JOURNAL CLUB SEMINAR INDIAN JOURNAL OF PHARMACOLOGY

1. WELCOME TO PG JOURNAL
PRESENTER-SANJAY
YADAV
M.PHARM
(PHARMACOLOGY)
II YEAR
SEP. 26.2014

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

4. CONTENTS
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFRENCES

5. 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 activetion of microgilia might be involved
in the pathogenesis of PD.

6.  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.

7.  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.

8.  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.

9. 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
 Copound Microcsope

10. STUDY DESIGN
Group
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

11. 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

12. ASSESSMENT OF MOTOR FUNCTIONS
OPEN 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.

13.  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 .

14. 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

15.  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.

16.  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

17.  DETERMINATION OF TUMOR NECROSIS
FACTOR-(TNF-ALPHA):-
TNF-ALPHA was determined using an elisa reader ,
according to mizutani et al. using biosourse
international kits.

18.  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.

19. RESULTS:-
 In the present study, repeated systemic administration
of rotenone to rats produced motor impairment,
histopathological changes and biochemical deficits.

20. 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.

21.  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.

22. 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.

27. 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.

29. Malondialdehyde:-

30. 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.

33. 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.

34.  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.

35. Refrences :-