NEURODEGENERATIVE DISEASE

NEURODEGENERATIVE
DISEASES
PRESENTED BY : ANAND SAGAR TIWARI
M.PHARM (FIRST SEMSTER)
DEPARTMENT OF PHARAMACOLOGY
GUIDED BY : Dr. NITIN MAHURKAR
PROFESSOR & HOD (DEPT. OF PHARMACOLOGY)

Overview
i. Basic concepts
ii. Neurodegenerative diseases
 Introduction
 Pharmacological screening models

Neuropsychopharmacology
Neuro-psychopharmacology is that branch of pharmacology
that deals with the study of neurodegenerative diseases( i.e. neurological
and psychiatric diseases)
This field seeks not only to understand how drugs can affect but also
how they can treat anxiety, mania , depression, schizophrenia and
seizures without altering consciousness in the CNS.
Thus drug can be employed in two strategies:
i. To understand the functional and structural systems that operate in a
normal CNS
ii. To provide the means to develop appropriate drugs to correct
pathophysiological events in the abnormal CNS .The research studies
are conducted at four hierarchical levels :molecular , cellular
multicellular and behavioral.

The entire concept of animal models of human
psychiatric diseases rests on the assumption that the
scientists can appropriately infer from observations of
animal behavior and psychology and that the
emotional states experienced by animals are
equivalent to the emotional states experienced by
humans expressing same psychological changes.
Animal studies have helped to understand human
behavior, elucidate the principles and mechanism of
drug action and in determining the dynamic
interactions between behavior and the mechanical
substrates influencing both behavior and drug action.

• While going for screening certain precautions need to be
taken :
i. The animals should be exposed to regular 12 hrs day/night
cycle.
ii. Standard laboratory chow and tap water should be provided ad
libitum unless mentioned otherwise in the protocol.
iii. The animals should be housed in clean and hygienically
maintained cages.
iv. Inbred animals of 3rd and 4th generation should be used to
reduce variation in results.
v. Preferable to use adult healthy male animals over female as the
hormonal variations in females may be an added source of
variation.
vi. To avoid bias in behavioral studies dependent on scoring
system the animals should be randomized and blindly scored.

Neurodegeneration
•Progressive loss of structure of the neurons.
•Such diseases are incurable, resulting in
progressive degeneration and death of neuron
cells.
•As research progresses there are many parallels
between different neurodegenerative disorders
including atypical protein assemblies as well as
induced cell death.

Neurodegenerative disease
• Neurodegenerative disease is an umbrella term for a range of
conditions which primarily effect the neurons in human
brain.
• Building block of the nervous system which primarily
includes brain and spinal cord.
• Since neurons don’t reproduce or replace themselves they get
damaged or they can’t be replaced by our body.
• Neurodegenerative diseases are incurable and results in
progressive degeneration of nerve cells.
• Dementias are responsible for the greatest percentage of
neurodegenerative disease.
e.g. Alzheimer’s disease, Parkinson’s disease, Huntington’s

Parkinsons disease
• It is a progressive neurodegenerative disorder marked by
sedative and extensive loss of dopaminergic neurons of
substantia nigra.
• The decrease in dopaminergic activity in the basal ganglia
results in a relative excess of cholinergic influence i.e.
biochemically there is a depletion of dopamine(DA) and
increment of Acetylcholine in the affected area.
• It is manifested as bradykinesia , muscular rigidity, resting
tremor and postural imbalance.
• Thus dopaminergic agonists as L- DOPA a precursor of
dopamine and cholinergic antagonists can be combined in the
treatment of Parkinson’s disease.

Compounds which were reported to have
adverse affects resembling parkinsonism in
patients were tested in laboratory animals for the
induction of extrapyramidal disorders.
Major tranquilizers reserpine and phenothiazine
derivatives have been successfully used to set-up
models of parkinsonism in animals.
The protective effect of the test drugs can then
be compared to that of standard in these animals
by assessing them on various behavioral
paradigms.

