Influences on an anti-inflammatory drug,
Ibuprofen, on spatial memory and N-Methyl D-
Aspartate receptor expression during aging
Undergraduate Researcher :
Alejandra Márquez Loza
BioResource Research
College of Agricultural Sciences
Project Mentor:
Dr. Kathy R. Magnusson
Department of Biomedical Sciences
College of Veterinary Medicine

Memory defines us
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Alejandra Márquez~ Memory and Aging

Memory decline with aging
 One of the first cognitive dysfunctions to arise with aging (Albert et al. 1992)
 40% of individuals within the fifth decade and 85% of elderly over the age of 80 suffer form AAMI (Larrabee et al. 1994)
Alejandra Márquez~ Memory and Aging
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World Population Ageing 1950-2050
I. DEMOGRAPHIC DETERMINANTS OF POPULATION AGEING
Underlying global population ageing is a process known as the “demographic transition“ in which mortality and then fertility decline from higher to lower levels. Decreasing fertility along children per woman in 1950-1955 to an extremely low level of 1.5 children per woman in 2000-
2005. Presently, the total fertility rate is below the replacement level in practically all with lengthening life expectancy (figure 1) has reshaped the age structure of the population in most regions of the planet by shifting relative weight from younger to older groups. The role of international migration in changing age industrialized countries. In 19 of those countries the rate is under 1.3 children per woman.
Fertility decline in the less developed regions started later and has proceeded faster
4 distributions has been far less important than that Future of memory decline
Figure 1. Total fertility rate and life expectancy at birth: w orld, 1950-2050
Major fertility reductions in the less developed regions occurred, in general, during the last three decades of the twentieth century. Over the last 50 years, the average total fertility rate in those regions dropped by more than 60 per cent,
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6
80 from 6.2 children per woman in 1950-1955 to 2.9 in 2000-2005 (figure 2).
Figure 2. Total fertility rate: world and development regions, 1950-2050 5
60
4
3
40
7
W orld
More developed regions
Less developed regions
Least developed countries
6
2
20
5
1
T otal fertility rate
Life expectancy at birth
4
0 0
1950 -55 1975-80 2000-05 2025 -30 2045 -50
(UN World Statistics, 2002)
A. F ERTILITY DECLINE
Fertility decline has been the primary determinant of population ageing. Over the last half century, the total fertility rate decreased globally by almost half, from 5.0 to 2.7 children per woman. Over the next half century, it is expected to drop to the replacement level of 2.1 children per women.
Fertility is well below the replacement level in the more developed regions
As a result of the sustained decline that occurred during the twentieth century, the average total fertility rate in the more developed regions has dropped from an already low level of 2.8
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1
0
1950-55 1975-80 2000-05 2025-30 2045-50
However, great disparities persist. In the least developed countries, the average total fertility rate is now 5.2 children per woman. In particular, in Eastern, Western and Middle Africa, it remains in excess of 5.5 children per woman.
Meanwhile, current rates are 2.5 children per woman or less in South-central Asia, South
America and the Caribbean. In 18 developing countries, the total fertility rate is estimated to be under replacement level already.
Population Division, DESA, United Nations 5

Biologically, memory can be defined as:
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Alejandra Márquez~ Memory and Aging

Analogous to our daily pathways
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Facilitating a neuronal pathway builds the strength of a memory
Alejandra Márquez~ Memory and Aging

 Neurotransmitter Release- through increased production and storage
 Formation of additional synaptic connections
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Long-term Potentiation 8
A mechanism underlying memory formation
Alejandra Márquez~ Memory and Aging

N-Methyl D-Aspartate Receptors
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One subtype of glutamate receptor that provide regulatory roles in neurotransmission associated with the performance of memory tasks
Alejandra Márquez~ Memory and Aging

NMDA receptors are made up of a combination of 3 major groups of subunit families
1. GluN1
 Has 8 different splice variants
 Aging particularly affects the
GluN1-1 and GluN1-3 variants
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2. GluN2A-D
 Aging particularly affects the
GluN2B subunit
3. GluN3A-B
 It is not known how aging affects these subunits
Alejandra Márquez~ Memory and Aging

