The presentation is about the alzheimer's disease and Parkinson Disease. In this presentation, various screening models are given for both of the diseases.

1. ALZHEIMER’S DISEASE AND
PARKINSON DISEASE:
In vitro and In vivo models

2. Alzheimer’s Disease
• Alzheimer’s disease is a disease which
swindles the memory of the people
making it most devastating
neurodegenerative disorder.
Alzheimer’s disease is disease
contrasting the normal dementia.
• Disease was first demonstrated by
Dr. Alois Alzheimer.
• The study was conducted on 51 year
woman Auguste Deter’s Brain.
Neuritic plaques, neurofibrillary
tangles & amyloid angiopathy was
observed in the new silver histological
technique.

3. Symptoms of AD
• Memory loss
• Poor judgment
• Cognitive decline
• Changes in personality

4. Histopathology
• Amyloid plaques
• Neuritic plaques
• Cerebral amyloid
Angiopathy
• Neurofibrillary tangles
and neuropil threads
• Granulovacuolar
degeneration
• Hirano bodies

5. AD Brain PET Scan
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6. Goals for treatment ofAlzheimer’s
Disease
• Improve memory
• Improve functional status
• Improve behavior
• Delay or prevent onset

7. Screening methods
Screening methods
In Vitro Methods In Vivo Methods

8. IN VITRO METHODS
• Inhibition of acetylcholinesterase activity in rat
straitum.
• Inhibition of Butyrylcholinesterase activity in
Human Serum.
• Stimulation of phosphatidylinositol turnover in
Rat brain slices.
• [3H]N-Methylcarbamylcholine binding to
Nicotinic cholinergic receptor in Rat Frontal
Cortex.
• Uncompetitive NMDA receptor Antagonism.

9. Inhibition of acetylcholinesterase activity
in rat striatum
Purpose: To screen drugs for inhibition of acetylcholinesterase activity.
Rationale:
• Physiological role of acetylcholinesterase is rapid hydrolysis and
inactivation of Ach.
• Thus, inhibitor of AChE show marked cholinomimetic effects in
cholinergically innervated effectors of organs.
Reagents:
• 0.05 M Phosphate buffer, pH 7.2
• Substrate in buffer (198 mg acetylthiocholine chloride)
• DTNB in buffer (19.8 mg 5,5-dithiobisnitrobenzoic acid)(0.5 mM)
• A 2mM stock solution of test drug is made up in a suitable solvent
and q.s. to volume with 0.5 mM DTNB.
• Drugs are serially diluted (1:10) such that final concentration 10-4
and screened for the activity.

10. Inhibition of acetylcholinesterase
activity in rat striatum
Tissue Preparation:
Rats Are Decapitated
Brains are removed and corpora striata dissected free
Weighed and Homogenized in 19 volumes (approx. 7 mg /ml
protein) of 0.05 M NaH2PO4, pH 7.2.
25μl Aliquot of this suspension is added to 1 ml of the vehicle
and various concentration of test drug are prepared.
Incubated for 10 min. at 37o C.

11. Inhibition of acetylcholinesterase activity
in rat striatum
Evaluation:
• For IC50 determinations: Substrate conc. Is 10 mM diluted 1:2
in an assay yielding a final concentration of 5 mM.
• DTNB conc. is 0.5mM yielding 0.25mM final concentration.
% inhibition = slope control - slope drug × 100
slope control
• IC 50 values are calculated.

12. Stimulation of phosphatidylinositol
turnover in Rat brain slices
Purpose and Rationale:
• The muscarinic cholinergic cascade in brain and other
tissues appears to involve the hydrolysis of
phosphoinositides to form diacylglycerol and inositol
phosphates which can serve as second messengers.
• Muscarinic cholinergic stimulation can increase
orthophosphate incorporation into phosphatidylinositol.

