This file includes the general introduction of Parkinson's, sign and symptoms of Parkinson's, treatment of Parkinson's and the main content that is the Preclinical Screening models for Neurodegenerative disease like Parkinson's

1. Preclinical Screening of Drugs
For Neurodegenerative
Diseases( Parkinsonism)
Presented By :- Janhavi Yashwant Burade
M.Pharm 1st sem
Vidyabharti College of Pharmacy,
Amravti

2. Content
 Introduction
 Sign & Symptoms of Parkinson’s
 Treatment of Parkinson’s
 Preclinical Screening of parkinsonism

3. Introduction
Que :- What are neurodegenerative diseases and how do they affect the
brain ?
 Neurodegenerative diseases result in neuron death .
 The word neurodegenerative can be split into neuron meaning brain &
degenerative meaning breaking down or dying .
 Neurodegenerative diseases are a great example of the devastating
effects of miscommunication between brain cells .
 Neurodegenerative diseases are a heterogeneous group of disorder
that are characterized by the progressive degeneration of the structure
& function of the central nervous system or peripheral nervous system.
 E.g.:- Parkinsonism & Alzheimer’s disease

4. Parkinson’s Disease  It is one of the type of neurodegenerative disease.
 When neurons die in a part of the brain called the
substantia nigra , movement problem appear.
 The substantia nigra ( lattin word which means black
substance ) is a region within the brain that contains a large
number of neurons that release a substance called
dopamine .
 By releasing dopamine , the neurons of the substantia
nigra communicate with movement – producing part of
the brain , like the frontal lobe and basal ganglia .
 The word ganglia means clusters of neurons.
 The basal ganglia are located deep in the center of the
brain and are made up of several different groups of
neurons.
 Neuronal death in the substantia nigra means that these
groups of neurons can no longer work properly,
 causing stumbling and shaking in the people that have this
disease .These individuals also experience problems
starting and maintaining their movements.

5. Sign & Symptoms of Parkinsonism

6. Treatment of Parkinsonism

7. Preclinical Screening of Drugs For
Parkinsonism
Screening models
In Vivo Model’s In vitro Model’s

8. Screening Models of Drugs For
Parkinsonism
( A ) In-vivo Model’s
1. Tremorine and Oxotremorine Antagonism
2. MPTP Model of Parkinson’s Disease
3. Reserpine Antagonism
4. Circling Behaviour 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 Transplantation into Lesioned Animals
12. Transfer of Glial Cell Line- Derived Neurotropic Factor
(GDNF)
( B) In-vitro Model’s
1. Experiment using rat atrial 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 Neuroprotective Efficacy

9. In-vivo Models
1.Tremorien & Oxotremorine Antagonism
PURPOSE & RATIONAL
 The Muscarinic agonists tremorine and
Oxotremorine induce parkinsonism-like sign such as
tremor , ataxia , spasticity , salivation, lacrimation &
hypothermia.
 These signs are antagonized by anticholinergic
drugs

10. PROCEDURE:-
1. Groups of 6-10 male NMRI mice weighing 18–22 g are used.
2. 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.
3. Rectal temperature is measured before administration of the
compound (basal value) and 1, 2 and 3 h after oxotremorine
injection.
4. Tremor is scored after oxotremorine dosage in 10 s
observation periods every 15 min for 1 h.

11. Observation
 TREMOUR
 Absent
 Slight
 Medium
 Severe
 Salivation and lacrimation are
scored 15 & 30 min after
Oxotremorine injection
 Absent
 Slight
 Medium
 Severe
 SCORE
 0
 1
 2
 3
 SCORE
 0
 1
 2
 3

12. Evaluation:-
 1. Hypothermia:- The differences of body temperature after
1,2&3hrs versus basal value are summarised for each animal in
the control group & the test group. The average values are
compared statistically .
 2.Tremor :- The scores for all animals in each group at the 3
observation periods are summarised. The numbers in the
treated groups are expressed as percentage of the number of
the control group.
 3.Salivation & Lacrimation:- The scores for both symptoms for
all animals in each group are summarised at the 2 observation
periods.The no. is the treated group are expressed as
percentage of the number of the control group.