Models for screening
• The models for screening of Parkinson’s disease can be
classified as:
in- vitro models
CULTURE OF SUBSTANTIA NIGRA
INHIBITION OF APOPTOSIS IN NEUROBLASTOMA IN SH-
SY5Y CELLS
in- vivo models
 ELEVATED BODY SWING TEST
SKILLED PAW REACHING IN RATS
TREMORINE AND OXOTREMORINE ANTAGONISM

in-vitro model
• Culture of substantia nigra:
Purpose and rationale : In 1993 Lardozo and
Smeyne and Smeyne(2002) described details of a
method for generating mixed and chimeric
neuron/glial cultures of postnatal Substantia Nigra
(SN) independent of other monoaminergic nuclei in
the ventral midbrain.
Since many toxins don’t effect regions of the
midbrain except of the SN use of whole ventral
midbrain from embryos can dilute any measurements
of cell death.

By specifically culturing ventrolateral midbrain
containing the SN, one can more directly target
one can more directly target the effects of
dopaminergic traits. In addition, this method can
be used to test potential therapies for
amelioration of Parkinson’s Disease.

Some concepts
•Dissociation media composition:
i. SODIUM SULFATE = 12.8 gm.
ii. POTASSIUM SULFATE = 5.2 gm.
iii. CALCIUM CHLORIDE( CaCl2. H2O) = 0.036 gm.
iv. MAGNESIUM CHLORIDE (MgCl2.6H2O) = 1.18 gm.
v. GLUCOSE ANHYDROUS = 1.8 gm
vi. HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid= 0.238
gm. in 1 lt. deionized water adjusted to pH 7.4 with NaOH.
MPTP = 1-Methyl-4-phenyl-1,2,3,6- Tetrahydropyridine

Procedures
1. Postnatal day 2-5 C57B1/6 mice or Swiss Webster mice are placed
on ice for 2-3 minute to achieve an appropriate plane of anesthesia.
2. Animals are quickly decapitated. 8-10 brains from P2-P5 C57BL/6
or SWR mating are removed and placed in a freshly prepared
dissociation media.
3. Brains are placed on their ventral surface and a slice of midbrain
rostral to the cerebellum and caudal to the hippocampus is isolated.
4. This removed brain slab is placed flat in DM(Dissociation Media)
and either the entire midbrain or the ventrolateral mid brain
containing the SN(Substantia Nigra) is dissected and minced into
small pieces.
5. The minced SN or midbrain is then incubated in papain and DNAase
for 30 min. at 37 degree Celsius. A second incubation with fresh
papain is followed by three rinses in DM and 1 rinse in plating
media.

Procedure contd….
6. Tissue is triturated in 5 ml plating media (PM) and the cell
suspension is added to 2 ml (1 ml of BSA stock and 1 ml of
ovalbumin stock).
7. The cell suspension is spun 1400 rev/min. for 8 min and the
pellet is then re-suspended in 1 ml plating media containing 2%
rat serum.
8. Once re-suspended the cells are counted using Trypan blue
(0.4%) to determine cell viability.
9. Cells are adjusted to 1.2*10^6 cells/ml and plated at 200,000
cells/cm in lab . Cells are maintained in an incubator at 37 degree
Celsius, 5% CO2 and fed 2-3 times/ week with feeding media
complete with 2% rat serum by exchanging approximately one-
fifth of the media.

Chimeric glial culture
SNpc cells are plated on pre-plated SNpc glia are produced
using a variation of the previously described methods. SNpc
glial feeder layers from P2-P5 C57BL/6 or SWR mice are
produced using the above described method but cells are
plated at 20,000- 50,000 cells/cm2 and fed with plating media
containing 2% rat serum and 8% fetal bovine serum to
promote glial proliferation and neuronal death.
For astrocyte culture this is the final step.
To generate mixed neuron/glial cultures ,3-4 weeks after the
initial SNpc cells are plated ,the glial feeder layers are rinsed
once again with plating media without serum and 0.5 ml of
plating media with 2% rat serum and 0.5 ml of plating media
with 2% rat serum is added.

