anti parkinson screening model (in vitro and in vivo model ) introduction about parkinson drug of parkinson symptoms of parkinson

1. ANTI PARKINSON SCREENING MODELS
PRESENTED BY:-
NITTAL VEKARIA
Dharal mistry
(KBIPER)

2. 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.

3. SIGN AND SYMPTOMS

4. ETIOLOGY

5. PATHOPHYSILOGY

7. MECHANISM OF ACTION OF DRUGS:

8. Screening Models of Drugs For Parkinsonism
(A) In-vivo Model's
1 . Tremorine and Oxotremorine Antagonism
2. MPT Modelfo Parkinson's Disease
3 . Reserpine Antagonism
4 . Circling Behaviour ni Nigrostriatal Lesioned Rsta
5. Elevated Body Swing Test
6. Skilled wPa Reaching in Rats
7. Stepping Test ni Rats
8. Gait Analysis
9 . Rotenone Induced Parkinsonism
10. Transgenic Animal Models foParkinson's Disease
11. Cel Transplantation into Lesioned Animals
12. Transfer of Glial Cel 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. tremorein & oxotremorine antagonism
PURPOSE &RATIONAL
The Muscarinic agonists(cholinergic agonist) tremorine and Oxotremorine induce parkinsonism-
like sign such as tremor, ataxia, spasticity, salivation, lacrimation & hypothermia.
These signs are prevented by centrally acting anticholinergic drugs like (benztropine,
trihexyphenidyl, biperidene, procyclidine).

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)
1h 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 10s observation periods every 15 min for 1h.
5. Lacrimation and salivation are scored 15-30 min after oxotremorine.

11. observation:-
Tremor, salivation & lacrimation Score
Absent 0
Slight 1
Moderate 2
severe 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. 2. MPTP MODEL IN ANIMALS
Principle -:
MPT (MPTP: - 1,2,3,6-Methyl-phenyl-tetrahydropyridine) act as a neutrotoxin

MPP+

which preferentially affected dopaminergic cells in substantia nigra par compacta.
MPTP shows toxicity due to conversion into MPP+ by MAO enzyme.
MPP+ inhibit mitochondrial oxidation reaction and due to this oxidative stress occur this will leads to
selectively destroy the nigrostriatal neuron.
Most Popular MPT Model:- (a) MPT Model in mice
(b) MPTP Model in monkey
Used to evaluate levodopa drug

14. (a) MPTP MODEL IN MICE
Principle:-
Neuro protective effect of test drug measured in MPT Model in mice
Substantia nigra area is especially rich in microglia activation release a variety 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

15. 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

16. 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).

17. (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

18. Procedure:-
1. Take 8 rhesus monkey (5-8 kg) of either sex.
2. Administered the N-MPT 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

19. EVALUATION:
The severity is rated by using scale of 0 (normal) to 17 (max)
Observation scoring
(1)Movement
Normal 0
Reduced 1
Sleepy 2
( 2 ) Checking movement
Present 0
Reduced 1
Absent 2

20. observation scoring
(3)Attention& blinking
Normal 0
Abnormal 1
(4) Balance &co-ordination
Normal 0
Impaired 1
Unstable 2
Falls 3

21. 3.RESERPINE ANTAGONISM
Principle:-
Reserpine induces depletion of central catecholamines & 5-HT etc.. 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(L-dopa, bromocriptine, dopamine)
Requirements:-
• Animal:- male rats (Wistar strain 280-300 gm)
• Drug :- Chloral hydrate (350g/kg i.p.)
Reserpine (5mg/kg i.p.)
• Apparatus:- photocell

22. Procedure:-
1. Male NMRI rast of either sex are taken 20-25g
2. reserpine (5mg/kg pi.). 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 proximaly 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-dopa is
given.
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 dopaminergie 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.

25. Requirements:-
• Animal :- Male Wistar rats(200-250gm)
• Dose :- 6-OHDA neurotoxin 6-hydroxydopamine(8l in 4 ml of 0.2 mg/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 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 8g of 6-OHDA in 4y/l of saline is injected at a rate of 1y/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 amphetamine injected i.p.
to rats.
 Control groups for base value of contra lateral rotation - 1mg/kg of Apomorphine injected i.p.
to rats.
 Test compounds are given i.p or sc. 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 1g/kg &placed in circling chamber for 1hour
 Test compound are given i.p. or sc. &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.
 7days after lesion, behavioral testing is performed.
Remaining 24 animal served ascontrol group. The animals are placed into a plexiglass box
(40x40x35.5 cm.)
 The rat is held about 2.5cm from the base of its tail and elevated 2.5cm above the surface on
which it has been resting. A swing is recorded whenever the animal movesits headout of the
vertical axis of either side

31.  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 segment

32. Observation:
A swing is recorded whenever the animal moves its head outof vertical axis. Swing are counted
for 60sec.With interval of 15sec.