13. Modification of the method:-
 Matthews & Chiou (1979 ) developed a method for quantifying resting
tremores 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.
 Johnson (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.
Administration of Oxotremorine,2.5mg/kg ,i.p. produced numerous high
– frequency, high-intensity peaks within 5min.

14. 2. MPTP MODEL IN ANIMALS
Principle :-
 MPTP acts as –a neutrotoxin which preferentially affected
dopaminergic cells in substantia nigra par compacta .
 MPTP shows toxicity due to conversion into MPP+ by MAO enzyme
 This ion acts by inhibiting the ET system of mitochondrial complex -
1
 Most Popular MPTP Model :- (a) MPTP Model in mice
(b) MPTP Model in monkey
( MPTP :- 1,2,3,6-Methyl-phenyl-tetrahydropyridine)

15. (a) MPTP MODEL IN MICE
Principle:-
 Neuro protective effect of test drug measured in MPTP Model in
mice
 Substantia nigra area is especially rich in microglia activation
,release a veriety of neurotoxic factors like superoxide, NO, cytokines
& eicosanoids
 Test drug reduces NADPH oxidase activity at extracellular and
intracellular level
Requirements:-
 Animal :- mice-wild strain (C57BL/6J). Mice-null strain.
 Drug :- MPTP ( 15mg free base / kg ) s.c.&test drug

16. Procedure:-
1. Take NADPH - Oxidase null & wild type mice
2. MPTP (15mg/kg) injected s.c. to mice daily for 6-consecutive days
3. Then test drug injected to mice twice daily for first 6-days& then inject once daily for
remainder study
4. After 6-days of last MPTP injection, mice are killed
5. Striatal tissue are rapidly dissected
6. Striatal cell viability estimation
NO release estimate by assays
ROS estimate by Fluorescence assay

17. Evaluation:-
 Test drug evaluated for showing neuroprotective action
 Reducing NADPH oxidase activity in PHOX+/+( wild strain )
is present but in PHOX-/- , NADPH oxidase are absent
 Reduces MPTP induced production of superoxide free
radicals extracellular level and intracellular reactive oxygen
species (ROS).

18. (b) MPTP MODEL IN MONKEY
Principle :-
Requirement:-
Animal:- Rhesus monkey (5-8kg)
Drug:- N-MPTP up to 10-18mg/kg i.v. for 5-8days. Test drug

19. Procedure:-
1. Take 8 rhesus monkey (5-8 kg) of either sex.
2. Administered the N-MPTP i.v. with cumulative dose up to 10-18
mg/kg for 5-8 days.
3. Produces PD like symptoms
4. Administered test drug
5. Symptoms are Evaluated

20. Evaluation:-
OBSERVATION
 The severity is rated by using scale of 0(normal) to 17 (max)
1. Movement
Normal
Reduced
Sleepy
2.Checking Movement
Present
Reduced
Absent
3.Attention & Blinking
. Normal
Abnormal
4.Balance & Co-ordination
. Normal
Impaired
Unstable
Falls
SCORING
 0
 1
 2
 0
 1
 2
 0
 1
 0
 1
 2
 3

21. 3.RESERPINE ANTAGONISM
Principle:-
Reserpine induces depletion of central catecholamine stores.
The sedative effect can be observed in mice shortly after
injection,followed by signs of eyelidptosis, hypokinesia, rigidity,
catatonia, and immobility. These phenomena can be antagonized by
dopamine agonists.
Requirements:-
Animal :- male rats (Wistar strain 280-300 gm
Drug :– Chloral hydrate (350mg/kg i.p.)
Reserpine (5mg/kg i.p.)
Apparatus:- photocell

22. Procedure:-
1. Male NMRI rats of either sex are taken 20-25g
2. reserpine (5mg/kg i.p.) injected to rats and tested 24hrs later
3. 30min. Prior to observation test compound is injected
4. Animals are placed singly on to floor of Perspex container (30x26x20 cm.) which situated
on panlab proximally sensor unit.
5. Horizontal movement are recorded for 10min.
6. Rearing & grooming episodes are registered