Contd……
SNpc cells from C57BL6 or SWR mice are isolated
and plated at 250,000 cells/well on the previously
generated glial feeding layers.
24 hours after plating of neurons onto the glial feeder
layers the cultures are fed with feeding media
complete with 2% rat serum by an exchange of
approx. one-fifth of the media and cytosine beta D-
arabinofuranosil (Ara- C,2 micro meter) to prevent
glial proliferation of the freshly plated cells.
Thereafter, Ara-C is added at each feeding.

MPTP treatment
MPTP is added to cultures 7-10 days after the cultured
neurons are plated into glia.
The MPTP is prepared by dissolving MPTP in feeding media
for a 1 mM stock then diluting to a 1 microM stock with
feeding media complete with 2% rat serum and adding this
directly to the cultures.
The final concentration of MPTP in media is 50nM.
Two feedings of MPTP in 2 days are necessary to achieve the
desired toxic affect.
7 days after MPTP is added the cultures are rinsed with 3*
with TBS, fixed in 4% buffered paraformaldehyde for 10
min.

Evaluation
To determine the no of SN cells cultures are immunostained
for expression of tyrosine hydroxylase.
Endogenous peroxidase activity is quenched by rinsing with
0.3% methanol in TBS solution for 30 minutes.
Cell cultures are covered in 400 microliter of a polyclonal
antibody directed against tyrosine hydroxylase and incubated
in this condition for overnight at 4 degree Celsius.
The next day cultures are rinsed 3 times with TBS followed
by application of secondary antibody and amplification with
Avidin –Biotin.
Final visualization of the immune-positive neurons is made
using diaaminobenzene as a chromogen.

All TH- positive cells having the previously described
characteristics of SNpc neurons from each culture are counted
at a magnification of 200X.
Once cells are counted in each well the no. of TH(+)ve cells in
the MPTP treated cultures from each genotype compared to the
adjacent non treated culture.
Since an identical number of cells generated from the same
brains was plated on a single slide these cultures are directly
compared to determine the % cell loss following MPTP.
The percentage cell loss data following MPTP are then pooled
from all C57B1/6, SWR or mixed cultures.

•The term Chromogen is applied to a colorless
chemical compound that can be converted by
chemical reaction into a compound which can be
described as colored.
•A substance capable of conversion into a pigment
or dye.
•Any substance without color giving origin to a
coloring matter.

Inhibition of apoptosis in
neuroblastoma SH-SY5Y cells
Purpose and rationale:
The original cell line called SK-N-SH from which it was isolated from
a bone marrow biopsy taken from a 4 years old female with
neuroblastoma .
These cells are often used as in vitro models of neuronal function and
differentiation.
They are adrenergic in phenotype but also express dopaminergic
markers and as such used to study Parkinson’s disease, neurogenesis
and other characteristics of the cells.
SH-SY5Y cells can easily interconvert between two phenotypes in
vitro the neuroblast like cells and epithelial cells.

Neuroprotection to halt progressive cell death of neurons has
been proposed as a future therapy for neurodegenerative
disorders.
Here apoptosis contributes to neuronal death. The well
regulated and relatively slow apoptotic process was proposed
as a target of neuroprotection.
Apoptotic signaling is a multistep pathway induced by
opening of a mitochondrial mega channel called permeability
transition pore(PT), followed by decline in membrane
potential, Δrelease of apoptosis inducing factors, activation of
caspases and fragmentation of nuclear DNA.
Mitochondrial pore is regulated by Bcl-2 protein family,
preventively by Bcl-2 and Bcl-XL family.

Procedure
SH- SY5Y cells were cultured in Cosmedium -001 tissue
culture medium supplemented by 5% fetal calf serum in
atmosphere of 95 % air and 5% CO2.
Assesment of apoptosis induced by NM(R) sal
and the protection by BPAP derivatives
Apoptosis was quantitatively measured by Fluorescence
Augmented Flow Cytometry (FACS).
Cells were cultured in a 6- wellpoly- 1 lysine coated culture
flask were incubated with or without 1µM – 1nM BPAP
analogs at 37 degree Celsius for 30 min. and then for 24 hr.

with 250µM NM(R) Sal in Cosmedium- 001
culture medium supplement with fetal calf serum.
The cells were treated with trypsin, gathered and
washed with culture medium and twice with the
phosphate buffered saline(PBS).
The cells were incubated with 10 nm test drugs
solution in an ice bath for 30 minutes washed and
suspended in PBS then subjected to FACS
analysis.

