Alzheimers disease symptoms pathophysiology screening models - in-vitro and in-vivo models

1. Screening Models of
Alzheimers
NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY
(PHARMACY INSTITUTE)
GREATER NOIDA
Presented By:
Archna Singh
M.Pharm (Pharmacology)
PTSM-1
Under Guidance:
Dr. Saumya Das
Professor (H.O.D. Pharmacology)
NIET (Pharmacy Institute)
Greater Noida

2. ALZHEIMER’S DISEASE
Neurodegenerative disease or Aging
Disease.
Progressive impairment of memory and
cognitive function.
Development of senile plalgues=
Neuronal Destruction
Etiology= Destruction of nerve=
Cholinergic= Ach
Treatment= Ach level by
Acetylcholinestrase

3. SYMPTOMS
Poor Judgement
Depression, Anxiety,
Insomnia
Misplacing objects

4. Pathophysiology
Cholinergic
Hypothesis Amyloid
Hypothesis
Tau
Hypothesis
Acetylcholine
Plaques
Beta- Amyloid

5. Screening Methods
In-Vitro Models
In-Vivo Models

6. In-Vitro Models
1. Inhibition of Acetylcholinestrase Activity in Rat
Striatum.
2. Inhibition of Butylcholinestrase Activity in Human Serum.
3. Molecular forms of Acetylcholinestrase from rat frontal
cortex and striatum.
4. Release of [H3] Ach and other transmitters from Rat Brain
Slices.
5. Ex-Vivo Cholinestrase Inhibition.
6. Stimulation of phosphatidylinositol Turnover in Rat Brain
Slices.
7. Uncompetitive NMDA Receptor Antagonism.

7. Inhibition of Acetylcholinestrase Activity
in Rat Striatum
PURPOSE AND RATIONALE
• The purpose of this assay is to screen drugs for inhibition of
acetylcholine-esterase activity. Inhibitors of this enzyme may be useful
for the treatment of Alzheimer’s disease.
• It is generally accepted that the physiological role of AChE is the rapid
hydrolysis and inactivation of acetylcholine.
• AChE inhibitors may also be beneficial in the treatment of
Alzheimer’s dementia.

8. PROCEDURE
Reagents
1. 0.05 M Phosphate buffer, pH 7.2
- 6.85 g NaH2PO4 · H2O/100 ml distilled H2O
2. Substrate in buffer
-198 mg acetylthiocholine chloride (10 mM)
3. DTNB in buffer
- 19.8 mg 5,5-dithiobisnitrobenzoic acid (DTNB) (0.5 mM)
4. A 2 mM stock solution of the test drug is made up in a suitable solvent and q.s. to volume with 0.5
Mm DTNB (reagent 3). Drugs are serially diluted (1 : 10) such that the final concentration (in
cuvette) is 10–4 M and screened for activity. If active, IC50 values are determined from the inhibitory
activity of subsequent concentrations.

9. Tissue Preparation
• Male Wistar rats are decapitated, brains rapidly re moved, corpora
striata dissected free.
• Weighed and homogenized in 19 volumes (approximately 7 mg
protein/ml) of 0.05 M NaH2PO4, pH 7.2 using a Potter-Elvejhem
homogenizer (Kontes, Vineland, NJ).
• A 25 µl aliquot of this suspension is added to 1 ml of the vehicle or
various concentrations of the test drug and reincubated for
10 min at 37 °C.

10. Assay
Enzyme activity is measured with the Beckman DU-50 spectrophotometer. This method can be used
for IC50 determinations and for measuring kinetic constants.
Reagents are added to the blank and sample cuvettes as follows:
Blank: 0.8 ml PO4 buffer/DTNB
0.8 ml buffer/Substrate
Control: 0.8 ml PO4 buffer/DTNB/Enzyme
0.8 ml PO4 buffer/Substrate
Drug: 0.8 ml PO4 buffer/DTNB/Drug/Enzyme
0.8 ml PO4 buffer/Substrate
Blank values are determined for each run to control for non-enzymatic hydrolysis of substrate and
these values are automatically subtracted by the kindata program available on kinetics soft-pac
module.

11. EVALUATION
For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2
in an assay yielding a final concentration of 5 mM. DTNB concentration
is 0.5 mM yielding 0.25 mM final concentration
%Inhibition = slope control – slope drug
slope control
IC50 values are calculated from log-probit analysis.
X 100

12. In-Vivo Methods
Passive Avoidance
• Step Down
• Step Through
• Two compartment Test
• Up Hill Avoidance
• Scopolamine induced
amnesia in mice
Active Avoidance
• Shuttle Box Avoidance
(Two-Way Shuttle Box)
• Jumping Avoidance (One-
Way Shuttle Box)
• Runway Avoidance

13. Step-Down Method
Purpose and Rationale
• An animal (mouse or rat) in an open field spends most of the time
close to the walls and in the 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.