23. Evaluation:-
 Locomotors activity & grooming scores of test group is
compared with control group.

24. 4. CIRCLING BEHAVIOUR IN
NIGROSTRITAL LESIONED RATS
Principle:-
 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,orthe dopamineprecursor L-dopaisgiven.
 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 antiparkinsoniandrugs.
 An imbalance of dopaminergic activity within the basal ganglia isassociated
with markedly asymmetric circling behavior (Rotationturning) which
measured by Rota meter.
 This model isused in drug induced rotating behavior andunderstanding of
extra pyramidal disorder & of their treatment bydopaminergic agents.

25. 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

26. 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-gaugestainless-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 4γ/l of saline is injected at a rate of 1γ/4min
 After the intracranial injection the wound is closed. The animal is allowed several
weeks for recovery and for development of the lesion.

27. Rats are divided into groups:-
 Control groups for base value of ipsilateral rotation -2.5mg/kg of
damphetamine injected i.p. to rats.
 Control groups for base valueof contra lateral rotation- 1mg/kg
of Apomorphine injected i.p. to rats.
 Test compounds are given i.p or s.c. and the animals placed into
the circling chambers. Circling is recorded over a 1-h period.
 Further studied test group as compared to control group.
 No. of full turns( either ipsilateral Or contra lateral turning to
lesion) are recorded an automatic print out counter every 15
min. for one or two hr. session.

28. Observation:-
 For ipsi-lateral turning : - administer 2.5 mg/kg
Amphetamine & placed in circling chamber for 2 hour
 For contra-lateral turning : - administer 1mg/kg & placed in
 circling chamber for 1hour
 Test compound are given i.p. or s.c. & record reading with 15
min. interval.
Evaluation :- % change of drug turns from control turns is
recorded.

29. 5.ELEVATION BODY SWING TEST
Principle :-
 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 hemi parkinsonian animals.
 High positive co-relations b/w swing & Apomorphine
induced rotational behavior.
Requirements:-
 Animal : - Sprague Dawley rats
 Drug :- 6-OHDA (8 mg in 4ml 0.9% saline containing 0.02%
ascorbic acid). Test drug

30. Procedure:-
 40 rats are taken as test group
 Anesthetised with sod.Pentobarbital(60mg/kg i.p.) mounted in stereotaxic
device
 Stereotaxically lesioned in left substantia nigra
 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 (40x40x35.5 cm.)

31.  The rat is held about2.5cmfrom the base of its tail and elevated 2.5cm above the
surface on which it has been resting. A swing is recorded wheneverthe animal movesits
headout of the vertical axis of either side
 Before attempting another swing, the animal must return to the vertical position for the
next swing to be counted. Swings are counted for 60s over four consecutive 15-s
segments

32. Observation:-
 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.

33. Evaluation:-
 %LEFT SWING= Σno. Of swing towards left side / Σ L+R
 %RIGHT SWING = Σno. Of swing toward right side / Σ L+R

34. 6.SKILLED PAW REACHING TEST
Principle :-
 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 :- Des methyl Imipramine (25 mg/kg i.p.) 6-OHDA (2μl of
4mg/ml in 0.95% saline with 0.02% ascorbic acid)
 Equipment :- Single pellet boxes (25x35x30cm) Food tray
boxes(10x18x10cm)

36. Procedure:-
 20 rats are taken
 30minute before surgery, desmethylimipramine administered
(25mg/kg i.p.)
 Rats anesthetized with pentabarbital(60mg/kg i.p.)
 12 rats received 6-OHDA lesions but 8 rats not received

37.  The apparatus consists of a clear Perspex chamber with a hinged
lid.
 A narrower compartment with a central platform running along its
length, creating atrough 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
45mgsaccharin-flavored pellets are place din each well.

38. 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. 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

39. 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 acidtwice over3min viaa
30-gaugestainless steel cannula at the stereotaxic coordinates:
Drug Treatment :-
 The animals are injected i.p.with the test drug or saline 30min
before the unilateral 6-OHDA lesion and 24h there after.

