Screening Models for Parkinson's Disease Ppt

1. Screening model of Parkinson Disease
Presented by:
Sanskriti
M.Pharm (Pharmacology)
PTSM-1
Under Guidance:
Dr. Saumya Das
Professor (HOD)
Pharmacology
NIET (Pharmacy Institute)
Greater Noida
NOIDA INSTITUTE ENGNEERING AND TECHNOLOGY
(PHARMACY INSTITUTE)
GREATER NOIDA

2. PARKINSON DISEASE
• Parkinson’s disease is a progressive generative disorder ,characterized by rigidity ,
tremor , and hypokinesia.
• Mostly affected the older people.
• First described by James Parkinson in 1817.
• Parkinson ‘s disease ,which is the second most common neurogenerative disorder
after a Alzheimer’s disease.

4. Parkinson’s disease is primarily associated
with the gradual loss of cell in substantia
nigra of the brain .This area is responsible
for the production of dopamine .
Dopamine is a chemical messenger that
transmits signals between two region of
the brain to coordinate activity

5. SCREENING METHOD OF PD
• In screening model two types
1. In vivo model
2. In vitro model

6. IN VIVO METHOD
1. MPTP model In monkey
2. Reserpine Antagonism
3. Stepping Tests In Rats
4. Tremorine and oxotremorne antagonism
5. Circling behavior In Nigrostriatal Lesioned Rats
6. Skilled Paw Reaching In Rats
7. Stepping Test In Rats
8. Elevated Body Swing Test

7. TREMORINE AND OXOTREMORINE
ANTAGONISM
PURPOSE AND RATIONALE
Muscarinic agonist tremorine and oxatremorine induced Parkinson
like disease like sign and symptom tremor, ataxia, tremor , spasticity,
salivation , lacrimation and hypothermia.
These signs are antagonized by anticholinergic drugs.

8. PROCEDURE
 Animal :Male NMRI Mice (18- 22gm)
 Std Drugs :5 mg/kg Benzatropine Mesilate
 Inducer:0.5 mg/kg oxatremorine sc.
Rectal temperature is measured
before administration of the
compound (basal value)
1,2 and 3hr after oxotremorine
injection
Tremor is scored after
oxotremorine dosage 10 sec
observation period every 15 min
for 1 hour

9. EVALUATION
• TREMOR SCORE
• Absent 0
• Slight 1
• Severe 3
• Medium 2
LACRIMATION AND SALIVATION
• Absent 0
• Slight 1
• Medium 2
• Severe 3
 Hypothermia
• The difference 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.

10. CONTD……
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 groups are expressed as
percentage of the number of the control group.

11. MPTP MODEL IN MONKEYS
Purpose and Rational
• N-MPTP (N- methyl-4-phenyl -1,2,3,6-tetrahydro pyridine)has been
shown to cause symptoms of Parkinson’s disease in exposed
individuals.
• Used to evaluate potential antiparkinsonian drugs.

12. PROCEDURE
8 adult rhesus monkeys (5-8kg)
N-MPTP 10-18mg/kg given by I.V route
Treatment period 5-8 days

13. CONTD…..
Develop parkinsonism :eyelid closure , kinesia
Symptoms are reversed by the administration of L-dopa

14. EVALUATION
• The severity is rated by using scale of 0 (normal)to 17 (max)
• Observation Scoring
• Movement
Normal 0
Reduced 1
sleepy 2
• Checking movement
Present 0
Reduced 1
Absent 2
 Attention and blinking
Normal 0
Abnormal 1

15. Observation Scoring
 Balance and co-ordination
Normal 0
Impaired 1
Unstable 2
Falls 3
 Vocalization
Normal 0
Reduced 1
Absent 2
Attention and blinking
Normal 0
Abnormal 1

16. 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 antagonised by dopamine agonist.

