WELCOME TO PG JOURNAL CLUB

PRESENTER

SANJAY YADAV

M.PHARM

(PHARMACOLOGY)

II YEAR

OCT.10.2014

OBJECTIVE

To evaluate the neuroprotective effect of the nootropic

drugs, piracetam (PIR) and vinpocetine(VIN), in

rotenone–induced Parkinsonism in rats.

CONTENTS

LIST OF ABBREVIATION

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

CONCLUSION

REFRENCES

LIST OF ABBREVIATION

PIR= Piracetam

VIN= Vinpocetine

SNpc= Substantia nigra pars compacta

PD= Parkinsons disease

TNF= Tumor necrosis factor

IL= Interleukin

IFN= Interferon

MDA= Malondialdehyde

GSH= Glutathione

INTRODUCTION

Parkinson’s disease(PD) is a chronic , progressive

neurodegenerative disease that is charaterised by irreversible

loss of doaminergic neurons in the substantia nigra pars

compacta (SNpc).

Motor features of PD include bradykinesia, rigidity , resting

tremors and abnormalities of balance, posture and giat.

There is a strong evidence that inflammation in the brain

mediated by the activation of microgilia might be involved in

the pathogenesis of PD.

Under abrnormal conditions microglial cells , the resident

macrophages in the brain are in the resting stage and serve

the role of immune survelliance.

When subject to abnormal stimulation such as neurotoxins

or traumatic brain injury, microglia become activated and

undergo significant morphological changes.

The activated microglia secrete a panel of pro-inflamtory

cytokines and prostaglandins , such as tumor necrosis

factor , interleukin, interferon and prostaglandin –E2.

Once activated , these cytokines receptors trigger

intracellular death- related signalling pathways and lead to

the production of iNOS , COX-2 and the activation of

NADPH oxidase.

Activation of these pro inflamatory and cytotoxic and

factors is directly deleterious to neurons and subsequently

induces further activation of microglia , leading to

progressive degeneration of dopaminergic neurons.

PIR and VIN belong to no-otropic ,this category of drugs

enhance memory , facilitate learning and protect memory

processes against conditions which tend to disrupt them.

PIR is used to treat cognitive impairment in ageing brain injury, dementia , cerebral stroke, hence posses variety of neuroprotective actions.

VIN is widely used as a neuroprotective agent improves blood circulation , oxygen uptake and glucose utilization by brain. In addition it is an effective scavenger of hydoxyl radicals and inhibit lipid peroxidation. It can help improve cognitive function and short term memory both in animals and humans.

The present study was designed to test the role of VIN and PIR in neuroprotection and improving the motor deficits in rat model of PD.

MATERIALS AND METHODS.

MATERIALS:- Rotenone dissolved in DMSO and PEG (1:1)

• PIR in saline

VIN in acidic saline

Animals – 60 male albino rats 200-260kg BW.

Uv spectrofluorometer

Uv spectrophotometer

GSH and TNF kits

Compound Microscope

STUDY DESIGN

Group

no.

Description Treatment

I Vehicle injected group Sc. Injected every 48hr

II Rotenone group(1.5mg) Sc. In 5ml/kg every 48hr

III PIR group(100mg) p.o 100mg/kg daily

IV PIR group(200mg) p.o. 200mg/kg daily

V VIN group(3mg) p.o. 3mg/kg daily

VI VIN group(6mg) p.o. 6mg/kg daily

PARAMETERS ASSESSMENT ASSESSMENT OF MOTOR FUNCTIONS

1. Open field test

2. Pole test

BIOCHEMICAL ASSESMENT

1. Striatal dopamine content

2. Malondialdehyde content

3. Glutathione content

4. TNF-α factor

HISTOPATHOLOGICAL ASSSESSMENT

ASSESSMENT OF MOTOR FUNCTIONS

OPEN FIELD TEST:-

The open field arena with the measurement 113 x 113 x 44cm. was made of fumy glass.

The floor was painted with white lines that formed a 5 x5 cm.pattern.

The rats were introduced individually to the open field arenaand observed for ten minutes.

The ambulation (the no. of squares crossed) and mobilityduration (time of paw movement ) were scored.

POLE TEST:-

To assess basal ganglia –related disorders in rodents, The rats were placed head up on top of vertical wooden pole of 50cm long .

The base of pole was placed in the home cage . The rats received 2 days of training that considered of 5 trials for each session.

On the test day the animals received 5 trials , and time to orient downward (t-turn) and total time to descend (t-total) were measured. The mean of 5 trial was used and compared .

BIOCHEMICAL ASSESSMENT:- After performing the behavioral tests the animals were

deeply anesthetised by ether , the brains are quickly removed and washed with ice- cold saline.

One hemisphere for each brain was perfused with 10% paraformaldehyde sol. and serial coronal sections were cut through SNPc and were prepared for staining with dye for histopathological stidies.

The striata of second hemisphare of each brain were isolated , weighed , using teflon hamogeniser . Hamogenisation is carried out as 10% (w/v) either in acidified n- butanol (supernatant A) or phosphate – buffered saline (supernatant B).