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 / no. of hit ×100
 Reaching posture
• Two point scale
• Scored as 0
• Scored as 1

41. 7.STEPPING TEST IN RATS
Principle :-
 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 contra lateral paw which can
be ameliorated by application of drug.
Requirement:-
Animal: Sprague Drawley rats ,
Dose:- 6-OHDA(3.6μg/μl in 0.2 μg/ml Ascorbate saline, test
drug

42. 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

43.  A smooth-surfaced table is used for measuring adjusted steps.
 During the first 3 days the rats are handled by the experimenter to
familiarize them with the experimenter’sgrip.
 During the subsequent1–2days 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 he animal was
moved side ways along the bench surface

44.  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.

45. 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.

46. 8. GAIT ANALYSIS
 Gait analysis is useful in objective assessment of walking
ability and identify causes for walking abnormality 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. Animal.

47. 9.ROTENONE INDUCED PARKINSONISM
Principle:-
 Chronic systemic complex 1st inhibition caused by Rotenone
exposure induces of parkinsonism in rats including selective
nigrostriatal dopanimergic degeneration & formation of
ubiquitin & α-synuclein inclusion .

48. 10. TRANSGENIC ANIMAL MODELS OF
PARKINSON’S DISEASE
Purpose & 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 alterations
were associated with a loss of dopaminergic terminals in the basal
ganglia and with motor impairments.

49. (b) IN-VITRO METHODS
1. EXPERIMENT USING RAT STAITAL SLICES
1. 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.
2. Male spargue dawley rats(150-250g)are decapitated,the skull is
opened.
3. Right and left striata are removed & placed in ice-cold krebs solution.
4. The striata is cut into 0.4mm thick slices using a tissue chopper.

50. 5. The slices are kept floating for 30 min in krebs solution & gassed with
95% o2 & 5% co2 at room temperature
6. The slices are labeled by incubating for 30 min at 37C with
[3H]dopamine (5μci/ml) &[14c] choline (2 μci/ml)in the presence of
0.15mM pargyline chloride & 0.1mM ascorbic acid.
7.Labeled slices are transferred to superfusion chambers & perfused with
krebs solutions at 37c at flow rate of 0.5ml/min
8. After washing & stabilization 5min fraction of superfusate are collecte
9.The perfusion buffer contains 1 μM nomifensine to inhibit dopamine
reuptake & 10μM hemicholinium to inhibit choline uptake.

51. 10.The slices are subjected to field strength to the current strength of 10-
15mA/cm2 & pulse duration of 2msec at stimulation frequency of 3Hz for
5min.
11.Drugs to be tested are present in the superfusion fluid.
12.The radioactivity in the superfusate samples & in the tissue is
determined by liquid scintillation counting.
13. 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.DOPAMINE STIMULATED ADENYLY
CYCLASE ACTIVITY
1. Male sprague- dawley(150-250g) are decapitated & right & left striata are
removed.
2. Striatal tissue is homogenized by teflon homogenizer in chilled buffer
containing 10mM imidazole, 2mM EDTA & 10% sucrose ph 7.3.
3. Homogenate is centrifuged at thousand g for10min & supernatant is
recentifuged at 27000g for20min.
4. The pellet obtained is washed twice & suspended in 10mM imidazole, ph
7.3. Membrane protein is determined by bradfords method using bovine
serum.

53. 5.Adenylyl cyclase activity is measured by calculating the conversion rate of
(32p) ATP to (32p) cAMP
.
6.The assay is perform in 250μl solution containing imidazole, mgcl2
7.papaverine dithiothreitol,ATP
,GTP
,phspocreatine,creatine phosphokinase.
8.The reaction mixture is preincubated at 30c for 5min, the reaction is
initiated by adding membrane proteins and incubated for 10min.
9.The reaction is terminated by adding stopping solution(ATP
,SDS,cAMP).
Formed (32p) cAMP is separated from (32p) ATP by chromatography