Inflammation may account for aging changes in the NMDA receptor
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 Lim. et al., 2000 study: transgenic mouse model for Alzheimer showed chronic oral ibuprofen treatment was capable of suppressing plaque pathology including amyloid deposition
 The Mesches et al., 2004 study: an antiinflammatory drug, sulindac, improved spatial short-term memory and reverse the effects of aging on protein expression of NMDA receptor subunits in old mice
Alejandra Márquez~ Memory and Aging

Purpose of this study
 It is not known whether previously seen effects are at the level of mRNA and/or protein and whether it would improve both long and short-term spatial memory and NMDA receptor expression at younger ages
 In the present study we analyze the effects of an anti-inflammatory drug, ibuprofen, on spatial working (short-term) and reference
(long-term) memory and NMDA receptor at the mRNA level
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 Targeting the NMDA complex may play a role in developing treatment options to prevent or improve age-related changes in memory
Alejandra Márquez~ Memory and Aging

Hypothesis
 If inflammation is playing a role during aging on NMDA receptors, we would expect to see:
1) Ibuprofen enhance memory compared to control groups
2) Improve mRNA expression of GluN2B and all GluN1 subunits as a whole and the GluN1-1 and GluN1-3 splice variants, but not affect GluN1-2 splice variants
3) Ibuprofen would reduce cytokine levels compared to control groups, especially at older ages
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This research required 48 male, C57BL/6 mice of four different age groups
Treatment 1
375ppm
Treatment 2
Number of Mice per Age Group
5 months of age
14 months of age
20 months of age
26 months of age
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6
6
6
Number of Mice per Age Group
5 months of age
14 months of age
20 months of age
26 months of age
6
6
6
6
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Testing Techniques:
1. Cognitive memory
 Morris Water Maze
2. mRNA densities of NMDA receptor subunits
 In Situ Hybridization
3. Cytokine levels in brain and spleen
 Reverse Transcriptase- Polymerase Chain
Reaction
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1. Behavioral Cognitive Testing
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The Morris Water Maze

Memory Assessment
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Memory is measured as cumulative and average proximities to the platform
Alejandra Márquez~ Memory and Aging

Morris Water Maze - spatial memory tasks

Figure 1.2
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1. Spatial Long Term Memory
• Place trials
• Probe trials
2. Cognitive Flexibility
• Reversal trials
3. Spatial Short-term memory
• Working memory trials: Naïve and Delay
4. Cued control trials
Alejandra Márquez~ Memory and Aging

2. In Situ Hybridization
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1.
One half was used for sectioning for in situ hybridization
2.
Using probes for genes of interest- cDNA labeled with 33P-dATP,
3.
Brain and standard images were captured autoradiography
4.
The different prefrontal and frontal cortex brain regions analyzed for mRNA expression were:
 deep (cortical layers IV–VI)
 superficial (cortical layers II–III) layers of ventral orbital cortex, lateral orbital cortex, prefrontal cortex
 insular cortex
Alejandra Márquez~ Memory and Aging

3. Reverse Transcriptase-
Polymerase Chain Reaction
1. Tissue samples from brain and spleen collected
2. Interleukin I (IL-1beta) cytokine RNA isolated
3. RNA was reverse transcribed into cDNA for quantitative real time polymerase chain reaction  technique used to amplify DNA
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Effect of Ibuprofen on:
1. Cognitive memory
2. mRNA densities of NMDA subunit and
GluN1 slice variants
3. IL-1beta cytokine levels in the brain and spleen
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Long-term memory
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Place trials showed effect of age but there was no significant effect of treatment across trial types
*p <0.05 for difference from 5 month old mice. Bracket indicates significant differences collapsed across treatments. Mean ± SEM, N = 6.
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Short-term memory 26
Oldest ibuprofen treated mice showed significantly better performance in the delayed trial, compared to naive, as did both treatment groups at 14 months of age * p < 0.05 for difference from naive trial of the same age and treatment. Mean ± SEM N = 6.
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Cognitive Flexibility
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There was a significant effect of age but no significant effect of treatment. *p < 0.05 for difference from 5 month old mice.
Bracket indicates significant differences collapsed across treatments. Mean ± SEM, N = 6.
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Cued Control Task 28
There were no significant effects of age or treatment on performances in the cued control task, an assessment of sensory/motor skills and motivation. Mean ± SEM. N = 6.
Alejandra Márquez~ Memory and Aging

