or tau,
and models of aging.
• Most of the Tg models are mice, whereas non-Tg models could also be rats, dogs and monkeys.
• Mice could be considered a good model of Aβ hyperproduction, they did not resemble other
features of an AD human brain. Thus, these are excellent models to better understand the
pathophysiologic role of Aβ in AD or to test drugs aimed to modulate or reduce Aβ levels, but they
might not be appropriate for the study of other aspects of AD since they lack other relevant yet
critical factors.
65

• The advantages with using these mice consist in:
• (i) Their well-known characterization (they have been used in several laboratories as a model of
AD for almost 20 years)
• (ii) The relatively simple management of the colony (good fertility when using Tg2576 males and
C57Bl/6 females, easier genotyping of a single transgene).
• The disadvantage is that the AD phenotype occurs late. Indeed, we usually wait the age of 12
months to perform experiments to be sure that animals present both synaptic and memory
dysfunction.
• Conversely, long-term memory and basal synaptic transmission (BST) were impaired at 6
months, as amyloid burden increases.
• As for single APP, there is conflicting literature on the emotional changes in APP/PS1 mice. Some
studies have demonstrated normal fear and anxiety levels, whereas others decreased anxiety in
APP/PS1 mice.
• These mice have the advantage of presenting the AD-related phenotype at early age. However,
they do not show some aspects of the disease such as neuronal loss and tau deposition.
• Histopathological changes, synaptic dysfunction, memory loss and other behavioral are not the only
features of the disease that can be mimicked using both Tg and non-Tg models of the disease. 67

• For instance there is extensive evidence from these animal models suggesting a key role of proinflammatory
cytokine overproduction as a possible driving force for progression of pathology in AD.
• Non-Tg models for the study of AD are mainly obtained by injecting Aβ or tau directly into the brain via
intracerebroventricular (i.c.v.) or intrahippocampal injections.
• Moreover both acute Aβ infusion models and transgenic APP models have limitations and, unfortunately,
resemble some but not all the features of the human disease.
• For instance, while Tg mice mostly reflect genetic forms of the disease because they overexpress mutated
forms of APP, AD is primarily a sporadic disorder. This can be partially mimicked in-vivo by icv or
intrahippocampal injections of Aβ, even if they do not reflect neither the concentration nor the time course of
changes seen in humans.
• For these reasons, we believe it is better to combine both Tg and non-Tg models to overcome limitations of
the different models.
• The use of acute injections, for instance, gives the possibility to better understand how Aβ impairs specific
signaling pathways leading to synaptic and memory dysfunctions, and this is crucial when designing new
therapeutic strategies.
• Additionally, acute injection could be used to identify the targets of specific soluble Aβ species (from monomers
to oligomers of different molecular weights) since they might exert a different role in synaptic plasticity and
memory impairment.
68

• In this case, Tg mice do not represent a good tool because they overproduce different Aβ forms
(monomers, dimers, trimers, oligomers, fibrils up to plaques) making very difficult the evaluation of
the specific pathogenic role of these aggregates.
• Intrahippocampal or icv injections of a specific Aβ species, in turn, are a more appropriate model
than transgenic models.
• Non-Tg models allows:-
 To investigate the effects of Aβ and tau in animals for which Tg models are not available.
 To exclude the confounding effects of overexpression of APP and its fragments.
 To investigate the different role of Aβ and tau species (monomers vs. oligomers vs. insoluble) at
different concentrations.
 To investigate the difference between an acute or a chronic administration (in this last case one
could also implant mini-pumps for a chronic delivery of the peptide).
 To clarify aspects of the molecular mechanisms underlying Aβ and tau pathology that cannot be
investigated using Tg models.
• Drawbacks :-
 Disease progression cannot be occurred at single administration.
 Time consumption and invasive nature make this model tedious.
69

Chemically induced animal models :
1. Scopolamine induced memory deficit
2. Colchicine induced memory impairment
3. Streptozotocin Induced Dementia
4. Alcohol induced memory deficit
5. Aβ induced memory deficit
6. L-Methionine induced dementia
7. Excitotoxins, neurotoxins, cholinotoxins induced memory deficit
8. Sodium azide induced dementia
9. Heavy metal induced dementia
10. Benzodiazepine induced memory deficit
11. Okadaic Acid-Induced Memory Impairment
Miscellaneous animal models
70

