About Author:
Richa Shrivastava
National Institute Of Pharmaceutical Education And Research
Niper, Guwahati
richashrivastava18888@gmail.com
Dementia is defined as a loss of intellectual abilities that is severe enough to interfere with social or occupational functioning.
Alzheimer’s disease (AD) is most common cause of dementia. 50-70% of all cases of dementia are due to Alzheimer’s disease.
AD is characterized by early memory deficits, followed by gradual erosion of other cognitive functions. The most severe neuropathological changes occur in the hippocampus, followed by association cortices or subcortical structures, including the amygdale and nucleus basalis of Meyhert.
REFERENCE ID: PHARMATUTOR-ART-1832
We have two types of models for screening of anti-Alzheimer activity:
1 ) Models to test learning and memory.
2) Transgenic mouse models of dementia.
1) MODELS TO TEST LEARNING AND MEMORY:
As there is no definite model for AD, we can use models based on learning and memory. Learning and acquisition of memory are complex behavioural phenomenon. Memory deficiency due to aging is difficult to design in animal models.
The most commonly used in vivo screening methods for drugs affecting learning and memory can be considered under following:
I) Inhibitory avoidance methods.
II) Active avoidance test.
III) Discrimination learning.
IV) Conditioned response.
2) TRANSGENIC MOUSE MODELS OF DEMENTIA
I) Tau models.
II) Aβ-tau axis
III) Secretase models
IV) APOE models
V) Axonal transport models.
3) BEYOND RODENT MODELS
4) NON-TRANSGENIC MODELS
I) INHIBITORY AVOIDANCE METHODS: In these test animal fails to imitate activites and learned habits. Here animal learn to avoid an unwanted events by suppressing a particular behaviour.
A) Step down test: In open field, rodents prefer to be closed to wall or in corners. If placed on an elevated platform, they come down to floor and reach wall or corner. In this both mice and rats of either sex can be used. Room should be free of noise. A rectangular box 50x50x50 with grid floor and movable platform (7.5x7.5) in the centre of box is used. Floor is connected to shock device. The animal is given a foot shock of 50Hz, 1.5mA, 1s as it descends from platform. Latency to descend is measured. Prolongation of step-down learning is defined as latency.
B) Step through test in rodents: Rodents prefer to be in dark. If placed in a brightly illuminated area they rapidly go to dark area. The apparatus has a light chamber connected to a large dark chamber through a small door. A bulb (7W/12V) is used in bright in light chamber.
As the animal enters the dark chamber from light chamber the small door is closed and in dark chamber animal receives a unavoidable foot shock of 1mA; 1s to mouse and 1.5mA;2s to rat. An increase in step through latency is defined as learning.
C) Uphill avoidance test in rats: On a slanted surface, rodents move toward the top known as negative geotaxis. Rats and mice placed on a tilted platform facing down hill, they turn around and move up the inclined.
The apparatus is 50x50cm box with 35cm high opaque plastic walls. The floor consists of 10mm diameter stainless steel grid bars placed 13mm apart. A shock of 2mA is given to rats when it turns 180° and I step of climbing. Prolongation of latencies by test drug indicates positive effect on learning.
D) Scopolamine –induced amnesia in mice : Scopolamine, an anticholinergic drug produces amnesia.
Scopolamine is given to mice in dose of 3mg/kg and animal is placed in dark and light apparatus. As the mice enters the dark room the door is closed and shock of 1mA, 1s is given latencies in mice after treatment with test compounds are expressed as percentage of latencies in mice treated with scopolamine only.
E) Ibotenic acid-induced impairment of memory: Bilateral injection of ibotenic acid into the basal forebrain of rats produce lesions which leads to memory impairment.
Water maze, Y-maze, habituation tasks and inhibitory avoidance tasks with a light and dark compartment apparatus and inhibition of decrease of cholinesterase activity in the cortex are used to screen anti Alzheimer drug.
II) ACTIVE AVOIDANCE TEST: To avoid an noxious stimulus, animal may escape to terminate it.
A) Runaway avoidance in rats and mice: Animal is placed in a box which is uniformly illuminated and has one small door. A loud speaker is mounted 50 cm above the start box, and provides acoustic conditioning stimulus (80db,2000Hz tone. 5min animal is allowed to explore the whole apparatus. The door is then closed, after 10s the acoustic condition stimulus is applied and door is opened. After 5 min the shock of 1mA;1s for mouse and 1.5mA;2s for rat is administered after 5s. The time required for reaching safe are is measured to assess the efficacy of drug.
B) Shuttle box avoidance test (two way shuttle box): Apparatus is rectangular box 50x15cm with 40cm high metal walls and a grid floor. It is divided into two 25x25 compartments by a wall and a small door. Each compartment is illuminated by 20W bulb. The animal is allowed to explore the box for 5 mins. Then the door is closed. 20 s later light is switched on in compartment in which animal is present, the door is opened, atone(60db) is produced and 5s later a shock is applied in the light chamber and continued till animal goes to dark chamber. The time required by the animal to reach each safe compartment is recorded.
