WELCOME TO PG JOURNAL

PRESENTER-

SANJAY YADAV

M.PHARM

(PHARMACOLOGY)

II YEAR

SEP. 26.2014

TITLE

INDIAN JOURNAL OF PHARMACOLOGY

PIRACETAM AND VINPOCETINE AMELIORATE ROTRENONE INDUCED PARKINSONISM IN RATS

OBJECTIVE

To evaluate the neuroprotective effect of the nootropic drugs, piracetam (PIR) and vinpocetine(VIN), in rotenone–induced Parkinsonism in rats.

CONTENTS INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFRENCES

INTRODUCTIONparkinson’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 activetion of microgilia might be involved in the pathogenesis of PD.

Under abrnormal condition s microgilaial cells , the resident macrophages in the brain are in the resting stagte and serve the role of immune suvelliance.

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 nad 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 nootropic ,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 salineAnimals – 60 male albino rats 200-260kg BW.Uv spectrofluorometerUv spectrophotometerGSH and TNF kits Copound Microcsope

9

STUDY DESIGNGroup no.

Description Treatment

1 Vehicle injected group Sc. Injected every 48hr

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

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

1v PIR group(200mg) p.o. 200mg/kg daily

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

v1 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. Malondialdihyde content

3. Glutathione content

4. TNF-a factor

HISTOPATHOLOGICAL ASSSESSMENT

ASSESSMENT OF MOTOR FUNCTIONSOPEN FIELD TEST:- The open field arena with the measurement 113 x 113 x 44

cm. 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 arena and observed for ten minutes.

The ambulation( the no. of squares crossed ) and mobility duration (time of paw movent ) were scored.

POLE TEST:-

To assess basal ganglia –related disorders in rodents, The rats

were placed head up om 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 fro each brain was perfused with 10% paraformaldehyde sol. And serial coronal sections wrer 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 nad 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-ALPHA was determined using an elisa reader , according to mizutani et al. using biosourse international 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 1v : 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 .groupI.

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 pir 100 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 group(1.5mg/kg/48h/6 doses s.c.) showed a significant decrese 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:-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 nootropic 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 throughly in clinical trials for use in parkinson’s disease.

Refrences :-