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PIRACETAM AND VINPOCETINE AMELIORATES IN ROTENONE INDUCED PARKINSONISM IN RATS
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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 :-