1. NEUROPROTECTIVE EFFECT OF
CYCLOOXYGENASE INHIBITORS IN
SPORADIC TYPE OF ALZHEIMER’S
DISEASE
Dinesh Kumar Dhull
B.Pharma.
Supervisor Co-supervisor
Dr. Satyanarayana S. V. Padi Mr. Saurabh Sharma
I.S.F. COLLEGE OF PHARMACY, MOGA, PUNJAB
2. cont….
Alzheimer Disease (AD) is a
Progressive, irreversible, age-related neurological and
psychiatric disorder characterized by
Progressive decline in memory
Cognitive performance
Loss of acquired skills leading to
Apraxia, Agnosia, Anomia and Aphasia (Stuchbury and Munch, 2005)
It was named after Alois Alzheimer who was the first to
described the disorder in a 51 year old women Auguste D., in
1907 (Maurer et al., 1997)
3. cont….
Basically, AD is of two types:
• Early onset or Familial AD
• Late onset or Sporadic AD
The prevalence of AD is rapidly increasing affecting more
than 26 million people around the world and this will
quadruple by 2050. (Magdalena et al., 2008)
4. cont….
CHARACTERISTIC FEATURES
Familial AD Sporadic AD
(Early onset) (Late onset)
Genetic abnormalities Insulin signaling and
Energy metabolism alterations
• Mutation in certain • Neuronal insulin receptor (IR) desensitization
genes like PS-1,PS-2 • Energy homeostasis imbalance in brain
or APP gene. • Decrease in ATP
• Decrease in GLUT-1 and GLUT-3 in brain
• Aβ peptide formation
• Neurofibrillary tangles
• Oxidative stress
(NFT) formation
• Neurotransmitter abnormalities
• Aβ peptide formation
• Neurofibrillary tangles (NFT)
formation
Taskasu et al, 1993; Salkovic et al., 2006; Emilia et al., 2008
5. NEUROPATHOLOGY
Sporadic Alzheimer’s disease is the most common type of
dementia which includes:
• Energy metabolism alterations
• Oxidative stress
• Neuroinflammation
• Excitotoxicity
• Neurotransmission abnormalities etc.
• Senile plaque formation
• Neurofibrillary tangles
6. Animal Model For Experimental Dementia
Intracerebro-ventricular (I.C.V.) administration of streptozotocin (STZ)
resulted in-
insulin receptor desensitization
behavioral,
biochemical,
histological and
neurochemical alterations
along with progressive deterioration of cognitive function similar to those
seen in sporadic type of alzheimer patients.
(Ding et al., 1992; Salkovic et al., 2003)
7. How I.C.V. STZ induces dementia?
ICV STZ
IR desentitization
↓ glucose uptake ↓ GLUT-1 & GLUT-3 ↓ glucose metabolism
Energy Homeostasis Imbalance
↓ ATP
↑ Ca2+ Mitochondrial dysfunction
Oxidative Stress
Neurodegeneration
Hellwag et al., 1992; Miller et al., 2006
8. How I.C.V. STZ induces dementia?
• Decreases glucose transporter proteins GLUT-1 and GLUT-3 (Simpson et al.,
1994; Harr et al., 1995)
• Decrease in cholineacetyltransferase activity (Hellwag et al., 1992; Blokland et al.,
1994)
• Insulin deficiency leads to rapid and large increases in tau phosphorylation
(Miller et al., 2006)
• Alters nerve growth factors (NGF) and monoaminergic neurotransmitter
(Ding et al., 1992)
• Increase in brain lactate and melondialdehyde and decrease in activities of
antioxidative enzymes
(Szkudelski et al., 2001; Sharma et al., 2002; Tahirovic et al., 2007)
• Decreases phospholipids, activates PLA2 and increases free fatty acids
(Hoyer et al.,1998)
11. CYCLOOXYGENASE cont….
• COX-1 expression is present in neurons and microglia (Hirst et al., 1999)
• COX-2 is expressed constitutively and inducible under pathological
conditions in the CNS including cortex, hippocampus, hypothalamus and
spinal cord. (Breder et al., 1995, 1996)
• COX-3 was first identified as a splice varient of COX-1 by
Chandrasekharan in dog brain and is biologically stable. (Chandrasekharan
et al., 2002)
• COX-3 is highly expressed in choroid plexus and spinal cord followed by
hypothalamus, hippocampus, medulla, cerebellum, cortex, in major brain
arteries and brain microvessels but not expressed in neurons.