33. Evaluation:-
 %LEFT SWING= no. Of swing towards left side /  L+R
 %RIGHT SWING = no. Of swing toward right side /  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 (2l of 4mg/ml in 0.95% saline with
0.02% ascorbic acid)
Equipment:- Single pellet boxes (25x35×30cm) 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
 The apparatus consists of a clear Perspex chamber with a hinged lid.
 A narrower compartment with a central platform running along its length, creating 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 45 mg saccharin-flavored pellets are
placed in each well.

37. 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

38. Lesions:-
 The mesotelencephalic (memory and learning) system is lesioned by a stereotaxic
unilateral injection of 6-OHDA into the medial forebrain bundle under equithesin
anesthesia.
 6 OHDA is injected in a volume of 1.5ml and at a concentration of 4ug/l of 0.9% saline
and 0.01% ascorbic acid twice over 3min viaa 30-gaugestainless steel cannula at the
stereotaxic coordinates.
Drug Treatment :-
The animals are injected i.p.with the test drug or sanile 30min before the unilateral 6-
OHDA lesion and 24h there after.

39. 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

40. 7.STEPPING TEST IN RATS
Principle:-
This model is clinically relevant to unilateral model for parkinsonism akinesia.
the 6-OHDA lesion induced marked and long lasting 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 3.6ug/l in 0.2 ug/ml Ascorbate saline, test drug

41. Procedure:-
 6-OHDA Lesion Surgery Female Sprague Dawley rats receive two stereotaxic injections of 6-OHDA
(3.6g/  l in 0.2  g/ml ascorbate-saline) into the right ascending mesostriatal dopamine pathway using a
10-  l 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.
 During the first 3 days the rats are handled by the experimenter to familiarize them with the
experimenter’s grip.
 During the subsequent 1-2 days 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

42. 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.

43. 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. 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.

45. 9. Rotenone induced parkinsonism
Principle:
Chronic systemic complex 1 st inhibition caused by Rotenone exposure induces of
parkinsonism in rats including selective nigrostriatal dopanimergic degeneration & formation of
ubiquitin & a-synuclein inclusion.

46. 10.Transgenic Animal Models of Parkinson's Disease
Purpose and rationale :
The most prominent models are related to a-synuclein. The first transgenic mice that express
human a-synuclein were generated by Masliah et al. (2000).
These mice displayed a progressive accumulation of a 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.

47. (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.
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 (5ci/ml) & [14c]
choline (2 ci/ml)in the presence of 0.15mM pargyline chloride & 01.mM ascorbic acid.
7.Labeled slices are transferred to superfusion chambers & perfused with krebs solutions at 37c at
flow rate of 0.5ml/min.

48. 8. After washing &stabilization 5min fraction of superfusate are collect
9. The perfusion buffer contains 1  M nomifensine to inhibit dopamine reuptake &10  M
hemicholinium to inhibit choline uptake.
10. The slices are subjected to field strength to the current strength of 10- 15 mA/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.

49. 2. DOPAMINE STIMULATED ADENYLYCYCLASE 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 for 10min & supernatant is recentifuged at 27000g for
20min.
4. The pellet obtained is washed twice & suspended in 10mM imidazole, ph7.3. Membrane
protein is determined by bradfords method using bovine serum.
5. Adenylyl cyclase activity is measured by calculating the conversion rate of (32p) ATP to (32p)
CAMP.
6. The assay is perform in 250 l solution containing imidazole, mgcl2
7. papaverine dithiothreitol, ATP, GTP, phspocreatine,creatine phosphokinase.

50. 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.

51. Refrences:-
• Drug Discovery and Evaluation Pharmacological Assays By H. Gerhard Vogel (Ed.) Page no. :
577- 585
• S.K Gupta, text book of screening methods and toxicology
• Tripathi KD, Essentials of medical pharmacology, 6th edition, page no: 381-389.
• Rang, P.H. et al (2003) "Nuerodegenerative Disease" Pharmacology, Elsevier Science limited,
5.497-499.
• Goodman &Gilman's, 1996, Treatment of central nervous system degenerative disorders,
Mcgrawl Hill, 9 , 506-512.
• Richard, A.H.(2000) "Treatment of Parkinsons Disease",Pharmacology, Lippincotts Williams
&Wilkins, 2.83-84