13. Procedure:
Rat Brain slice preparation:
Rats were decapitated, and brains were rapidly removed and bathed in warm
Krebs-Ringer bicarbonate buffer (KRB) (118 mM NaCl,4.7 mM KCl, 0.75
mM CaCl2 , 1.18 mM KH2PO4, 1.18 mM MgSO4 24.8 mM NaHCO3, 10
mM glucose) which had been bubbled with O2/CO2, (95:5).
Slices from two rat cortices were pooled and then further sliced using a
tissue chopper to a thickness of 350 pm in two perpendicular directions. The
minced tissue was rapidly transferred to a flask containing KRB at 37°C and
dispersed. The tissue was continually agitated gently while being bubbled
with O2/CO2.
After the slices had been dispersed, they were washed four times in KRB for
30 min. Washing was performed by allowing the slices to settle, removing
the buffer with a pipette, and rapidly replacing it with fresh oxygenated
KRB.

14. Incubation with [3H]Inositol:
Following the wash period, the slices were allowed to settle,-excess buffer
was aspirated, and the volume of packed slices was estimated (usually 1.5
ml/cortex). The slices were transferred to a 50.ml conical tube and then
diluted with 4 volume of KRB to which had been added to give a final
concentration of 0.1 to 0.3 μm. The tube was gassed with O2/CO2 capped
tightly, and then incubated for 1h.
To measure the amount of label incorporated into lipids, 0.2 ml of the slices
was added to 0.8 ml of ice cold water to stop the incorporation. The slices
were then homogenized for 15 sec at a medium setting.
Aliquots were then taken for determination of protein by the method of Lowry
and for lipid extraction.

15. Lipid Extraction:
To tubes containing 0.25ml slices, either homogenized or intact, 1.0
ml of chloroform : methanol (1:2, v/v) was added.
An additional 0.35 ml of distilled water and 0.35 ml of chloroform
was added, and the tubes were capped tightly and shaken for 10 min.
The tubes were then briefly centrifuged at low speed to separate the
phases and 0.75 ml of the upper aqueous phase was taken for Dowex
chromatography.
The remaining upper phase and interface were aspirated, and 200 μ1
of the chloroform layer were taken for determination of radioactivity
incorporated into membrane lipids.
5 ml of OCS scintillation fluid were used as the scintillate, and
radioactivity was determined using a Beckman LS7500 liquid
scintillation counter.

16. Results:
Incorporation of [3H]inositol into phospholipids.
• For the first 15 min, there was a slow linear incorporation
of [3H]inositol into lipids which increased in rate after 15
min . This rate was linear for approximately 60 min, when
it began to slow. Incorporation reached a maximum at
about 240 min.
• To determine into which lipids [3H]inositol was
incorporate extracted and separated the various lipid
components of the membrane.
• Analysis by one-dimensional TLC indicated that 88.5% of
the radioactivity recovered co-migrated with PI. A small
proportion of the radioactivity remained at the origin,
suggesting the presence of labeled
polyphosphoinositides.

17. IN VIVO METHODS
Passive Avoidance
• Step Down
• Step Through
• Scopolamine induced
amnesia in mice.
• Two Compartment test.
• Up Hill Avoidance
Active Avoidance
• Shuttle Box Avoidance
(Two way shuttle box)
• Jumping Avoidance (One
way shuttle box)

18. IN VIVO METHODS
Discrimination
Learning
• Open Field Test
• Radial Arm Maze
• Y-Maze
• Morris water Maze
Conditioned
Responses
• Conditioned Nictitating
membrane response in
rabbits.
• Automated Learning and
memory model in mice
Genetic models
• Tau models
• Aβ tau axis
• Secretase models
• APOE Models
• Axonal transport models

19. Passive avoidance
• In the passive avoidance test, the laboratory animal (rat or
mouse) learns to avoid an unpleasant stimulus by inhibiting
locomotion and exploration. It is also called the inhibitory
avoidance test.
Step Down Test:
Purpose and Rationale:
• An Animal in an open spends a most of the time close to the
walls and in corners.
• When placed on an elevated platform in the centre of a
rectangular compartment, it steps down almost immediately to
the floor to the floor to explore the encloser and to approach
the wall.