1. Scopolamine induced memory deficit :-
• Scopolamine is an anti-cholinergic drug commonly used experimental drug for cognition
impairments.
• Scopolamine blocks the binding site of acetylcholine (Ach) muscarinic receptors in cortex and
excessive ACh causes damage of hippocampus receptors in cortex. It causes memory and
learning impairment in dose dependent manner.
• Scopolamine induced memory deficit is widely used because of the noninvolvement of any
surgical procedures.
• Infusions of scopolamine into the hippocampus blocks LTP and impairs spatial encoding and
infusion into the medial septum impairs spatial learning and reduces ACh release in the
hippocampus.
• Biological effects of scopolamine on tasks evaluating learning and memory were seen at higher
(0.03 mg/kg) systemic doses.
• When higher doses (>0.03mg/kg) are used, deficits in other cognitive and non-cognitive functions
(e.g., learning and memory, locomotor activity) are reported.
• Several behavioral processes (taste aversion, anxiety, short-term memory, attention) are found to
be affected after intracerebral injections of scopolamine. 72

• Effects on learning and memory performance which are observed after higher doses of
scopolamine are mediated by
1. primary effects on attention and sensory/stimulus discrimination,
2. non-specific effects on behavior (e.g., locomotor activity, anxiety),
3. peripheral side-effects (e.g., pupil dilation, salivation)
73

2. Colchicine induced memory impairment :-
• Colchicine induces dementia by loss of cholinergic neurons either by destruction of cholinergic
pathways or by decrease in cholinergic turnover. It also causes decline in dopamine, nor-
adrenaline, serotonin in cerebral cortex, caudate nucleus and hippocampus.
• In animal models of central nervous system damage, colchicine, a microtubule disrupting agent,
is used as a neurotoxin.
• ICV (intracerebral ventricularly) infusion 0.3 µg of colchicine significantly impaired the memory with
decrease in norepinephrine, Dopamine and serotonin level in cerebral cortex and hippocampus.
• Increased level of protein carbonyls, lipid peroxidation leading to oxidative stress may also be
a reason for memory loss by colchicine administration.
• Increased expression of cyclooxygenase [COX]-1 and 2 also contribute to the colchicine induced
memory deficit.
• Dopamine, norepinephrine and Serotonin is involved in plural process supporting learning and
memory.
• Decreased level of Dopamine and Serotonin is associated with AD. Central administration of
colchicine also causes loss of cholinergic neurons and cognitive dysfunction that is associated with
excessive free radical generation. 75

• The major benefit of this model is that it simulates definite features of sporadic dementia of
Alzheimer’s type (SDAT) in humans such as time dependent alterations in behavioral, biochemical
pattern and onset.
• The drawback of this model is that it is time-consuming, necessitates the use of many animals due
to high mortality rate.
76

3. Streptozotocin Induced Dementia:-
• STZ is a glucosamine nitrosourea compound present in the strain of Streptomyces achromogenes. It
is an alkylating agent mimicking some properties of nitrosourea, an anticancer agent and has
hyperglycaemic effect independent of its role in memory impairment.
• Streptozotocin, when injected intracerebroventricularly (ICV) in a subdiabetogenic dose (3mg/kg)
in rat, causes prolonged impairment in learning and memory.
• After ICV administration, STZ it reaches the fornix and passes into the 3rd ventricle because of the
flow of CSF in a rostrocaudal direction. STZ when administered ICV damages the
septohippocampal system whereby memory impairment in rat could occur due to direct damage
to the system.
• After ICV administration of STZ severe abnormalities in brain glucose and energy metabolism
have been found.
• Tau protein is hyperphosphorylated as a long-term consequence of STZ ICV administration and it
also causes neuronal damage and cell loss as well as the accumulation of Aβ in the brain.
• STZ impairs the glycolytic enzyme activity in brain which leads to decline in ATP and creatine
phosphate level. This impaired energy system and reduced acetyl CoA synthesis leads to the
defects in cholinergic transmission. STZ in rats have also shown the increased activity of AChE
and lesser ACh.
77

