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M. Pharm 1st Semester 2012-13




• A group of chronic neurological disorders • Characterized by progressive loss of motor function • Caused due to degeneration of neurons in the area of the

brain that controls voluntary movement


Parkinson’s disease
• Most common form

• First described in 1817 by the British physician, James

Parkinson • Also called primary parkinsonism, paralysis agitans, or idiopathic parkinsonism


Secondary parkinsonism
• Group of disorders similar to Parkinson’s disease with known causes
• Viral encephalitis, a rare brain inflammation that follows a flu-like infection • Degenerative disorders, such as dementia, multiple system atrophy,

corticobasal ganglionic degeneration, and progressive supranuclear palsy
• Structural brain disorders, such as brain tumors and strokes • Head injury, particularly the repeated injury that occurs in boxing • Drugs,







antihypertensives (methyldopa and reserpine)
• Toxins, such as manganese, carbon monoxide, and methanol • Use of the substance MPTP, previously found in contaminated heroin

• Usually refractory to all forms of treatment


Diagnostic Features
• Four Cardinal Signs • Tremor • Rigidity

• Akinesia & Bradykinesia
• Postural instability


Characteristic Symptoms
• Micrographia – small handwriting • Hypomimia – decreased facial animation • Hypophonia – soft speech • Dysarthia – unclear pronunciation • Dyspnea – labored breathing • Festination – shuffling gait


• The annual incidence increases with age
• Usual age of onset is at about age 60 • 20 per 100,000 persons in the fifth decade

• 90 per 100,000 persons in the seventh decade

• Men and women are effected equally

• Caucasians are more susceptible than Asians

and Africans


• Not precisely known but appears to be multifactorial • Oxidative damage due to impaired protective mechanisms • Environmental factors such as rural living, drinking well

water, and heavy metal, MPTP and hydrocarbon exposure
• Genetic predisposition particularly if the disease begins

before age 50
• Aging may induce defect in energy metabolism


• Most dopamine in the brain comes from the Substantia •

• •

Nigra It is composed of dopaminergic neurons whose cell bodies are located in the substantia nigra pars compacta (SNpc) These neurons project to the basal ganglia and synapse in the striatum (i.e., putamen and caudate nucleus). Degeneration of neurones in the SNpc and the nigrostriatal tract results in deficiency of dopamine in the striatum which controls muscle tone and coordinates movements. Clinical signs of PD appear when straital dopamine is reduced by about 80%


• Loss of pigmentation of the

SNpc, produced by neuromelanin within the dopaminergic neurons • Degeneration of the neural connection between the substantia nigra and striatum • Presence of filamentous cytoplasmic inclusions, termed “Lewy Bodies” in dopaminergic neurons of SN • A major component of Lewy bodies is α-synuclein gene




Neurotoxins based Models
• Compounds that produce both reversible (reserpine) and

• •

• •

irreversible (MPTP, 6-OHDA, paraquat, rotenone) effects have been used effectively Recent studies have focused more on irreversible toxins Models produced by 6-hydroxydopamine (6-OHDA) and 1methyl-1,2,3,6-tetrahydropyridine (MPTP) administration are the most widely used Paraquat and rotenone are more recent additions to the stable of toxic agents A common feature of all toxin-induced models is their ability to produce an oxidative stress and to cause cell death in Dopamine neuronal populations that reflect what is seen in PD Drawbacks to the use of these models are the time factor in these models versus the time factor in the human condition


Genetic Models
• Recently, the identification of different genetic mutations

(α-synuclein, parkin, LRRK2, PINK1, DJ-1) has led to the development of genetic models of PD
• Limitations are that the vast majority of PD cases arise

from unknown origins, with only ∼10% of PD cases are due to genetic mutations
• Importance is that they may reveal specific molecular

events that lead to the death of the DA neurons and potential therapeutic targets


Common Screening Methods
In Vivo
• Reserpine Antagonism • MPTP Model of Parkinson’s Disease • Circling Behavior in Nigrostriatal

In Vitro
• Culture of Substantia Nigra • Inhibition of Apoptosis in

• • • •
• • •

Lesioned Rats Elevated Body Swing Test Skilled Paw Reaching in Rats Stepping Test in Rats Tremorine and Oxotremorine Antagonism Transgenic Animal Models of Parkinson’s Disease Cell Transplantations into Lesioned Animals Transfer of Glial Cell Line-Derived Neurotrophic Factor (GDNF)

• • •

Neuroblastoma SH-SY5Y Cells Experiment using rat striatal slices Dopamine stimulated adenyl cyclase activity Radioligand binding studies for D1 & D2 dopamine receptors Dopamine release from synaptosome


In Vivo Methods
1. MPTP Model of Parkinson’s Disease
2. 3. 4. 5. 6. 7.