Evaluation
Experiments were repeated 4-8 times and the
results were expressed as the mean and SD.
Differences were statistically evaluated by
analysis of variance (ANOVA) followed by
Shelfe’s F-Test.
A p- value less than 0.05 was considered to be
statistically significant.

Modifications of the method
•Maruyama et al. found that dopaminergic
neurotoxins 6,7- hydroxy-1- (3,4-
dihydroxybenzyl)isoquinolines cause different
cell death in SH-SY5Y cells i.e. apoptosis was
induced by oxidized by papaverolines and
necrosis by reduced tetrahydrolpapaverolines.

in-vivo models
1)Elevated body swing test:
Purpose and rationale : Borlongan and Sanberg and
Borlongan et.al proposed the elevated body swing test
as a measure of asymmetrical motor behavior of
hemiparkinsonian animals in a drug free state.

Procedure
Male, 8 week old Sprague Dawley Rats are anesthetized with
Sodium pentobarbital (60mg/kg i.p.) and mounted in a Kopf
stereotaxic frame.
They are lesioned by injection of 8 mcg 6-Hydroxydopamine
in 4 microliter saline containing 0.02% ascorbic acid in the
left Substantia nigra.
The solution is injected over a 4 min. period and the needle
is left in place for an additional 5 min. before retraction.
Seven days after the lesion behavioral testing is performed .
The animals are allowed to habituate in a Plexiglas box and
attain a neutral position having all four paws on ground.

Procedure contd…….
The rat is held about 2.5 cm from the base of its tail and
elevated 2.5 cm above the surface on which it has been
resting.
A swing is recorded whenever the animal moves its head out
of the vertical axis to either side.
Before attempting another swing, the animal must return to
the vertical position of the next swing to be counted.
Swings are counted for 60 sec over four consecutive 15 sec
segments. The total no of swings made to each side is divided
by the overall total number of swings made to both sides to
get %age of left and right swings.

The criterion of biased swing is set at 70% or higher.
At 30 and 45 sec, 6-OMDA lesioned rats exhibit right
biased swings of 70% or higher compared to normal
rats.
Evaluation: A two way of ANOVA is used to analyze
behavior data across 15-s segments.

2) Skilled paw reaching in rats
Purpose and rationale: The skilled paw reaching test
as a model of Parkinson’s disease in the rat was used by
Montoya et al. (1990,91), Abrous et al.(1993) , Abrous
and Dunnett (1994), Nikkhah et al.(1993) and Barneoud
et al. The term “Staircase” test mentioned in this context
has nothing to do with the Staircase test described by
Thiebot et al(1973) for evaluating anxiolytic activity in
rats.
Unilateral injection of 6-OHDA
(HYDROXYDOPAMINE) into the medial forebrain
bundle results in an impairment of paw reaching on both
sides.

This effect can be ameliorated by drug
treatment or transplantation of a nigral cell
suspension.
The apparatus has been developed after earlier
studies by Wishaw et al. who investigated the
contributions of motor cortex , nigrostriatal
dopamine and caudate putamen to skilled
forelimbs used in the rat.

Apparatus
The apparatus consists of a clear Perspex chamber with a
hinged lid. A narrower compartment with a central platform
running along its length and creating a trough on either side is
connected is connected to the chamber.
The narrowness of the side compartment prevents rats from
turning around so that they can only use their left paw for
creating into the left paw for reaching into the left trough and
their right paw for reaching in 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 contain a small well and
two 45 mg 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 paw reaching task.
For 4 weeks they are placed in the test boxes once/day
for 10-15 minutes . The no. of pellets eaten during the
test period indicates the rat’s ability of grasping and
retrieving the pellets.

Procedure contd……..
In addition to these parameters it is noted which
forepaw the rat used for the first movement to reach
the pellet on each test day.
A first choice score of +1 corresponds to the paw
contralateral to the lesions, a score of -1, to the paw
ipsilateral to the lesion.
Because rodents exhibit a pawedness it must be noted
whether there is a preference to the paw(one).