4. Alcohol induced memory deficit :-
• Ethanol has been reported to cause hippocampus and cholinergic neurons impairment, affect
sensory-motor system, disrupts memory and learning.
• Acute ethanol treatment produces NO excessively which impairs memory and learning,
whereas higher doses of ethanol interfere with glutamatergic system and enhances GABAergic
transmission in memory related areas of brain. It also increases the extracellular level of
adenosine leading to memory impairment.
• Neonatal model of ethanol has also been reported in which memory impairment is induced in
pregnant animal feeding them on ethanol mixed diet.
• This model does not require any surgical procedure but it is very long and time consuming.
• Exposure to ethanol at high doses have been documented to produce retrograde amnesia and
disruption of encoding, storage, consolidation, retrieval potential and impair memory.
• Ethanol might induce destructions of hippocampus-dependent learning and memory and impair of
the cholinergic neuronal system by oxidative stress via the generation of ROS and lipid
peroxidation.
• Acute (0.5–1 g/kg) and chronic (15 w/v%, 2 g/kg, for 24 days) exposure to ethanol also displays
deficits in memory. 79

• The drawback of this model is that the method is very long and time consuming since pregnant
female rats are used.
• Ethanol-mediated memory impairment is significantly related to state dependency, which can also be
affected by the quantity of its exposure.
80

5. Aβ induced memory deficit :-
• Aβ plaque is the major pathological hallmark in AD and direct injection or continuous infusion of
Aβ into the brain causes brain dysfunction, neurodegeneration and learning and memory
impairment.
• Aβ is infused for 14 days into the 3rd ventricle of rat brain causing its accumulation in
hippocampus and cortex.
• Intracerebral or intra-cerebro ventricular (ICV) infusion of Aβ peptides in the rodent brain.
• Aβ oligomer can be administered accurately, using a single stereotactic injection, or chronically,
using injections through an implanted cannula.
• To better simulate the progressive nature of AD, chronic and continuous administration of Aβ peptide
can be achieved by linking an implanted cannula to an osmotic mini-pump or by using a micro-
infusion pump, or with microdialysis.
• It has been established that continuous infusion or acute injection of Aβ into the brain leads to
brain dysfunction followed by neurodegeneration and impairment of learning and memory very
similar to that seen in AD.
• This model is highly specific for screening of drugs used in AD.
81

6. L-Methionine induced dementia :-
• Chronic homocysteine level causes changes in cerebral blood vessels producing impaired cerebral
perfusion, oxidative stress and decrease in nitric oxide (NO) bioavailability.
• Hyperhomocysteinemia also causes vascular dementia and also neurotoxicity by NMDA hyper-
excitation leading to tau hyperphosphorylation and Aβ deposits.
• High doses of L-methionine (8.2 g/kg) administered parenterally.
82

7. Excitotoxins, neurotoxins, cholinotoxins induced memory deficit :-
• Ibo is a heterocyclic amino acid obtained from mushrooms of the Amanita genus and
structurally related to glutamate.
• Ibotenic acid is an excitotoxin as it is a NMDA receptor agonist causing calcium overload in
neurons and neuronal toxicity. Lesion of NBM (nucleus basalis of meynert) (unilateral) by ibotenic
acid is a validated model for AD.
• Ibotenic acid also damages cholinergic neurons in NBM lesions.
• Unilateral injection of Ibo (10 µg/kg /rat, dissolved in 5 µL of artificial cerebrospinal fluid) to
NBM produces considerable memory loss.
• In comparison to kainic acid, Ibo less toxic to the animals and of developing more distinct
lesions, perhaps due to other fundamental biochemical differences and/or faster metabolism.
• Several other cholinotoxin and neurotoxin which produces AD like symptoms are kainic acid,
intracerebroventricular (i.c.v) infusion of quinolinic acid, intraseptal infusion of anti-NGF, NMDA
antagonist dizocilpine infusion, selective cholinergic toxin AF-64A (ethylcholine aziridinium
ion), quisqualic acid, 3-nitropropionic acid and AMPA.
84

• MPTP is a well-identified neurotoxin that destroys dopaminergic neurons and has been used
since decades to study models of PD.
• 6-OHDA cannot cross the BBB, this neurotoxin needs to be administered directly into the target
brain region.
85