Circling Behavior in Nigrostriatal Lesioned Rats (6-OHDA) Elevated Body Swing Test (6-OHDA) Skilled Paw Reaching in Rats (6-OHDA) Stepping Test in Rats (6-OHDA) Reserpine Antagonism Tremorine and Oxotremorine Antagonism


Purpose and Rationale
• MPTP(N-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine) has

been shown to cause symptoms of Parkinson’s disease in exposed individuals.
• When administered to primates this compound causes a

partial destruction of basal ganglia and a syndrome that resembles Parkinson’s disease.
• Used to evaluate potential antiparkinson drugs.


• 8 adult rhesus monkeys(5–8 • •

kg) Treatment period 5–8days N-MPTP Cumulative I.V doses of 10–18 mg/kg is administered Develop parkinsonism-like disorder (akinesia, rigidity, postural tremor, flexed posture, eyelid closure, drooling) Symtoms are reversed by the administration of L-dopa


• The severity of parkinsonian symptoms is rated by trained

observers using a scale of 0 (normal) to 17 (maximum severity)
Scores Assesses movements Checking movements Attention and blinking Posture Balance and coordination Reactions Vocalizations 0 Normal Present Normal 1 Reduced Reduced Abnormal Abnormal trunk Abnormal trunk and tail Abnormal trunk, tail, and limbs Flexed posture 2 Sleepy Absent 3 4


Normal Normal

Reduced Reduced

Slow Absent



In Vivo Methods

MPTP Model of Parkinson’s Disease

2. Circling Behavior in Nigrostriatal

Lesioned Rats (6-OHDA)

5. 6. 7.

Elevated Body Swing Test (6-OHDA) Skilled Paw Reaching in Rats (6-OHDA) Stepping Test in Rats (6-OHDA) Reserpine Antagonism Tremorine and Oxotremorine Antagonism


Purpose and Rationale
• 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, or the dopamine precursor Ldopa is given. • Used for the study of central dopamine function and the evaluation of dopamine antagonists and agonists, particularly the activity of novel antiparkinsonian drugs.


• Male Wistar rats (200–250g) housed individually in a controlled • • • • •

environment with free access to food and water. Animals are anesthetized with sodium pentobarbital. Head is placed in a stereotaxic device (DKI 900) and positioned according to the atlas of König and Klippel (1963). After a sagittal cut is made in the skin of the skull, a 2-mm-wide hole is drilled with an electrical trepan drill. Care is taken not to lesion the meninges. A 30-gauge stainless-steel cannula connected to a Hamilton syringe is aimed at the anterior zona compacta of the substantia nigra (coordinates anterior 4.1, lateral 1.0 and dorso-ventral –2.5 from instrument zero). A total of 8 μg of 6-OHDA in 4 γ /l of saline is injected at a rate of 1 γ /4min. After the intracranial injection the wound is closed.


• The animal is allowed several weeks for recovery and for • •

• •

development of the lesion. Specially constructed opaque plastic spheres attached to solidstate programming equipment serve as test chambers. The number of full turns, either ipsilateral or contralateral to the lesion, is recorded on an automatic printout counter every 15min for 1- or 2-h test sessions. To determine the control values for ipsilateral turning, each subject is administered 2.5mg/kg of damphetamine and immediately placed in the circling chamber for 2 h. Control values for contralateral circling are determined by injecting apomorphine at 1mg/kg s.c. and recording the rat’s circling for 1 h. Test compounds are given i.p or s.c. and the animals placed into the circling chambers. Circling is recorded over a 1-h period.


• Percent change of drug turns from control turns is

recorded. • Using various doses ED50 values can be calculated.


In Vivo Methods
1. 2.