20. Procedure:
• Requirements:
1. Mice of either sex.
2. A rectangular box (50×50) with electrifiable grid floor.
3. Grid floor is connected to shock device.
• Procedure:
The typical process consists of:
1. Familiarization
2. Learning
3. Retention test

21. Familiarization:
Animal is placed on the platform.
Released after the cylinder
Latency to descend is measured.
After 10 seconds of exploration, it is
returned to the home cage.

22. Learning:
Immediately the animal has descended from the
platform
An avoidable shock is applied (foot shock 50Hz, 1.5 mA for 1 sec.)
Animal is returned to home cage

23. Retention test:
The animal is again placed on the platform.
Step down latency is measured.
The test is finished when the animal steps down
or remain on the platform (cut off time: 60 sec.)
24 hrs. after the learning the trial.

24. Evaluation:
• The time of descent during the phase and
the time during the retention time is
measured.
• A prolongation of the step down latency is
defined as learning.

25. Scopolamine induced amnesia in
mice
Purpose and Rationale:
1. Scopolamine impairs memory retention when given to mice.
2. The ability of different cholinergic drugs agonist to reverse
the amnesic effect of scopolamine is now well documented.
Procedure:
Requirements: 10 male NMRI mice weighing 26-32 g, Scopolamine
hydrobromide.
Each mouse is placed in bright part of two chambered apparatus after
administration of 3 mg/kg of scopolamine hydrobromide.
After brief orientation period, mouse enters the second i.e. dark
chamber.
Doors are closed to avoid escape. A 1mA shock is applied trough grid
floor.
Then, mouse is return to home cage. After 24 hours, test is performed

26. Evaluation:
• After treatment with various doses of test drugs
latencies obtained were expressed as % latencies.
• In some cases, dose response curve obtained is
straight. Whereas, with other drugs inverse U
shaped curve is obtained.

27. Parkinson Disease
• Parkinson’s disease (PD)
was first described by Dr.
James Parkinson in 1817 as
a ‘Shaking palsy’.
• It is a chronic, progressive
neurodegenerative disease
characterized by both
motor and non motor
features.
• Progressive degeneration of
dopaminergic neurons in
the substantia nigra pars
compacta.
Symptoms:
• Tremor (trembling) in
hands, arms, legs, jaw, or
head.
• Stiffness of the limbs and
trunk.
• Slowness of movement.
• Impaired balance and
coordination, sometimes
leading to falls.

28. Pathology of Parkinson Disease
• Cerebral atrophy
• loss of dopaminergic neurons
from the substantia nigra
associated with the presence
of intraneuronal inclusions
called Lewy bodies.
• Significant neuronal loss also
occurs in the locus ceruleus,
dorsal motor nucleus of the
vagus, raphe nuclei, and
nucleus basalis.

29. IN VITRO METHODS
• Experiments using rat striatal slices.
• Dopamine stimulated Adenylyl cyclase activity.
• Radio ligand binding studies for D1 and D2
Dopamine receptors
• Dopamine release from synaptosomes.

30. Experiments using rat striatal slices
Purpose And Rationale:
Striatum in Brain is primarily affected in parkinsonism. The
release of dopamine and acetylcholine in response to test agent
serve as good in vitro marker.
Procedure:
Male rats 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.

31. The slices are kept floating for 30 mins. in Krebs solution & gassed with
95% & 5% CO2 at room temperature.
The slices are labeled by incubating for 30 min. at 37o C with [3H]
dopamine (5μci/ml) in the presence of 0.15mM pargyline chloride &
0.1mM ascorbic acid.
Labeled slices are transferred to superfusion chamber & perfused with
Krebs solution at 37o C at flow rate of 0.5 ml/min.

32. Drugs to be tested are present in the superfusion fluid.
The radioactivity in the superfusate samples and in the tissue
is determined by liquid scintillation counting.
After washing and stabilization for 5 min., superfusate are collected.
The perfusion buffer contains 1μM nomifensine to inhibit dopamine
reuptake & 10μM hemicholinium to inhibit choline uptake.
The slices are subjected to field strength to the current strength of 10-
15 A/cm2 & pulse duration of 2 m/sec at stimulation frequency of
3Hz for 5 min.