Lesions:
The mesotelencephalic system is lesioned by a by a
stereotaxic unilateral injection of 6-OHDA into the medial
forebrain bundle under equithes in anesthesia.
6-OHDA is injected in a volume of 1.5 microliter and at a
concentration of 4 mcg/ml of 0.9 % saline and 0.01%
ascorbic acid twice over 30 min via a 30- gauge stainless
steel cannula at the stereotaxic co-ordinates : L=1.6 mm, AP=
0 mm, V= -7.6 mm and L=1.6 mm , AP= -1mm, V= -8mm.
The coordinates AP and L are estimated relative to the
bregma , and V is measured from the level of dura, with the
incisor bar set 5 mm above the interaural line.
Following each injection the cannula is left in place for the
neurotoxin away from the injection site.

Drug treatment : The animals are injected i.p. with
the test drug or saline 30 minutes before the unilateral
6-OHDA lesion and 24 hour thereafter.
Evaluation : Test lesions are performed 4,5,7 and 8
weeks after 6-OHDA lesion. The parameters success,
attempts and sensorimotor co-ordination are subjected
to a two way ANOVA with group as the independent
measure and weeks as the dependent measure.

Modifications of the method
Fricker et al. investigated the effect of unilateral ibotenic acid lesions
in the dorsal striatum placed at anterior , posterior, medial or lateral
loci. In the staircase test of skilled forelimb use.
Nako et al. studied paw reaching ability in rats with unilateral
quinolinic acid of the lesions of the striatum as an animal model for
Huntington’s disease.
Barneoud et al.(1996) evaluated the neuro-protective effects of
riluzole using impaired skilled forelimb use, circling behavior and
altered dopaminergic metabolism of the mesotelencephalic system in
unilaterally 6-OHDA lesioned rats.
Meyer et al.(1997) described a revolving food pellet test for
measuring sensorimotor performance in rats.

3) Tremorine and Oxotremorine
antagonism
Purpose and rationale : The muscarinic
agonists tremorine and oxotremorine induce
parkinsonism like signs as tremor, ataxia,
spasticity, salivation, lacrimation and
hypothermia. these signs are antagonized by
anticholinergic drugs.

Procedure
NMRI (NAVAL MEDICAL RESEARCH INSTITUTE) groups of 6-10 mice
weighing 18-22 gm are used.
They are dosed orally with the test compound or the standard 95
(mg/kg) benzatropine mesilate 1 hour prior to 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 hour
after oxotremorine injection.
Tremor is scored after oxotremorine dosage in 10 sec observation
periods every 15 min for 1 hour.
TREMOR: Absent score = 0, Slight = 1, Medium = 2, Severe = 3
Salivation and lacrimation are scored 15 and 30 minutes
after oxotremorine injection . Scoring same as tremors.

Evaluation
• Hypothermia : The differences of body temperature after 1,2
and 3 hours 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 three
observation periods are summarized . The numbers in the
treated groups are expressed as %age of the number of the
control group.
• Salivation and lacrimation: The scores for animals in each
group are summarized at two observation periods. The
numbers in the treated groups are expressed as a %age of the
number of the control group.

 Critical assessment of the method: The
oxotremorine antagonism has been proven to be
a reliable method for testing control
anticholinergic activity . The overt isomorphism
between the animal models and the symptoms of
Parkinson’s disease recommend this test for
screening of anti-Parkinson drugs. However, the
model measures only central anticholinergic
activity.

Modifications
Matthews and Chiou developed a method for quantifying
resting tremors in a rat model of limb dyskinesia. 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 DRC for tremor
inhibition by potential anti-parkinsonian drugs.
At the same time Coward et al. recommend N-carbamoyl-2-
(2,6-dichlorophenyl) acetanilide hydrochloride , a
tremorigenic agent as an alternative to oxotremorine for the
detection of anti- Parkinson drugs.

Reserpine antagonism
Purpose and rationale:
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
agonists.

Evaluation
•Locomotor activity and grooming scores of
drug- treated animals are compared with
controls treated with reserpine and vehicle
only by analysis of vaccine.