17. PROCEDURE
Male mice of either sex (20-25 g)
Inject intraperitoneally with 5mg/kg
Tested 24 h later injected 30 min

18. CONTD…..
The animals are placed singly floor of a Perspex container
(30×26,cm,20cm high)
Horizontal movements are recorded for 10 min
Moreover, rearing and grooming episodes are recorded by an experiences
observer

19. EVALUATION
• Locomotor activity and grooming scores of drug- treated animals are compared
with controls treated with reserpine and vehicles only by analysis of variance.

20. ELEVATION BODY SWING TEST
• Principle
EBST measures asymmetrical motor behaviour of hemiparkinsonian animals in a drug free
state and drug induced state.
Drug induced motor behaviour widely used as – behavioural index of hemiparkinsonian
animals .
High positive co-relations b/w swing and Apomorphine induced rotational behaviour.
 Requirements
Animal – Sprague Dawley rats
Drug : 6-OHDA(8 mg in 4 ml 0.9% saline containing 0.02% ascorbic acid). Test drug

21. PROCEDURE
40 rats are taken as test group
Anesthetised with sod.phenobarbital (600mg/kg i.p.) mounted in stereotaxic device
Stereotaxically lesioned in left substantia nigra
6-OHDA solution are injected over 4min and needle left in place for an additional 5 min before retraction
7 days after lesion, behavioural testing is performed
Remaining 24 animal served as control group.
(The animals are placed into a plexiglass box (40 ×40 × 35.5 cm)

22. CONTD….
• 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 moves its head out of the
vertical axis of either side.
• Before attempting another swing ,the animal must return to the vertical position for the next
swing counted .swings are counted for 60s over four consecutive 15 –s segments.

23. OBSERVATION
• A swing is recorded whenever the animal moves its head out of vertical axis .swing are
counted for 60 sec. with interval of 15 sec.
• The total number of swings made to each side is divided by the overall total number of
swings made to both sides to get percentages of left and right swings.
• At 30 and 45 s,6-OHDA- lesioned rats exhibit right biased swings of 70% or higher compared
to normal rats.
• A two way ANOVA is used to analyse swing behaviour data across the 15 segments.

24. IN VITRO METHODS
• Experiment using rat straiatal slices

25. EXPERIMENTAL USING RAT STRIATAL SLICES
Purpose and Rationale
• 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.

26. PROCEDURE
Male rats (150-250g) are decapitated the skull
Right and left striata are removed and placed in ice-cold kerb's solution
The striata is cut into 0.4mm thick slices using a tissue chopper
The slices are kept floating for 30 min in kerb's solution and gassed with 95% O2and CO2 at room temp.
The slices are labelled by incubating for 30 min at 37 C with dopamine (5 µg/ml)and choline (2µg/ml)in the
presence of 0.15ml pargyline chloride and 0.1ml ascorbic acid.
Labelled slices are transferred to super fusion chamber and per fused with kerb's solutions at 37C at flow rate
of 0.5ml/min.
The suprachiasmatic nucleus (SCN)

27. CONTD……
After washing and stabilization 5min fraction of superfusate are collected.
The perfusion buffer contains 1µm nomifensine to inhibit dopamine reuptake and 10 µm
hemicholinium to inhibit choline uptake.
The slices are subjected to field strength of 10-15mA/cm2 and pulse duration of 2 sec at
stimulation frequency of 3Hz for 5min.
Drugs to be tested are present in the super fusion fluid.

28. EVALUATION
• The radioactivity in the superfusate samples and in the tissue is determined by
liquid scintillation counting .
• The radio labeled choline method makes it possible to study Ach release in vitro
without inhibiting cholinesterase, thus minimizing auto inhibition of transmitter
release caused by accumulation of unhydrolyzed of Ach.

29. REFERNCES
• Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, Schrag AE,
Lang AE, Parkinson disease. Nature reviews Disease primers .2017 Mar 23;3:17013.
• A, Scharge , P. Barone ,R.G. Brown et al.,” Depression rating scales in Parkinson's
disease : critique and recommendations,” Movement
Disorders,vol.22,no.8,pp.1077-1092,2007.
• Bishoyi Arup k”slide share of screening model for Parkinson’s disease”2008 may
18.