The homogenate was sonicated and centrifused at 2000 x g for 10 min. The supernatant were kept at -80 deg. C. until the analysis of dopamine , MDA,GSH and TNF were done

DETERMINATION OF DOPAMINE:-

The supernatant A was subjected to specrtrofluorometric assay of dopamine following the principle of ciarlone and juras.

DETERMINATION OF MALONDIALDEHYDE

Tissue MDA level was assessed according to the spectrophotometric method of ohkawa et al. based on the reaction with thiobarbituric acid using 1,1,3,3-tetramethoxypropane as standard the color intensity was

measured uing a UV-visible spectrophotometer.

DETERMINATION OF REDUCED GLUTATHIONE:-

Concentration of total Glutathione and oxidised

Glutathione were measured spectrophotometrically using

commercial kits according to instructions of the

manufacturer. total GSH content was expressed in

micromole per gram of protein

DETERMINATION OF TUMOR NECROSIS FACTOR-(TNF-ALPHA):-

TNF-α was determined using an ELISA reader , according to mizutani et al. using biosourseinternational kits.

NEURONAL CELL QUANTIFICATION AND IMAGE ANALYSIS:-

Neuronal cells were quantified steriologically on three

regularly spaced sections covering the entire surface of the

SNpc as described previously by hoglinger et al. each section

was viewed at a low power whereas the cell number were

counted at high power.

After determination of the cell number in each slide, the

percentage increase in the cell number relative to rotenone

group was calculated and compared.

RESULTS:-

In the present study, repeated systemic administration of

rotenone to rats produced motor impairment,

histopathological changes and biochemical deficits.

ASSESSMENT OF MOTOR FUNCTIONS:-

OPEN FIELD TEST:-

It was observed that the injection of vehicle in

group 1 did not cause deterioration of the motor

performance in the rats in the open field test whereas ,

rotenone group showed a significant decrease in

locomotion, a lower rearing frequency and a shorter

mobility duration.

PIR (group iv : 200mg/kg/day, p.o.) and VIN (group v

and v1, 3 or 6 mg/kg/day,p.o.)significantly enhanced

the motor activity of the rats in the open field test as

compared to rotenone group(p<0.05).

Treatment with high dose of PIR 200mg/kg increased

the ambulation as compared to rotenone group.

further, treatment with both doses of VIN improved

the ambulation and the mobility duration as compared

to rotenone group.

The low dose of VIN 3mg/kg significantly improved

the ambulation and increased the mobility duration in

to both doses of PIR.

Pole test:-

Rotenone treated rats groupII showed higher t-turn and t-total compared to vehicle treated rats group I.

Treatment with PIR 100 or 200 mg/kg as well as vin 3 or 6mg/kg ameliorated both of the measurements t- turn and t-total in the pole test as compared to rotenone group.

VIN 6mg/kg group shows significantly lowered t-turn and t-total compared to PIR100 mg and 200 mg /kg groups p<0.05.

Biochemical analysis:- Biochemical analysis of stress biomarkers in the tissue

homogenate of rotenone group demonstrated significant changes as compared to vehicle –injected group

Striatal dopamine content :-Rotenone group(1.5mg/kg/48h/6 doses s.c.) showed a

significant decrease in dopamine levels compared to vehicle injected group.

Treatment with PIR (200mg/kg) and VIN(3 or 6mg/kg) significantly improved the striatal dopamine level as compared to Rotenone group.

Malondialdehyde:-

HISTOPATHOLOGICAL STUDIES :- Histopathological assessment demonstrated thet vehicle-

injected rats showed normal SNpc neurons with obvious nuclei. However, rotenone treated rats showed marked degeneration ; neurons appeared with low number/field and with indistinct neuronal boundaries .

The percent increase in number of dopaminergic neurons per eye field was 244% in the vehicle group compared to 100% in rotenone group .

PIR (100 or 200 mg/kg) and VIN (3 or 6 mg/kg ) groups showed improved histopathological picture for the SNpc and greater increase in dopaminergic neurons per eye field compared to rotenone group.

DISCUSSION AND CONCLUSION:- Overall , the results of the present study suggest that PIR may have role in

inhibition of neuronal loss mediated by pro-inflamottory cytokine, which

may initiate a new aspect of the role of no-otropic drugs in the treatment of

chronic parkinsonism.

A unique mechanism of VIN is its ability to alter the rheological properties

of red blood cells by increasing the electrolytes’s deformability, and also

inhibiting platelet aggregation.

These two actions combine to enable the blood cells to better penetrate the

small vessels of the cerebromicrovasculature , thus delivering adequate

supplies of glucose ,oxygen and energy substrates and cell nutrients which

can improve neurocognitive health and function.

VIN has aslso been shown to facilitate the release of oxygen from

hemoglobin and increase blood oxygenation. PIR also changes the physical

properties of membranes , and inhances membrane fluidity.

These results suggest thatr chronic treatment with PIR and VIN exhibit neuroprotective effect in rotenone –induced parkinsonian rats.

The effect of PIR has been suggested to arise from its inflammatory properties whereas VIN is likely to have an antioxidant action .

Therefore these agents can be investigated throughlyin clinical trials for use in parkinson’s disease.

Refrences :-