Modifications
•Rats treated with reserpine develop
spontaneous orofacial dyskinesia that has
features similar to tardive dyskinesia in
humans (Abbott et al. 1991). The incidence
of tongue protrusions was recorded to
quantify the occurrence of oral dyskinesia.

PHARMACOLOGICAL
SCREENING MODELS FOR
ALZHEIMER’S DISEASE

Introduction
Alzheimer’s disease is a progressive disorder that
causes brain cells to waste away (degenerate) and die.
It is the most common cause of Dementia – a
continuous decline in thinking, behavioral and social
skills that disrupt person’s ability to function
independently.
The early signs of the disease may be forgetting
recent events conversations.
As the disease progresses , a person with Alzheimer’s
disease will develop severe memory impairment and
lose the ability to perform everyday tasks.

Hypothesized to be a protein misfolding disease
(proteopathy) caused by accumulation of abnormally folded
A-beta and tau proteins in the brain.
Plaques are made of small peptides, 39-43 amino acids in
length, called ß-amyloid.
Beta amyloid is a fragment from a large protein called
amyloid precursor protein (APP).
APP is critical to the growth , survival and post injury repair.
In Alzheimer’s disease APP is divided into smaller fragments
by enzyme through proteolysis.
One of these fragments give rise to ß-amyloid which forms
clump that deposit outside neurons.

Symptoms
Repeat statements and questions over and over.
Forget conversations, appointments or events and not
remember them later.
Routinely misplace possessions often putting them in
illogical locations.
Making judgments and decisions.
Social withdrawal, mood swings and distrust in others.
Irritability and aggression , changes in sleeping habits ,
wandering, loss of inhibitions, delusions such as believing as
stolen.
Get lost in familiar places and have trouble in the finding the
right words.

Screening models
•The screening methods for Alzheimer’s
disease can be divided into two types:
I. in-vitro models
II.in-vivo models

Inhibition of acetylcholine-esterase
activity in rat striatum
Purpose and rationale:
To screen drugs for inhibition of acetylcholine esterase
activity
As it is generally accepted that the physiological role of
AChe(Acetylcholine esterase) is rapid hydrolysis and
inactivation of ACh .
Thus inhibitor of AChe show marked cholinomimetic effects
in cholinergically- innervated effectors organs.
Recent studies have suggested that AChe inhibitors nay be
beneficial for the treatment of Alzheimer’s Disease.

Procedure
•Reagents
a) 0.05 M Phosphate buffer, ph-7.2
b) Substrate in buffer ( 198 mg acetylthiocholine
chloride).
c) DTNB( Ellman’s Reagent) in buffer (19.8 mg 5,5-
dithiobisnitrobenzoic acid) (0.5mM).
d) A 2mM stock solution of test drug is made up in a
suitable solvent and q.s. to volume with 0.5mM
DTNB. Drugs are serially diluted(1:10) such that the
final conc. in cuvette is 10 -4 m and screened for the
activity.

Tissue preparations
RREINCUBATED FOR 10 min. AT 37 DEGREE CELSIUS.
25µl ALIQUOT OF THIS SUSPENSION IS ADDED TO 1 ml OF THE VEHICLE AND
VARIOUS CONCENTRATION OF TEST DRUG ARE PREPARED.
WEIGHED AND HOMOGENIZED IN 19 VOLUMES (Approx. 7 mg
protein/ml OF 0.05 M NaH2 PO4 ph-7.2
BRAINS ARE RAPIDLY REMOVED, CORPORA STRIATA DISSECTED FREE
RATS ARE DECAPITATED

Evaluations
For IC 50 determinations: Substrate
concentration is 10mM diluted to 1:2 in an assay
yielding a conc. Of 5mM.
DTNB conc. is 0.5 mM yielding 0.25 mM final conc.
% inhibition = slope control-slope drug × 100
slope control
IC 50 values are calculated from log- probe analysis.