8. Sodium azide induced dementia :-
• Sodium azide is an inhibitor of mitochondrial respiratory chain, causing excitotoxicity due to
generation of free radicals, inhibition of aerobic energy metabolism leading to
neurodegeneration and APP dysfunctioning.
• It affects the enzyme acetylcholine transferase (AChT) causing lesser cholinergic inputs but no
loss of cholinergic neurons.
• It increases the AChT, GAP-43 and transferring receptors producing the memory and learning
impairment.
• NaNO3 dose of 12.5 mg/kg per day caused cognitive deficit. The chronic Na azide-induced
mitochondrial poisoning is suitable for producing AD-like symptoms in rats and testing
neuroprotective drug candidates by preventive or curative applications.
86

9. Heavy metal induced dementia :-
• Heavy metals are reported to cause formation of reactive oxygen species (ROS) in brain leading
to development of AD and other neurodegenerative disease.
• Heavy metals known to cause neurotoxicity by ROS generation are Aluminium (Al), Zinc, Cobalt,
Chromium, Iron and Copper. Heavy metals like cadmium, lead and arsenic causes depletion
of glutathione by binding to the sulfhydryl group.
• Zinc causes the dimerization of Aβ while Al interferes with the metabolism of Aβ peptide and
insulin degrading enzyme (IDE).
• Al also causes tau hyperphosphorylation and apoptosis leading to neuronal toxicity in the
hippocampus as in case of AD.
• Pb has been revealed to dysregulate the activity of serine/threonine protein phosphatases in
human neurons.
• Al in drinking water have been reported to induce AD by interfering with Aβ metabolism as it
interacts with insulin degrading enzymes. It has been observed that exposure of Al leads to
accumulation of tau and apoptosis that further causes neuronal dysfunction.
87

10.Benzodiazepine induced memory deficit :-
• Benzodiazepine has been reported to cause suppression of Long Term Potentiation (LTP) which
is involved in maintaining learning and memory.
• Benzodiazepine receptor agonists such as diazepam and lorazapam have been reported to
produce anterograde amnesia, whereas tribenzodiazipine such as alprazolam and triazolam
produce both anterograde and retrograde amnesia in mice.
88

11.Okadaic Acid-Induced Memory Impairment :-
• Okadaic acid (OKA) is one of the chief polyether toxins obtained from marine microalgae which
induces diarrhetic shellfish poisoning.
• ICV administration of OKA-induced memory impairment in rat serves to be a useful test model to
screen anti-dementia drugs.
• Memory impairment induced by intra-hippocampal injection of OKA has been associated with
significant neuropathological changes including a paired helical filament-like phosphorylation of
tau protein, formation of Aβ containing plaque-like structures and hippocampal
neurodegeneration.
• OKA is an exceptionally useful tool for investigating the cellular processes that are modulated by
reversible phosphorylation of proteins as cell division, signal transduction, and memory.
89

• Miscellaneous animal models :-
• Bioactive lipid lysophosphatidic acid (LPA) has been reported to cause neurite retraction in
neuronal cells and produces tau hyperphosphorylation.
• N-nitro-L-arginine and NG-methyl-L-arginine are the nitric oxide inhibitors and have been
reported to show memory impairment and cognitive loss as nitric oxide is involved in the
regulation of learning and memory and through generation of reactive nitrogen species (RNS).
91
NO diffuses to the presynaptic terminal, leading
to enhanced transmitter release.

1. The Morris Water Maze
• The Morris water maze (MWM) is a particularly sensitive task to examine age-related AD like deficits
because it is highly specific for hippocampal function, one of the first and most affected brain regions
in AD.
• A task was developed where rats learn to swim in a water tank to find an escape platform hidden
under the water (Morris 1984).
• As there are no proximal cues to mark the position of the platform, the ability to locate it efficiently
will depend on the use of a configuration of the cues outside the tank.
• Learning is reflected on the shorter latencies to escape and the decrease on the length of the path.
Procedure:-
• Rats are generally used. The apparatus is a circular water tank filled to a depth of 20 cm with 25°C
water.
• Four points equally distributed along the perimeter of the tank serve as starting locations.
• The tank is divided in fourequal quadrants and a smallplatform (19 cm height) is located in the centre
of one of the quadrants.
93