14. Procedure
Mice or rats of either sex are used.
Training is carried out in 2 sessions
Animal is placed on the elevated platform
Animal steps down on to the grid ( innate exploratory
behavior)
Electric shock (15sec) is given on the grids
Step down latency (SDL) is noted

15. EVALUATION
• The time of descent during the training phase and the time during
retention test is measured.
• Prolongation of the step-down latency is defined as learning.

16. MODIFICATION
• Induce amnesia to animals.
• Different methods to do so-
1. Electroconvulsive shock
2. Scopalamine
3. alcohol

17. STEP-THROUGH METHOD
Purpose and Rationale
• This test uses normal behavior of rats and mice.
• These animals avoid bright light and prefer dim illumination.
• When placed into a brightly illuminated space connected to a dark
enclosure, they rapidly enter the dark compartment and remain
there.

18. Procedure
Mice and rats of either sex are used.
The test apparatus consists of a small chamber connected to a larger dark
chamber via a guillotine door.
The test animals are given an acquisition trail followed by a retention trail
24hr later. In the acquisition trail the animal is placed in the illuminated
compartment at the maximal distance from the door, and latency to enter
the dark compartment is measured.
Animals that do not step through door within a cut-off time: 90 sec(mice) or
180sec(rats) are not used.

19. .
• Immediately after the animal enters the dark compartment, the door
is shut automatically and footshock is delivered.
• The animal is then quickely removed (within 10sec) from the
apparatus and put back into its home cage.

20. EVALUATION
• The time to step-through during the learning phase is measured and
the time during the retention test is measured.
• In this test a prolongation of the step- through latencies is specific to
the experimental situation.
• An increase of the step-through latency is defined as learning.

21. UP-HILL AVOIDANCE METHOD
PURPOSE AND RATIONALE
• Many animal species exhibit a negative
geotaxis, i. e. the tendency to orient and
move towards the top when placed on a
slanted surface.
• When placed on a tilted platform with
head facing down-hill, rats and mice
invariably turn around and move rapidly
up the incline.

22. PROCEDURE
Rats of both sex were used and maintained under standard conditions.
The experimental apparatus is a 50 x 50 cm box with 35 cm high
opaque plastic walls
The box can be inclined at different angles. The floor consists of 10
mm diameter stainless steel grid bars placed 13 mm apart
To deliver the tail-shock-a- tail electrode is constructed, consisting of a wire clip
connected to a constant current shock source. The animal is first fitted with the
tail-electrode and then placed onto the grid with its nose facing down
• .

23. .
During baseline-trails the animal’s latency to make a 180 degree turn
and initiate the first climbing response is measured. Thereafter the
animal is returned to its home cage.
During the experimental trails the latencies are measured and additionally
a tail-shock (1.5 or 2 mA) is administered contingent on the first climbing
response after the 180 degree turn.
Immediately after the shock the animal is placed in its home cage. Retest
is performed 24hr later.

24. EVALUATION
• The latencies are measured.
CRITICAL ASSESSMENT OF THE METHOD
• The up-hill avoidance technique promises to provide a useful addition
to the existing resource of inhibitory (passive) avoidance methods.
• Its most obvious advantage is that it can be administered to animals
weakened in sensory motor coordination by pharmacological or
surgical treatments.

25. RUNWAY AVOIDANCE
Purpose and rationale
• A straightforward avoidance situation features a fixed aversive
gradient which can be transversed by the animal.
• The shoc can bee avoided when the safe area is reached within the
time allocated.

26. PROCEDURE
Mice or rats of either sex are used and handled for several days before the
experiment.
The same box as used in step-through model can be used in this experiment.
The apparatus is uniformly illuminated by an overhead light source. A
loudspeaker, mounted 50cm above the start-box, serves for presenting the acoustic
conditioned stimulus

27. .
• .
The footshock is employed
Animal is allowed to explore whole apparatus for 5 min. The
guillotine door is then closed and the animal is closed and animal is
placed into the light starting area.
After 10sec the acoustic CS is applied and the door is opened. Shock
is turned on after 5 sec.
The procedure starts again. The training is continuous until the animal
attains the criterion of 9 avoidance in 10 trails. The time needed to
reach the safe area is measured.

28. EVALUATION
• The time animal required to reach the safe area on both days is
measured.
• In addition, the number of errors (not reaching the safe area) is
recorded.

29. REFERENCE
 H. Gerhard Vogel (Ed.) (2002) “Drug Discovery and Evaluation-
Pharmacological Assays”, 2nd edition. Springer-Verlag Berlin
Heidelberg Publishers, New York, pp 619-626
 Gupta SK. (2016), Drug Screening Method, 3rd edition, Jaypee
Brothers Medical Publishers (P) Ltd, pp 514-532
 https://www.slideshare.net/roohna/screening-of-anti-alzheimers
 https://www.slideshare.net/DrxBurade/preclinical-screening-for-
alzheimers
 https://www.slideshare.net/mohammadmuztaba/alzheimer-models