C) Jumping avoidance in one way shuttle box: Apparatus is a rectangular box of 40x25 and a grid floor and one goal area with narrow walls. Animal is allowed to explore goal area for 5 min. After that goal is blocked for 2s an acoustic conditional stimulus (100Hz;85db) is applied then after 5 min shock of imA;50Hz;0.5s)to animal. Animal jumps on the platform. Retention is tested on the second day until the animal reaches the criterion.
III) DISCRIMINATION LEARNING:
A) Spatial habitual learning: Spatial habitual learning means a decrease in reactivity to anovel environment after repeated exposure to that now familiar environment.
Apparatus: Rectangular chamber of 60x60x40cm for rats and 26x26x40cm for mice made of painted wood or gray PVC. A 25W green or red light electric bulb is placed directly above the maze to achive an illuminated density of 0.3lx at the centre. Rat is placed in the centre or in corner for 5-10 min session and record the following:
i) rearings or vertical activity: the no. of times an animal stood on its hind legs with foreleg in air or against the wall,
ii) duration of single rearing,
iii ) locomotion
B) Spatial learning test in the radial arm maze: In radial arm maze the study of spatial reference and working memory process in rats can be done.
Apparatus: is a wooden elevated 8arm radial maze with arms extending from a central platform having a diameter of 20cm. Each arm is 56cm long and 5cm wide with height of 2cm. Food pellets which serve as reward at the end of the arm. Animal are trained daily to collect food pellets. The session terminates after 8 choices and the rat has to obtain the maximum no. of rewards with least no. of errors.
C) Spatial learning test in water maze: Rats learn to swim in water tank to find to escape platform hidden under the water.
A circular water tank filled to a depth of 20cm with water at 25°C. Tank is divided into 4 equal quadrants and a small platform at 19cm height is located in one quadrant. Rat has to find the platform to escape. Well trained rats take less than 10s.
D) Olfactory learning in rats: Animal is deprived of water for 48h.
Apparatus is box 30x30x55 with a photo sensitive cell on the top of water spout. Rats are trained to approach the water spout and to break the light beam. Responses to the positive order are awarded. Session terminates when rat makes 90% correct choices.
IV) CONDITIONED RESPONSES:
Conditioned nictitating membrane responses in rabbit:
A small loop of surgical nylon is sutured in to the right nictitating membrane. After one day the rabbit is placed in a restrainer. Rabbit is filled with a headmount that helps to record the nictitating membrane response by physical coupling with a length of thread to nylon loop in the nictitating membrane.
2. TRANSGENIC MOUSE MODELS OF DEMENTIA
I) Tau models: Pre-tangled formation and hyperphosphorylation of tau was observed.
Suppression of P301L tau expression in rTg4510 tau transgenic mice, which normally express the mutant protein at a high level, reverses behavioural impairments in these mice although NFT formation continues.
This suggests that soluble tau rather than NFT is neurotoxic. Both oligodendrocytes and astrocytes contain filamentous tau inclusions in patient with FTD.
Neuronal loss is lower in the B301L tau models than in P301S tau mouse, consistent with the early onset.
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II) Aβ-tau axis: Aβ can promote tau pathology, although recent data show that tau reduction blocks Aβ- mediated toxicity.
Crossing the APD transgenic strain Tg2576 with the P301L tau transgenic strain JNPL3, or intracerebral injection of the long form of Aβ, Aβ42, into a second transgenic strain PR5 can be helpful in understanding pathophysiology of AD.
III) Secretase models: By genetically interfering with β and γ-secretase activity the role of these enzymes in APD processing, Aβ deposition and memory impairment has been established, with implication for treatment strategies (supplementary information S1).
Altering γ-secretase activity by expression of MI46L PSEN1 in an APP transgenic background increased Aβ42 formation and deposition.
Behavioural deficients and neuronal loss were also observed, even before Aβ was deposited.
Surprisingly, this effect was even more pronounced upon removal of endogenous mouse PSEN1 in PSEN1 knock in mice, suggest that PSEN1 is protective.
IV) APOE model: The allele APOEε4 is major risk factor for AD.
Crossing APP transgenic PDAPP (platelet –derived growth factor promoting expression of APP) mice onto an APoE-/- background strongly reduced Aβ formation.
The state of APOE4 lipidation and solubility also impacts on amyloidogenesis.
V) Axonal transport models: Axonal transport along microtubules is mediated by kinesis and dynein protein.
Disrupted axonal transport has been implicated in the pathology of AD and axonal transport defects have been observed in tau and APP transgenic mice.
Moreover, reduction of kinesis light chain in k/k+/- mice increased axonal defect and amyloidogenic mice.
Both tau and APP might be directly involved in axonal transport. Tau regulates motor proteins to cargoes. This increases tau binding to microtubules contributing to the transit defects in AD, is unclear. In AD, tau is hyperphosphorylated, which reduces its association with microtubules. Another possibility is that tau interacts directly with proteins of the motor complex, thereby altering axonal transport.
3) BEYOND THE RODENT MODELS:
Studies in fruit fly: Invertebrates model, and Drosophila melangaster and round worm, Caenorhabaditis elegans has been done to detect easy mutation at genetic level that are cause of AD.
Two types of fish, the sea lamprey and the zebra fish are also used.
4) NON-TRANSGENIC MODELS:
SAM(senescence-accelerated mice)
Chemically induced lesion
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