(Kis et al., 2003,; 2004)
12. CYCLOOXYGENASE cont….
COX-1 COX-2
gene gene
CNS
COX-1 pCOX1 COX-3 COX-2b ?? COX-2 constitutive COX-2
constitutive constitutive inducible
pCOX1a pCOX1b
Chandrasekharan et
al.,2002 Nerve Neuroinflammation
Modulation of sensory Pain & Fever Transmission
process Neuronal Death
Yamagata et al.,1993
Adaptive Protective
function function
(Normal (Stressed neurons)
neurons)
Turini et al., 2002
13. CYCLOOXYGENASE cont….
• Non-selective COX inhibitors showed a reduced number of activated
microglia (Jin et al., 2008)
• Selective COX-2 and non-selective COX inhibitors prevent the
hypoperfusion induced memory impairment but not by protecting
hippocampal neurons. (Institoris et al., 2007)
• COX-2 inhibitor protected the brain against Aβ peptide induced memory
disturbances. (Cakala et al., 2007)
• Non-selective inhibitors reduced the excitotoxic neurodegeneration.
(Silakova et al., 2004)
14. CYCLOOXYGENASE cont….
• Non-selective COX inhibitor i.e. Mefenamic acid attenuates the I.C.V. STZ
induced cognitive deficit in rats.
(Mojarad et al., 2006)
• However, the differential role of COX isoforms-1, -2 and -3 in the
pathophysiology of sporadic Alzheimer’s disease is not clearly known.
• Further, the doses employed in the previous studies were selected
randomly that may inhibit two or more isoforms.
16. AIMS AND OBJECTIVES
To evaluate the neuroprotective effect of
cyclooxygenase-1, -2 and -3 inhibitors in
pathophysiology of sporadic Alzheimer’s disease.
17. MATERIALS AND METHODS
:
• Animals
Strain Wistar rat
Weight 200-250g
Sex Male
Food and Water ad libitum
• Materials
Streptozotocin (3mg/kg i.c.v.) dissolved in 0.1M citrate buffer pH 4.5
Valeryl salicylate (Selective COX-1 inhibitor)
Etoricoxib (Selective COX-2 inhibitor) and
Phenacetin (Selective COX-3 inhibitor) dissolved in 0.5% sodium CMC.
18. TREATMENT SCHEDULE
Day 1 2 3 Day 4 – 20 21 22
ICV ICV
STZ STZ Behavioral Day of
analysis sacrifice
Administration of COX-1 or -2 or -3 inhibitor
19. TREATMENT GROUPS
• Sham control
• ICV STZ control (3 mg/kg, i.c.v.)
• ICV STZ + Etoricoxib (5 mg/kg; i.p.)
• ICV STZ + Etoricoxib (10 mg/kg; i.p.)
• Etoricoxib per se (10 mg/kg; i.p.)
• ICV STZ + Phenacetin (20 mg/kg; i.p.)
• ICV STZ + Phenacetin (40 mg/kg; i.p.)
• Phenacetin per se (40 mg/kg; i.p.)
• ICV STZ + Valeryl salicylate (5 mg/kg; i.p.)
• ICV STZ + Valeryl salicylate (10 mg/kg; i.p.)
• Valeryl salicylate per se (10 mg/kg; i.p.)
20. BEHAVIOURAL STUDIES
Assessment of cognitive function:
• Spatial learning and memory using Morris water maze task
(Duckworth et al., 1999)
2. Spatial memory using Modified Elevated plus maze (Gupta et al., 2001)
Assessment of behavioral activity:
• Motor activity using Actophotometer
(Gupta et al., 2001)
21. OTHER PARAMETERS
Biochemical parameters
Acetylcholine esterase (AChE) (Ellman method, 1961)
Lactate dehydrogenase (LDH) (Racon Diagnostic Kit)
Superoxide dismutase (SOD) (Mishra and Fridovich, 1972)
Nitrite (Green et al, 1982)
Lipid peroxidation (LPO) (Will method, 1966)
reduced Glutathione (GSH) (Ellman, 1959)
Total protein (Biuret method)
Histopathological Studies
22. STATISTICAL ANALYSIS
• Results are expressed as mean ± S.D.
• Data for behavioral analysis was analyzed by two way
ANOVA followed by Bonferroni’s posthoc-test for
multiple comparisons.
• Data for biochemical analysis was analyzed by one
way ANOVA followed by posthoc Tukey’s test for
multiple comparisons.
• P < 0.05 considered statistically significant
23. DISCUSSION
Intracerebroventricular (ICV) administration of streptozotocin (STZ)
induces cognitive impairment, cholinergic deficit, oxidative stress,
neuroinflammation and neuronal death.
In the present study, administration of selective COX-1 or -2, but not
COX-3, inhibitor attenuated ICV-STZ induced behavioral and
biochemical alterations.
24. DISCUSSION cont…..
Accumulating data indicates that central administration of STZ causes
• Neuroinflammation
o ATP depletion and uncontrolled leakage of ions causing membrane
depolarization
o (Ter Horst and Korf, 1997)
o Overproduction of neurotransmitters such as glutamate that lead to
excessive Ca2+ influx (Adibhatla and Hatcher, 2005)
o Further activates PLA2, phospholipid hydrolysis and release of AA
o -a substrate for COX and is converted into final products i.e PGs
o (Lipton, 1999; Phillis and O’Regan, 2003)
o Ultimately leading to apoptotic or necrotic cell death. (Ho et al., 1998)
It has been reported that lercanidipine improved the cognitive dysfunction by
reducing oxidative stress and AChE activity suggesting the involvement of Ca2+
influx in ICV-STZ induced neurotoxicity and subsequent neuroinflammation.