Evaluation:-
• The latency to reach the escape platform is measured during the training days.
• A free-swim trial is generally performed after the training days where the escape platform is removed
and the animal is allowed to swim for 30 s.
94

2. Radial Arm Maze
• This maze consists of 8–17 equally spaced arms radiating from a central platform, which the rodent
has to enter in order to attain a food or water reward placed in some of the arms.
• In this task, the animals guide themselves using spatial cues around the room, with the goal to enter
each arm only once to receive the maximum amount of food or water rewards in the shortest period
of time and with the least amount of effort.
• This maze requires the use of working memory to retain information that is important for a short time
(within trial information), as well as the use of reference memory to retain the general rules of the
task across days.
• Specifically, the animal must be able to remember which arms were baited as well as which it
already entered (working memory), but it also must know to avoid non-baited arms across trials
(reference memory), all of which takes place by being able to successfully encode spatial
information.
• However, while this task permits the examination of both reference and working memory, major
limitations are the use of food or water deprivation in this task, as well as the presence of odor
confounds.
95

3. Radial Arm Water Maze
• A relatively new spatial memory task, the RAWM, has been designed to eliminate the limitations of
the above-mentioned tasks by combining the positive aspects of the MWM and RAM.
• The difference between the MWM and RAWM is that performance in the RAWM entails finding a
platform that is submerged in water located in one of several arms (6–8) in the water bath, compared
to the classic MWM which only has an open swim field.
• This makes the task a bit more difficult, but forces the animal to use spatial cues and working
memory (keeping track of the arms it has already visited) to remember where the platform is located.
97

4. Passive-Avoidance Learning
• In the passive-avoidance learning task, the animal must learn to avoid a mild aversive stimulus
(Punishment), in this case darkness, by remaining in the well-lit side of a two-chamber apparatus
and not entering the dark where it receives the aversive stimulus.
• Note that since rodents innately gravitate to darkness, the animal has to suppress this tendency
through pairing the negative stimulus with the desired compartment.
• Animals that do not remember the aversive stimulus will cross over earlier than animals that
remember. Dependent measures include the median step through latency (latency to cross into the
unsafe side) and the percentage of animals from each experimental group that cross the threshold
within an allocated time.
98

5. Y-Maze
• This test is based on the innate preference of mice to alternate arms when exploring a new
environment. Various modifications are available with different levels of difficulty and different
demands on specific types of cognition.
• In this instance, test animals are placed in a Y-shaped maze for 6–8 min and the number of arms
entered, as well as the sequence of entries, is recorded and a score is calculated to determine
alternation rate (degree of arm entries without repetitions
• A short-term memory version can also be carried out in which one arm of the Y-maze is blocked and
the subject is allowed to explore the two arms for 15–30 min. The animal is then removed from the
maze for a few minutes or up to several hours, depending on the experimental manipulation, and
then placed back into the maze, this time with all arms open, to explore for 5 min.
• Animals with preserved cognitive function will remember the previously blocked arm and will enter
that one first on the second trial. This test can also be repeated a week after the last trial with a delay
time of only 2 min between the trials in order to test long-term memory and the time it takes the
animal to relearn the task.
• Typically measured parameters include the first arm entered, amount of time spent in each arm, and
total number of arm entries. 99

6. T-Maze
• T-maze tasks are incredibly well characterized and are widely used for cognitive behavioral testing in
both mice and rats. Animals are started at the base of the T and allowed to choose one of the goal
arms abutting the other end of the stem.
• If two trials are given in quick succession, on the second trial the rodent tends to choose the arm not
visited before, reflecting memory of the first choice. This is called “spontaneous alternation.”
• This tendency can be reinforced by making the animal hungry and rewarding it with a preferred food
if it alternates. Both spontaneous and rewarded alternations are very sensitive to dysfunction of the
hippocampus, and hence are sensitive to AD-like symptoms, but other brain structures are also
involved.
• Each trial should be completed in less than 2 min, but the total number of trials required will vary
according to statistical and scientific requirements
101