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mRNA density of GluN1 subunit and splice variants in lateral frontal cortex
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No effect
Ibuprofen decreased mRNA density for GluN1-pan and GluN1-1-containing subunits, but increased mRNA for GluN1-3-containing subunits across ages. No effect on GluN1-2
Alejandra Márquez~ Memory and Aging

mRNA densities of GluN2B subunit in lateral frontal cortex
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No effect
Ibuprofen decreased mRNA density for GluN2B in all but the oldest group
Alejandra Márquez~ Memory and Aging

mRNA densities of NMDA subunits in lateral frontal cortex
32 mRNA densities for NMDA receptor subunits and splice variants in layer II-III of lateral frontal cortex
Glun1-pan GluN1-1 GluN1-2 GluN1-3
Age- Receptor Subunits
GluN2B
= Expected to increase with ibuprofen
= Actual results
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IL-1B cytokine levels in the brain and spleen
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IL-1beta levels, as a fold change from 5-month-old controls, in the brain
( a) and spleen ( b) from different ages and treatments. There was a significant overall effect of age (p<0.02), but no effect of ibuprofen in IL-
1beta levels in the brain or spleen. N = 5-6. Mean ± SEM.
Alejandra Márquez~ Memory and Aging

Average subject weight (g) averaged across weighing sessions
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 There was a significant overall effect of age ( p <0.01), but no significant main effect of treatment on individual mouse weights when averaged across age groups and different weighing days
( p =0.12). N = 5-6. Mean ± SEM.
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Expected Results
 If inflammation is playing a role during aging on NMDA receptors, we would expect to see:
1) Ibuprofen enhance memory compared to control groups
✔
2) Improve the expression of GluN2B and all GluN1 subunits as a whole and the GluN1-1 and GluN1-3 splice variants, but not ✖ affect GluN1-2 splice variants
3) Ibuprofen would reduce cytokine levels compared to control groups, especially at older ages
✖
Alejandra Márquez~ Memory and Aging

What study results suggest:
1) The effects of ibuprofen on cognitive behavior and mRNA expression did not appear to be through an anti-inflammatory mechanism
2) Ibuprofen may be affecting NMDA subunit gene expression through a negative feedback mechanism
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Ibuprofen may not be enough to override epigenetic mechanisms regulating cytokine expression
 Rao et al., 2012 study: cyclooxygenase-2 (COX-2) neuroinflammatory markers- IL-1beta
Due to the hypomethylated state of the COX-2 CpG promoter region
 Chronic treatment of drugs for Alzheimer's and Bipolar Disease acting at the cellular level may provide transient protection correcting neuroinflammatory and synaptic remodeling, but may not provide full recovery by not targeting epigenetic regulation
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Limitations of this Study
 It may be possible there were toxicity effects. To rule out this possibility we had to have retained some tissue samples for analysis
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 To determine if ibuprofen was really acting on the brain, it would be necessary to analyze ibuprofen in the brain using mass spectroscopy. This is currently something we are looking into incorporating into this study
Alejandra Márquez~ Memory and Aging

Future of this study
 Currently still ongoing study
 Analyzing the effects of ibuprofen on protein may reveal new information
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Acknowledgements
 Funding for this project was provided in part by the Center for Healthy Aging Research of Oregon
State University through the LIFE Scholars
Program and by AG016332 grant from National
Institutes of Health to KRM.
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