MPTP Model of Parkinson’s Disease Circling Behavior in Nigrostriatal Lesioned Rats (6-OHDA)

3. Elevated Body Swing Test(6OHDA)
4. 5. 6. 7.

Skilled Paw Reaching in Rats (6-OHDA) Stepping Test in Rats (6-OHDA) Reserpine Antagonism Tremorine and Oxotremorine Antagonism


Purpose and Rationale
• Measure

of asymmetrical motor behaviour hemiparkinsonian animals in a drug-free state



• Male, 8-week-old Sprague Dawley rats are anesthetized

• •

with sodium pentobarbital (60 mg/kg i.p.) and mounted in a Kopf stereotaxic frame. They are lesioned by injection of 8 μg 6-hydroxydopamine in 4 μl saline containing 0.02% ascorbic acid in the left substantia nigra (AP – 5.0, ML+1.5, DV – 8.0). The solution is injected over a 4-min period and the needle left in place for an additional 5 min before retraction. Seven days after the lesion, behavioural testing is performed. The animal is allowed to habituate in a Plexiglas box and attain a neutral position having all four paws on ground.


• The rat is held about 2.5 cm from the base of its tail and • • • •

• •

elevated 2.5 cm above the surface on which it has been resting. A swing is recorded whenever the animal moves its head out of the vertical axis to either side. Before attempting another swing, the animal must return to the vertical position for the next swing to be counted. Swings are counted for 60 s over four consecutive 15-s segments. 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. The criterion of biased swing is set at 70% or higher. 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 analyze swing

behavior data across the 15-s segments.


In Vivo Methods
1. 2. 3.

MPTP Model of Parkinson’s Disease Circling Behavior in Nigrostriatal Lesioned Rats (6-OHDA) Elevated Body Swing Test (6-OHDA)

4. Skilled

Paw Reaching in Rats

5. 6. 7.

Stepping Test in Rats (6-OHDA) Reserpine Antagonism Tremorine and Oxotremorine Antagonism


Purpose and Rationale
• Unilateral injection of 6-OHDA into the medial forebrain

bundle results in an impairment of paw reaching on both sides • Can be ameliorated by drug treatment or transplantation of a nigral cell suspension


Procedure: Apparatus
A Perspex chamber with a hinged lid.

A narrower compartment with a central platform running along its length, creating a trough on either side,

A removable double staircase is inserted into the end of the box, sliding into the troughs on either side of the central platform.

• 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.
• Each of the steps of the staircase contains a small well, and two 45-mg

saccharin-flavored pellets are placed in each well.


Procedure: Learning
• 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 10–15 min.


Procedure: Learning
• 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 The forepaw used for the first movement to reach the pellet on each test day is noted A first choice score of +1 corresponds to the paw contralateral to the lesion, a score of –1, to the paw ipsilateral to the lesion. Because rodents exhibit a “pawedness,” it must be noted whether there is a preference for one paw.


Procedure: Lesions
• The mesotelencephalic 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 1.5 μl and at a concentration of 4 μg/μl of 0.9% saline and 0.01% ascorbic acid twice over 3 min via a 30gauge stainless steel cannula at the stereotaxic coordinates: L = 1.6mm, AP = 0 mm, V = –7.6mm and L = 1.6 mm, AP = –1 mm, V = –8 mm. The coordinates AP and L are estimated relative to the bregma, and V is measured from the level of the dura, with the incisor bar set 5mm above the interaural line. Following each injection, the cannula is left in place for an additional 4 min to allow the diffusion of the neurotoxin away from the injection site. The sham-operated group receives sham lesions by identical injection of ascorbate-saline solution alone.


Procedure: Drug Treatment
• The animals are injected i.p. with the test drug or saline

30 min before the unilateral 6-OHDA lesion and 24 h thereafter.


• Test sessions are performed 4, 5, 7, and 8 weeks

after 6-OHDA lesion. • The parameters success, attempts and sensorimotor coordination are subjected to a twoway ANOVA with group as the independent measure and weeks as the dependent measure.


In Vivo Methods
1. 2. 3. 4.

MPTP Model of Parkinson’s Disease Circling Behavior in Nigrostriatal Lesioned Rats (6-OHDA) Elevated Body Swing Test (6-OHDA) Skilled Paw Reaching in Rats (6-OHDA)

5. Stepping Test in Rats (6-OHDA)
6. 7.

Reserpine Antagonism Tremorine and Oxotremorine Antagonism


Purpose and Rationale
• A clinically relevant unilateral model of Parkinsonian

akinesia • The 6-OHDA lesion induced marked and long lasting impairments in the initiation of stepping movements with the contralateral paw which can be ameliorated by the systemic application of drugs


Procedure: 6-OHDA Lesion Surgery
• Female Sprague Dawley rats receive two stereotaxic

injections of 6-OHDA (3.6 μg/μl in 0.2 μg/ml ascorbatesaline) into the right ascending mesostriatal dopamine pathway • A 10-μl Hamilton syringe is used at the following coordinates (in mm, with reference to bregma and dura):1. 2.5 μl at AP –4.4, L 1.2, V 7.8, tooth bar –2.4; 2. 3.0 μl at AP –4.0, L 0.8, V 8.0, tooth bar +3.4 at an injection rate of 1 μl/min. • The cannula is left in place for an additional 5 min before slowly retracted.