Inhibition of butyrylcholine-esterase
activity in human serum
This method is used in conjunction with the acetylcholine-
esterase assay to determine the enzyme selectivity of various
cholinesterase inhibitors.
Butyrylcholine-esterase preferentially hydrolyses
butyrylcholine.
This enzyme is found in the highest amount in serum, but its
physiological role is not known.
Ethopropazine and Tetra-isopropyl Pyrophosphoramide
(ISO- OMPA) are selective inhibitors of butyrylcholinesterase.

Procedure
Reagents:
0.05 M Phosphate buffer, pH 7.2
Substrate in buffer (225.8 mg s- butrylthiocholine
chloride)
DTNB in buffer (19.8 mg 5,5- dithiobisniytroenzoic
acid)(0.05mM)
A 2mM stock solution of test drug is made up in a
suitable solvent and q.s. too volume with 0.5mM
DTNB. Drugs are serially diluted (1:10) such that
determined from the inhibitory activity of subsequent
conc.

Enzyme preparation
PRE- INCUBATE FOR 10 MIN AT 37 DEGREE CELSIUS.
ADDED TO 1 ml OF THE VDEHICLE & VARIOUS CONC. OF TEST DRUG
ARE PREPARED.
25 ml ALIQUOT OF THIS SUSPENSION.
RECONSTITUTED IN 3ml OF DISTILLED WATER.
A VIAL OF LYOPHILIZED HUMAN SERUM

in- vivo methods
Some of the in-vivo methods are :
Inhibitory avoidance :
• Step-down
• Step- through
• Two-compartment test
• Scopolamine induced amnesia in mice.
• Up- hill avoidance
Active avoidance:
• Runway avoidance

Step down
Purpose and rationale
An animal (mouse or rat) in an open spends most of
the time close to the walls and in corners.
When placed on an elevated platform in the center of
a rectangular compartment, it steps down almost
immediately to the floor to explore the enclosure and
to approach the wall.
This technique is employed in different
modifications.

Procedure
•Requirements
a) Mice or rats of either sex are used
b) A rectangular box(50×50) with
electrifiable grid floor 35 fits over the
block.
c) Grid floor is connected to shock
device.

A typical paradigm consists of
:
•Familiarization
•Learning
•Retention test

Familiarization
AFTER 10 SECONDS OF EXPLORATION IT IS RETURNED TO
THE HOME CAGE.
LATENCY TO DESCEND IS MEASURED
RELEASED AFTER RAISING THE CYLINDER
ANIMAL IS PLACED ON THE PLATFORM

Learning
ANIMAL IS RETURNED TO THE HOME CAGE.
AN AVOIDABLE SHOCK IS APPLIED(FOOT SHOCK: 50Hz:
1.5mA; 1 sec)
IMMEDIATELY THE ANIMAL IS DESCENDED FROM THE
PLATFORM.

Retention test
THE TEST IS FINISHED WHEN THE ANIMAL STEPS DOWN OR
REMAIN ON THE PLATFORM ( CUT-OFF TIME: 60 SECS).
STEP-DOWN LATENCY IS MEASURED
THE ANIMAL IS AGAIN PLACDED ON THE PLATFORM
24 HRS. AFTER THE LEARNING TRIAL

Evaluation
The time of descent during the learning
phase and the time during the retention
test is measured.
A prolongation of the step-down
latency is defined as learning.

Scopolamine induced amnesia in
mice
• The administration of anti-muscarinic agent
scopolamine to young human volunteers produces
transient memory deficits.
• Similarly, scopolamine impairs memory retention
when given to mice.
• The ability of different cholinergic drug agonist to
reverse the amnesia affects of scopolamine is now well
documented in animal and human volunteers.

Procedure
The scopolamine test is performed in groups of 10
male NMRI mice weighing 26-32 g in one trial.
Five min. after i.p. administration of 3 mg/kg of
scopolamine hydrobromide each mouse is placed
individually in bright part of two chambered
apparatus.
After brief orientation period , mouse enters the
second or dark chamber.
Once inside the second chamber, the doors are closed
to avoid escape.
A 1mA, 1-sec foot shock is applied through grid floor
. The mouse then return to the home cage.