7. Object Recognition
• The object recognition test is based on the natural tendency of rodents to investigate a novel object
instead of a familiar one, as well as their innate tendency to restart exploring when they are
presented with a novel environment.
• The choice to explore the novel object, as well as the reactivation of exploration after object
displacement, reflects the use of learning and recognition memory processes.
• The available object-recognition tasks to test cognition in rodents use different numbers of available
objects and environments in which the animals are tested, as well as types of configuration aimed to
test spatial recognition and novelty, among other things.
• One particular object recognition task that is sensitive to age-related deficits is very suitable to test
AD-related deficits.
• In this task, a rodent is placed in a circular open field filled with different objects (i.e., various plastic
toys of different sizes and shapes) for 6 min. After a series of trials, during which the animal has
habituated to the configuration and properties of the different objects, some of the objects are
switched from one location to another to assess spatial recognition.
• Subsequently, some of the objects are replaced with new ones to evaluate novel object recognition.
The time spent exploring the open field (movement/inactivity) as well as number of times and length
of time inspecting each object over the different trials is calculated.
103

8. Fear Conditioning
• Freezing response, defined as a complete lack of movement, is the innate response of rodents to
fear. In a fear conditioning paradigm, the animal is placed in a box containing a grid that delivers a
mild aversive stimulus for two minutes.
• In the box, the animal is presented with a tone (usually 80 dB) (conditioned stimulus) that is paired
with a mild shock (unconditioned stimulus) at the end of the trial with the result that the tone elicits
the freezing response. Repeated exposures are sometimes necessary depending on the strain used
or the interval time between the tone and the shock.
• Some researchers use trace fear conditioning, which increases the time gap between the tone and
the shock in order to investigate prefrontal cortical activity. Here, the animal is taken out of the box
and returned 24 hr later to evaluate its learned aversion for an environment associated with a mild
aversive stimulus (context-dependent fear) by measuring freezing behavior in the absence of tone or
aversive stimulus.
• Cue-dependent fear can be measured by placing the animal in a new box that is different in color,
shape, etc., and presenting it with the tone as it explores the new environment; freezing behavior
associated with the tone is measured.
104

• Fear conditioning is a widely used test to measure hippocampal_x0002_dependent associative
learning. This test is thought to be sensitive to emotion-associated learning and therefore is a useful
measure of amygdalar–hippocampal communication.
• Many of the transgenic mouse models of AD display impairments in fear and anxiety, which is
primarily a function of the amygdala. The hippocampal function used in fear conditioning may be
different from learning in a spatial task.
105

Multiple Sclerosis
Introduction
• Multiple sclerosis is one of the neurodegenerative disorder in central nervous system that
include brain, spinal cord and nerves.
• Including problem with vision, arm or leg movement, sensation or balance.
Symptoms of MS
 The symptoms of MS vary widely from person to person and can affect any part of the body.
 The main symptoms include:
 fatigue difficulty walking
 vision problems, such as blurred vision problems controlling the bladder
 numbness or tingling in different parts of the body
 muscle stiffness and spasms
 problems with balance and co-ordination problems with thinking, learning and planning
106

 Types of MS
• 1. Relapsing-remitting
• 2. Primary progressive
• 3. Progressive relapsing
• 4. Secondary progressive relapsing
 Pathophysiology of MS
• MS is an autoimmune condition.
• The immune system attacks axon, leads to destruction of myelin sheath in brain and Spinal
cord resulting in Conduction block which leads to permanent loss of function.
• The attacks cause the myelin sheath to become inflamed in small patches (plaques or lesions)
which can be seen on an MRI scan.
• These patches of inflammation can disrupt the messages travelling along the nerves.
107

 CAUSES OF MS
• It's not clear what causes the immune system to attack the myelin sheath.
• It seems likely that it's partly caused by genes you inherit from your parents and partly by outside
factors that may trigger the condition.
• Some of the factors that have been suggested as possible causes of MS include:
I. Genes
II. Lack of sunlight and vitamin D
III. Smoking
IV. Teenage obesity
V. Viral infections(Epstein-Barr virus, responsible for glandular fever)
109

 MEDICATIONS
• Azathioprine
• Cyclophosphamide
• Dalfampridine
• Dexamethasone
• Dimethyl fumarate
• Fingolimod
• Glatiramer
• Mitoxantrone
• Natalizumab
• Prednisolone teriflunomide
110