33. Dopamine stimulatedAdenylyl
cyclase activity
Male Sprague dawley rats(150-200 g) are decapitated and striata is
removed from both sides.
Striatal tissue is homogenized with homogenizer in chilled buffer
containing 10 mM imidazole, 2mM EDTA & 10% Sucrose, pH 7.3.
Homogenate is centrifuged at 1000 g for 10 min. & supernatant is
recentrifuged at 2700g for 20 min.
The pellet obtained is washed twice and suspended with 1mM Imidazole, pH
7.3.Membrane protein is determined by Bradford assay.

34. Dopamine stimulatedAdenylyl
cyclase activity
Adenylyl cyclase activity is examined by calculating the conversion
rate of (32p) ATP to (32p) cAMP.
The assay is perform in 250μl soln. containing imidazole, MgCl2
papaverine dithiothreol, ATP, GTP, phosphocreatine, creatine
phosphokinase.
The reaction mixture is pre incubated at 30o C for 5min. The reaction
is initiated by adding membrane proteins and incubated for 10 min.
The reaction is terminated by adding stopping solutio (ATP, SDS,
cAMP). Formed (32p)cAMP is separated from (32p)ATP by
Chromatography.

35. IN VIVO METHODS
• Tremorine and oxotremorine antagonism.
• Reserpine antagonism
• Circling behavior in nigrostriatal lesioned rats.
• Elevated body swing test
• Skilled paw reaching in rats
• Stepping test in rats.

36. Tremorine and oxotremorine
antagonism
Purpose and Rationale:
• The muscarinic agonists tremorine and oxotremorine
induce parkinsonism like signs such as tremors, ataxia,
spasticity, salivation, lacrimation and hypothermia.
• These signs are antagonized by anticholinergic drugs.

37. Tremorine and oxotremorine
antagonism
Procedure:
Take Group of 6-10 NMRI male mice weighing 18-22 g.
They are dosed with the test compound or the standard (5mg/kg benzatropine
mesilate) 1 h prior the administration of 0.5 mg/kg oxotremorine s.c..
Rectal temperature is measured for administration of the compound (basal
value) and 1, 2, 3 h after oxotremorine injection.
Tremor is scored after oxotremorine dosage in 10s observation period every 15
min for 1 h.

38. Evaluation:
• Hypothermia:
The differences of body temperature after 1, 2, 3 h versus basal values
are summarized for each animal in control group and test group.
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 all animals in each group at the 2 observation periods
are summarized.
The numbers in the treated groups are expressed as percentage of the
number of the control group.

39. 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 sign of eyelid ptosis, hypokinesia, rigidity,
catatonia and immobility.
• These Phenomena can be antagonized by dopamine agonists.
Observation:
• Rats treated with reserpine develop spontaneous orofacial
dyskinesia that is similar to tardive dyskinesia in humans.
• The incidence of tongue protrusions was recorded to quantify
the occurrence of oral dyskinesia.

40. References:
• https://www.slideshare.net/mohammadmuztaba/alzheimer-
models. Accessed on 6 Feb. 2022.
• https://www.slideshare.net/SONALPANDE5/screening-of-
antiparkinson-agent Accessed on 7 Feb. 2022.
• Gonzales R. A., Crews F. T., Characterization Of The Cholinergic
Stimulation Of Phosphoinositide Hydrolysis In Rat Brain Slices,
The Journal of Neuroscience, Dec. 1984, Vol. 4, Florida.
• Bondi M. W. et al., Alzheimer’s Disease: Past, Present, and
Future, Journal of the International Neuropsychological
Society, 4 Dec. 2017, Vol. 23, Special issue 9-10, Cambridge
online press.
• Demaagd G., Parkinson’s Disease and Its Management, A
peer-reviewed journal for managed care and hospital
formulary management, Aug. 2015, Vol. 40(8).