24 hours later , testing is performed by placing the animal
once again in the bright chamber.
The latency in entering the dark chamber within 5 min. test
session is measured electronically.
Whereas, untreated control animals enter the darker
chamber in second trial with latency about 250 sec.
Treatment with scopolamine reduces the latency 50 sec.
Test compounds are administered 90 min. before training.
The prolonged latency indicates that animal remembers that
it has been punished and , therefore, avoids darker chamber.

Evaluation
•After treatment with various doses of test drug
latencies obtained were expressed as % latencies.
•In some cases , straight dose-response curve is
obtained whereas with other drugs inverse U-
shaped dose responses are observed.

Multiple sclerosis
Multiple sclerosis is a complex inflammatory disease of the
brain and spinal cord characterized by focal lymphocytic
infiltration, leading to the damage of oligodendrocytes(which
synthesis myelin) and axons.
Initially inflammation is reversible also re-myelination but
later it can cause permanent damage.
Inflammation is driven by IL-17 secreting T-lymphocytic-
subtype which allows penetration of BBB by T-helper 17(TH-
17) cells into the brain, where they attack the neurons.
This results in inflammation, demyelination,
oligodendrocytes depletion and finally neuronal degeneration.

Multiple sclerosis (MS) is widely considered to be the
result of an aggressive autoreactive T cell attack on
myelin.
How the autoimmune response arises is unclear but it
may be due to viral infection.
Thus viral and autoimmune diseases in animals have
been used to investigate the possible pathogenic
mechanisms operating in MS.
The autoimmune model , experimental autoimmune
encephalomyelitis is the most widely used animal
model and has greatly influenced therapeutic
approaches targeting autoimmune system.

Symptoms
Visual impairment optic neuritis, spasticity.
Unstable bladder
Increased mechanical sensitivity
Electric sensation in the spine or limbs or neck
flexion.
Causes disability and leads to secondary
progressive ms.

Therapy
a) Rest , physiotherapy family/social support
b)Symptomatic treatment : e.g. Carbamazepine
and Gabapentin.
c) Treatment with disease modifying drugs like
Methylprednisolone.
d)Second line drugs include Natalizumab,
Alemtuzumab , Mitoxantrone.

Screening models
The screening for multiple
sclerosis can be carried out by:
in-vitro models
in-vivo models

Experimental Autoimmune
Encephalomyelitis(EAE)
•Purpose and rationale: The most studied animal model
for MS is EAE in which autoimmunity to CNS components is
induced in susceptible mice through immunization with self-
antigens derived from basic myelin protein.
• EAE may be induced in mice with different genetic
backgrounds such as SJL/J, C57BL/6 and NOD, through
either active immunization with protein or peptide or by
passive transfer of encephalitogenic T cells.
• In all cases the relevant immunogen is derived from self-CNS
proteins such as Myelin basic proteins (MBP), proteolipid
protein (PLP) or myelin oligodendrocyte
glycoprotein(MOG).

Procedure: Preparation of
Encephalitogen
On day 0 EAE is induced by subplantar injection of 0.1 ml of
the encephalogen into the left hind paw.
Groups of 6-12 male Wister-Lewis rats with an initial body
weight of 130-200 g are used.
3g of spinal cord from guinea pig rats are homogenized with
7.5 ml distilled water , 3.8 ml phenol and 7.5 ml Freund’s
adjuvant under cooling.

Animals showing any of these changes are considered
positive for the purpose of evaluation.
The clinical signs of EAE are manifested by ataxia or paresis,
urinary incontinence, fecal impaction and abdominal wall
flaccidity.
From days 1-2 the animal receive the test compound or
vehicle only or the standard drug by oral administration once
a day.
An equal volume of Bordetella pertussis vaccine concentrate
( organism/ml) is injected into the same foot.

Evaluation
Starting from day 7 the severity of clinical signs and
mortality are determined daily and scored according to the
following :-
 The total score is recorded for both control and treatment
groups.
PARAMETER SCORE
Per 20 g loss of body weight 1
Paralysis of tail 1
Paralysis of the hind paw 3
Complete analysis 5
Death 6

Reference
Drug discovery and evaluation:
Pharmacological assays-Fourth
Edition– Gerhard H. Vogel
Internet

NEURODEGENERATIVE DISEASE

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