(Sonkusare et al., 2005)
25. DISCUSSION cont…..
• COX activaton occurs secondary to NMDA receptor activation and increased
Ca2+ influx. (Miettinen et al., 1997)
• PGs produced, are neurotoxic in action contributing to neuronal and synaptic
loss followed by neurodegeneration. (Kukreja et al., 1986; Bezzi et al., 1998)
• It is known that the induction of PGs formation by central administration of
PGE2 or COX inducers such as endotoxin LPS or IL-1β is involved in loss of
working and spatial memory. (Jain et al., 2002; Matsumoto et al., 2004; Hein et al., 2007)
• Previous evidence indicates that non-selective COX (COX-1/-2) or selective
COX-2 inhibitors improved the memory performances in both EPM and MWM
paradigms and also reduce increased AChE activity in various neurotoxin
induced models of dementia. (Kumar et al., 2006; Kumari et al., 2007)
• Based on the present data, it is possible that COX-1 and/or COX-2 are
activated following ICV-STZ injection.
26. DISCUSSION cont…..
• Oxidative/Nitrosative stress (Shoham et al., 2007; Tiwari et al., 2009)
• During the peroxidase activity of COX, one superoxide free radical is generated
that results in ROS production which is an important mediator of neuronal injury.
(Pepicelli et al., 2002; Candelario-Jalil et al., 2003; Madrigal et al., 2003)
• ROS formation and subsequent O.S. may cooperate in a series of molecular
events that link to memory impairment.
(Beal et al., 1995; Bruce-Keller et al., 1998; Kumar et al., 2006)
• Also, COX and iNOS are co-localized in brain cells and COX regulates the
expression of iNOS. (Nogawa et al., 1998)
• Induction of COX expression further incresae the expression of iNOS that results
in NO-• free radicals and peroxynitrite generation. Indeed, COX inhibition is
reported to decrease iNOS activity. (Goodwin et al., 1999)
• Also, the antioxidant defence mechanism including endogeneous GSH and
SOD, is altered.
27. DISCUSSION cont…..
Previous study indicate that activation of COX is required for execution of
oxidative neuronal death. (Lee et al., 2001)
However, NSAIDs administration prevents glutamate mediated excitotoxicity and
subsequent O.S. (Fass et al., 2000; Jain et al., 2002; Scali et al., 2003; Silakova et al., 2004)
Thus, it is plausible that ICV-STZ induces activation of COX-1 and/or -2 that
results in oxidative and nitrosative stress mediated neurotoxicity.
In the present study, the selective COX-1 and/or -2, but not COX-3, inhibitor
significantly attenuated the increased oxidative stress markers i.e.
malondialdehyde and nitrite and improved antioxidant defences such as SOD
and GSH in the brain of ICV-STZ treated rats.
28. DISCUSSION cont…..
Prostaglandins such as PGE1 and PGE2 cause neurotoxicity that results in
neuronal death. (Kukreja et al., 1986; Hewett et al., 2000; Mana et al., 2002)
LDH – a well known marker of cell death and pathological changes in cortex and hippocampus.
ICV-STZ administration in rats markedly increase the LDH activity and neuronal
death in cortex and hippocampus (Hoyer and Lannert, 2007)
Also, COX-2 inhibitor show their cerebroprotection action by reducing infracts in
rats following cerebral ischemia (Sugimoto and Iadecola, 2003).
In another study, selective inhibition of COX-1 and/or -2 significantly increases the
number of healthy neurons in the CA1 hippocampus. (Sasaki et al. 1988; Nakagomi et al.
1989; Hall et al. 1993 Candelaria-Jalil et al., 2002)
29. DISCUSSION cont…..
These data along with the present study suggest the neuroprotective effect of
COX-1 and/or -2 inhibitors in ICV-STZ-treated rats.
It further suggest that COX-1 and/or -2, but not COX-3, activity is involved in the
morphological changes in neuronal cells and the progression of their damage
following ICV-STZ administration.
One possibility, that COX-3 inhibitor fail to show protection, is that COX-3 might be
sparsely distributed and/or COX-3 might not be induced in the brain areas that are
prone to neuronal damage following central STZ injection.
30. CONCLUSION
Selective COX-1 or -2, but not COX-3, inhibitor
showed the neuroprotective effect in ICV-STZ
induced experimental model of SAD.
They significantly-
• improved impaired cognitive performance,
• attenuated oxidative stress,
• improved altered cholinergic function and
• reduced neuronal damage
31. “A theory is something nobody believes, except the person who made it.
An experiment is something everybody believes, except the person who made it”
Albert Einstein