Procedure: Experimental Setup
• 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. • The stepping test comprises two parts:


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 The initiation of adjusting steps by each forelimb when the animal was moved sideways along the bench surface.

• Each test consists of two tests per day for three consecutive

days and the mean of six subtests is calculated.


Procedure: Initiation Time, Stepping Time, and Step Length
• The rat is held by the experimenter with one hand fixing the • •

• • •

hindlimbs and slightly raising the hind part above the surface. The other hand fixes the forelimb not to be monitored. Time is measured until the rat initiates movement with the forelimb not fixed by the experimenter, using 180 s as break-off point. Stepping time is measured from initiation of movement until the rat reaches the home cage. Step length is calculated by dividing the length of the ramp by the number of steps required for the rat to run up the ramp. The sequence of testing is right paw testing followed by left paw testing, repeated twice.


Procedure: Adjusting Steps
• The rat is held in the same position as described above

• •

• •

with one paw touching the table, and is then moved slowly sideways (5 s for 0.9m) by the experimenter, first in the forehand and then in the backhand direction. The number of adjusting steps is counted for both paws in the backhand and forehand directions of movement. The sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions. The test is repeated twice each day. The paw contralateral to the lesion is passively dragging when the rat is moved in the forehand direction, while the ipsilateral paw performs frequent stepping movements.


Procedure: Drug Application
• Stepping tests are repeated to determine the baseline

weekly after the 6-OHDA lesion.
• The drug tests are administered for 1 day only. • Various drugs can be evaluated in weekly intervals.


• Results are expressed as means ±SEM. • For statistical evaluation, the data are subjected to one-

factor analysis of variance (ANOVA) and Fisher post hoc



In Vivo Methods
1. 2. 3. 4. 5.

MPTP Model of Parkinson’s Disease Circling Behavior in Nigrostriatal Lesioned Rats (6-OHDA) Elevated Body Swing Test (6-OHDA) Skilled Paw Reaching in Rats (6-OHDA) Stepping Test in Rats (6-OHDA)

6. Reserpine Antagonism

Tremorine and Oxotremorine 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 antagonized by dopamine agonists.


• Male NMRI mice of either sex (20–25 g)

• They are injected intraperitoneally with 5mg/kg reserpine
• •

• •

and tested 24 h later The test compounds are injected 30 min prior to observation The animals are placed singly onto the floor of a Perspex container (30 × 26 cm, 20 cm high), situated on a Panlab proximity sensor unit Horizontal movements are recorded for 10 min Moreover, rearing and grooming episodes are recorded by an experienced observer


• Locomotor activity and grooming scores of drug treated

animals are compared with controls treated with reserpine and vehicle only by analysis of variance.
• Locomotor activity of drug treated group must be more

than reserpine treated control.


In Vivo Methods
1. 2. 3. 4. 5.


MPTP Model of Parkinson’s Disease Circling Behavior in Nigrostriatal Lesioned Rats (6-OHDA) Elevated Body Swing Test (6-OHDA) Skilled Paw Reaching in Rats (6-OHDA) Stepping Test in Rats (6-OHDA) Reserpine Antagonism

7. Tremorine




Purpose and Rationale
• The muscarinic agonists tremorine and oxotremorine

induce parkinsonism-like signs such as tremor, ataxia, spasticity, salivation, lacrimation and hypothermia.
• These signs are antagonized by central anticholinergic




• Groups of six to ten male NMRI mice (18–22 g)

• Dosed

orally with the test/ standard (5 mg/kg benzatropine mesilate) 1 h prior to the administration of 0.5mg/kg oxotremorine s.c. • Rectal temperature is measured before administration of the compound (basal value) and 1, 2 and 3 h after oxotremorine injection. • Tremor is scored after oxotremorine dosage in 10-s observation periods every 15min for 1 h. • Salivation and lacrimation are scored 15 and 30min after oxotremorine injection.