 Screening methods:
 Invivo Models
• Experimental autoimmune encephalomyeltis
• Viral
• Toxin models
• Transgenic, mutant and parabiotic
• mice
 Invitro Models
• Microglia
• Oligodendrocytes
• Astrocytes
• Neurons
• Brain Slice and Aggregate Systems
111

IN-VIVO TESTS
1. Experimental Autoimmune Encephalomylitis
• EAE is a spectrum of neurological disorders induced in laboratory animals following immunisation
with CNS.
• Antigens emulsified in an adjuvant to augment the immune response.
• These models generally use purified myelin, recombinant proteins or encephalitogenic peptides of
myelinproteins.
• EAE studies used myelin basic protein (MBP) since it is a major protein component of the myelin
sheath and highly soluble, and can therefore be purified relatively easily.
PROCEDURE
Active EAE relies on CNS-reactive T cells that are induced following immunisation by an autoantigen
Emulsified in an adjuvant, while passive or adoptive transfer EAE is induced by transferring the
autoreactive T cells to native recipient animals.
112

Adjuvants used for EAE induction frequently contain killed mycobacteria that elicit T cell responses as
well as antibody production due to innate immune activation via Toll-like receptor triggering
The first adjuvant for EAE studies was an oil-in-water emulsion called Incomplete Freund's Adjuvant
(IFA). Addition of inactivated dried mycobacteria such as M. butyricum, M. tuberculosis or Bordetella
pertussis.
This has led to the development of secondary progressive EAE, models of cortical demyelination and
experimental inflammatory neurodegenerative and spastic diseases.
113

2. Viral
• several mechanisms have been proposed to explain how viruses can induce demyelination, and
may thus be involved in MS. Damage may result either from a direct effect on neurons, in which
case myelin damage occurs as a secondary event (the so-called insideout'model or from a direct
attack on myelin, in which case neurons die due to the lack of trophic support by mvelin (the so-
called 'outside-in' model).
PROCEDURE
• Some of the viruses are used to induce the disorder, virus infection could additionally induce or
augment autoimmunity to myelin and neurons via several pathways.
1. Semliki Forest Virus
2. Japanese Macaque Encephalomyelitis
3. Theiler's Murine Encephalomyelitis Virus
114

I. Semliki Forest virus
• Semliki Forest virus (SFV) does not cause demyelination in humans.SFV is an enveloped Togavirus,
first isolated from mosquitoes in 1942.The common strains used to induce myelin damage in mice
are the mutant M9 and avirulent A7. SFV is neuroinvasive. once inoculated peripherally and after
crossing the BBB, the virus infects neurons and oligodendrocytes. While the M9 strain is highly
virulent, causing death in 10-20% of adult mice or paralysis as a result of neuronal damage
II. Japanese Macaque Encephalomyelitis
• A spontaneous inflammatory demyelinating disease was reported in a colony of Japanese macaques
in Oregon from which a gamma-herpes virus was isolated.
III. Theiler's Murine Encephalomyelitis Virus
• Theiler's Murine Encephalomyelitis Virus, first identified by Max Theiler, is a natural pathogen of
mice, causing paralysis and encephalomyelitis.
• Theiler's Murine Encephalomyelitis Virus infection induces clinical neurological disease in
immunocompetent mice, along with atrophy of the brain and spinal cord.
115

3. Toxin models
 In these models, demyelination is induced after focal application or systemic administration of the
toxin.
 Agents for focal demyelination used so far are:
• Lysolecithin, also called LPC
• Ethidium bromide (EB)
• Antibodies to oligodendrocyte-related proteins
• Bacterial endotoxins
• 6-aminonicotinamide
• Electrolytes or cocktails containing complement and antibodies against galactocerebroside
116

In-vitro tests
1. Microglia
I. Microglia activation is observed in actively demyelinating MS lesions, pre-active lesions, areas of
remyelination as well as the normal-appearing white matter.
II. Primary microglia cultures are derived from embryonic or early post-natal animals.
PROCEDURE
This method is simple and allows for relatively high yields of cells.
Microglia can also be separated from confluent primary mixed glial cultures by agitation on a rotary
shaker, producing a highly enriched (> 95%) cell culture.
Given that microglia in the brain, The initial trigger of microglia activation.
microglia phenotype supportive of regeneration is observed at the earliest stages of demyelination.
117