Absent Slight Medium Severe

0 1 2 3

Test drug & standard should reduce the score


Hypothermia • The differences of body temperature after 1, 2 and 3h versus basal values are summarized for each animal in the control group and the test groups. The average values are compared statistically. Tremor • The scores for all animals in each group at the 3 observation periods are summarized. The scores in the treated groups are expressed as percentage of the of the scores in control group. Salivation and lacrimation • The scores for both symptoms for all animals in each group are summarized at the 2 observation periods. The numbers in the treated groups are expressed as percentage of the number of the control group.


Critical Assessment
• The oxotremorine antagonism has been proven to be a

reliable method for testing central anticholinergic activity.
• The overt isomorphism between the animal model and the

symptoms of Parkinson’s disease recommend this test for
screening of anti-Parkinson drugs.
• However, the model measures only central anticholinergic

activity & cannot be used to screen dopaminergic drugs.


In Vitro Methods
1. Experiment




2. 3.

Dopamine stimulated adenyl cyclase activity Radioligand binding studies for D1 & D2 dopamine receptors

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.



Male spargue dawley rats(150250g) 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. The slices are kept floating for 30 min in krebs solution & gassed with 95% O2 & 5% CO2 at room temperature.

The slices are labeled by incubating for 30 min at 37C with [3H]dopamine (5μci/ml) &[14c] choline (2 μci/ml)in the presence of 0.15mM pargyline chloride & 0.1mM ascorbic acid. Labeled slices are transferred to superfusion chambers & perfused with krebs solutions at 37c at flow rate of 0.5ml/min

After washing & stabilization 5min fraction of superfusate are collected.

The perfusion buffer contains 1 μM nomifensine to inhibit dopamine reuptake & 10 μM hemicholinium to inhibit choline uptake

Drugs to be tested are present in the superfusion fluid.

The slices are subjected to field strength to the current strength of 10-15mA/cm2 & pulse duration of 2msec at stimulation frequency of 3Hz for 5min.


• The radioactivity in the superfusate samples & in the

tissue is determined by liquid scintillation counting.
• 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.


In Vitro Methods

Experiment using rat striatal slices

2. Dopamine

stimulated cyclase activity


Radioligand binding studies for D1 & D2 dopamine receptors


Male spraguedawley(150-250g) are decapitated & right & left striata are removed. Striatal tissue is homogenized by teflon homogenizer in chilled buffer containing 10mM imidazole, 2mM EDTA & 10% sucrose ph 7.3.

Homogenate is centrifuged at thousand g for10min & supernatant is recentrifuged at 27000g for20min.

Adenylyl cyclase activity is measured by calculating the conversion rate of (32p) ATP to (32p) cAMP.

The pellet obtained is washed twice & suspended in 10mM imidazole, pH7.3. Membrane protein is determined by bradfords method using bovine serum. The reaction mixture is preincubated at 30c for 5min, the reaction is initiated by adding membrane proteins and incubated for 10min. The reaction is terminated by adding stopping solution(ATP,SDS,cAMP). Formed (32p) cAMP is separated from (32p) ATP by chromatography.

The assay is perform in 250μl solution containing imidazole, mgcl2 papaverine dithiothreitol, ATP,GTP,phosphocreatine,creati ne phosphokinase.


In Vitro Methods
1. 2.

Experiment using rat striatal slices Dopamine stimulated adenyl cyclase activity

3. Radioligand binding studies for

D1 & D2 dopamine receptors


Male Sprague Dawley (150-200 g) are cervically dislocated, the right and left striata removed. straiatal tissue is homogenized with Teflon glass homogenizer in 20 volumes of ice cold buffer containing 50 mM trisHcl, pH7.4, 2 mM EGTA, and 10 % sucrose.

Homogenate is centrifuged for 5 min at 800 gms, and the supernatant is centrifuged for 20 mins at 49,000 gms.

Reaction mixtures containing 50 mM Tris-Hcl buffer, pH 7.4, 120mM NaCl , 5 mM KCl , 2mM CaCl2, 1mM MgCl2 and [3H] SCH 23390 (0.1-6.4nM) or [3H] Raclopride (0.25-16nM) in a total volume of 250µl is incubated at 37 C for 30 minutes. Terminated by vaccum filtration through whatmann filter. Further, washing with cold 50 mM TrisHcl buffer, pH 7.4, may be given.

The pellet is washed and suspended in 50 mM Tris- Hcl buffer, pH 7.4.

Nonspecific binding is defined as binding in the presence of 10µM cis-flupenthixol for D1 dopamine receptor binding or 1 µM haloperidol for D2 dopamine receptor binding.

The receptors densities and affinities are calculated by scathard analysis.


Teena Kunwar