REFERENCES
1. https://www.brainkart.com/article/Parkinson-s-Disease_32540/
2. H. Gerhard Vogel, “Drug Discovery And Evaluation” Pharmacological Assays; published by Springer; 2nd
edition. page no. 577-585.
3. KD TRIPATHI;“Essentials of Medical Pharmacology”, JAYPEE The Health Science Publisher; 8th edition. page
no. 452-453.
4. Animal Models in Drug Discovery of Alzheimer’s Disease: A Mini Review, Anil Kumar1*, Archi Aggarwal1, Arti
Singh1, Pattipati Sreenivaslu Naidu21Pharmacology Division, Punjab University, India Department of
Pharmaceutical and Biomedical Sciences, California Health Sciences University (CHSU), USA.
5. IJPSR (2010), Vol. 1, Issue 8 (Review Article) Received on 10 April, 2010; received in revised form 14 June,
2010; accepted 01 July, 2010 EXPERIMENTAL MODELS FOR ALZHEIMER’S DISEASE: A MECHANISTIC
VIEW Vivek Kumar Sharma Department of Pharmacology, Govt. College of Pharmacy, MECHANISTIC VIEW
Vivek Kumar Sharma Department of Pharmacology, Govt. College of Pharmacy, Rohru, Shimla, Himachal
Pradesh, India.
6. Patterns of memory failure after scopolamine treatment: Implications for cholinergic hypotheses of
dementia,North Dakota State University, Fargo, North Dakota 58105, USA David S.Janowsky2. Numbers and
letters correspond to the affiliation listhttps://doi.org/10.1016/S0163-1047(86)90772-7.
7. Attenuation of scopolamine-induced amnesia in mice..,,Stone WS1, Croul CE, Gold PE.
8. Review Toxin-Induced Experimental Models of Learning and Memory Impairment by Sandeep Vasant More
Hemant Kumar, Duk-Yeon Cho, Yo-Sep Yun and Dong-Kug Choi. 118

9. The validity of scopolamine as a pharmacological model for cognitive impairment: a review of animal behavioral
studies, Klinkenberg I1, Blokland A.
10.Research report,A chronic Alzheimer’s model evoked by mitochondrial poison sodium azide for pharmacological
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11.Am J Nucl Med Mol Imaging. 2012; 2(3): 386–404, Published online 2012 Jul 10,PMCID: PMC3477739.
12.Current neuroimaging techniques in Alzheimer's disease and applications in animal models,Linda Zhang,1
Raymond Chuen-Chung Chang, 2,3,4 Leung-Wing Chu, 3,4,5 and Henry Ka-Fung Mak1,3,4.
13.Am J Neurodegener Dis. 2013; 2(2): 108–120. Published online 2013 Jun 21. PMCID: PMC3703123.
14.Alzheimer’s disease biomarkers in animal models: closing the translational gap Jonathan J Sabbagh,1
Jefferson W Kinney,1 and Jeffrey L Cummings2 Published online 2015 Apr 30. doi:
10.1517/17460441.2015.1041913, PMCID: PMC4592281 Daniela Puzzo, MD, PhD,1 Walter Gulisano, BSc,1
Agostino Palmeri, MD, PhD,1 and Ottavio Arancio, MD, PhD2.
15.Br J Pharmacol. 2011 Oct; 164(4): 1285–1300. 11. Br J Pharmacol. 2011 Oct; 164(4): 1285–1300. doi:
10.1111/j.1476-5381.2011.01299.x PMCID: PMC3229762, Animal models in the drug discovery pipeline for
Alzheimer's disease Debby Van Dam1 and Peter Paul De Deyn.
16.Baukje J. van der Star etal., In Vitro and In Vivo Models of Multiple Sclerosis CNS & Neurological Disorders -
Drug Targets, 2012, 11, 570-588.
17.Text book of pharmacology by lippincott illustrated reviews 6th edition.
18.www.nhs.uk 119

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