This document summarizes a dissertation investigating the interaction between donepezil and rosiglitazone on cognition and neurochemistry in young and middle-aged rats. Donepezil is an acetylcholinesterase inhibitor used to treat Alzheimer's disease, while rosiglitazone is a PPARγ agonist with cognitive enhancing effects. Rats underwent object recognition testing after treatment with donepezil, rosiglitazone, or their combination. Molecular analyses included ELISAs of insulin, corticosterone, adiponectin, and ACh levels, as well as RT-PCR of genes related to cognition. Results found the drug combination did not enhance memory as expected, suggesting they may inhibit each other's
The current slide focuses on different screening models for neurodegenerative diseases along with a brief description of the diseases where the slides are to the points and brief with detailed evaluation.
Feature story from the Garvan Institute of Medical Research's August 2012 issue of Breakthrough newsletter. More at https://www.garvan.org.au/news-events/newsletters
THE ENDOCANNABINOID SYSTEM - Cady Wellness InstituteLouis Cady, MD
This lecture, presented on February 2, 2018 at the Cady Wellness Institute in Newburgh, IN, represented Dr. Cady's review of the current peer-reviewed medical literature current as of that morning. The concepts of "endocannabinoids" vs. "phytocannabinoids" were reviewed. Themes in the medical literature were explored, including the relationship of the endocannabinoid system and CB! and CB2 receptors to modulate hippocampal neurogenesis, cause apoptosis in breast cancer cells, and many other interesting correlations. A careful review of legal vs illegal options for obtaining plant phytocannabinoids in the state of Indiana was emphasized.
The current slide focuses on different screening models for neurodegenerative diseases along with a brief description of the diseases where the slides are to the points and brief with detailed evaluation.
Feature story from the Garvan Institute of Medical Research's August 2012 issue of Breakthrough newsletter. More at https://www.garvan.org.au/news-events/newsletters
THE ENDOCANNABINOID SYSTEM - Cady Wellness InstituteLouis Cady, MD
This lecture, presented on February 2, 2018 at the Cady Wellness Institute in Newburgh, IN, represented Dr. Cady's review of the current peer-reviewed medical literature current as of that morning. The concepts of "endocannabinoids" vs. "phytocannabinoids" were reviewed. Themes in the medical literature were explored, including the relationship of the endocannabinoid system and CB! and CB2 receptors to modulate hippocampal neurogenesis, cause apoptosis in breast cancer cells, and many other interesting correlations. A careful review of legal vs illegal options for obtaining plant phytocannabinoids in the state of Indiana was emphasized.
Molecular Substrates of Drug Abuse in a Schizophrenic PopulationAlan Lesselyong
This presentation is a Work In Progress (WIP) covering experiments examining the molecular correlates of drug abuse in human brains diagnosed with schizophrenia
Schizophrenia is a chronic and disabling mental illness affecting millions of people worldwide. The symptoms of schizophrenia are classified into positive, negative and cognitive symptoms. New receptor targets and drugs have being evaluated for addressing the multifaceted syndrome of schizophrenia.
is a chronic and disabling mental illness affecting millions of people worldwide. The symptoms of schizophrenia are classified into positive, negative and cognitive symptoms. New receptor targets and drugs have being evaluated for addressing the multifaceted syndrome of schizophrenia.
Molecular Substrates of Drug Abuse in a Schizophrenic PopulationAlan Lesselyong
This presentation is a Work In Progress (WIP) covering experiments examining the molecular correlates of drug abuse in human brains diagnosed with schizophrenia
Schizophrenia is a chronic and disabling mental illness affecting millions of people worldwide. The symptoms of schizophrenia are classified into positive, negative and cognitive symptoms. New receptor targets and drugs have being evaluated for addressing the multifaceted syndrome of schizophrenia.
is a chronic and disabling mental illness affecting millions of people worldwide. The symptoms of schizophrenia are classified into positive, negative and cognitive symptoms. New receptor targets and drugs have being evaluated for addressing the multifaceted syndrome of schizophrenia.
1. THE BEHAVIOURAL NEUROPHARMACOLOGY OF
PPAR AND CHOLINERGIC NEUROTRANSMISSION
INTERACTIONS IN THE AGING BRAIN.
Julie Corrigan
This dissertation was submitted to The University of Dublin, Trinity College,
in partial fulfilment of Bachelor of Arts
(Moderatorship in Neuroscience)
8th
April 2011
Name of Supervisor: Prof. Shane O’Mara
2. i
I
Abstract
The aim of this project was to investigate the relationship between two known cognitive
enhancing drugs. Donepezil (Aricept), is an acetylcholinesterase inhibitor used worldwide in
the treatment of Alzheimer’s Disease and dementia. Rosiglitazone (Avandia), is a PPARγ
agonist previously used as an anti-diabetic drug as it is involved in glucose uptake, disposal
and lipid metabolism, but is now also known to have cognitive enhancing effects. The
combination of these two drugs has not previously been researched. An object recognition
task was performed on young (3-4 months) and middle aged (13-14 months) rats. Each age
group was divided into 4 groups with 8 rats in each group (control, donepezil, rosiglitazone,
rosiglitazone and donepezil). Treatment was carried out for 5 days with testing commencing
on day 3 and animals sacrificed on day 6, 1-2 hours after the last test. Dosing was not
received on the last day of testing. Each donepezil rat received 0.2ml of a 0.3mg/kg/day of
donepezil and maple syrup suspension. Rats receiving a combination of the two drugs
received 0.2ml of a mixed 6mg/kg/day rosiglitazone and 0.3mg/kg/day donepezil with maple
syrup suspension. The object recognition task revealed MA rats did not learn the task but
rosiglitazone treatment improved recognition of the novel object. YA rats were able to
identify the novel object but when rosiglitazone and donepezil were given combined YA rats
were unable to identify the novel object to a significant level. After sacrifice, the brain was
dissected and frozen for further use in ELISA and PCR. The corticosterone, adiponectin and
insulin ELISA’s were carried out using plasma obtained from blood. Donepezil decreased
corticosterone levels in MA rats, rosiglitazone increased adiponectin levels in YA and MA
rats while drugs did not have a significant effect on insulin levels. An ACh ELISA was
performed on rat hippocampal tissue. Donepezil increased levels of ACh in YA rats. Real
time PCR was used to examine AChE, α-7, GLUT3, PPARγ, IL-1β and IL-4. There were no
significant changes in the levels of these primers. In conclusion, giving these drugs in
conjunction does not enhance memory. The drugs do not complement each other. They do
not appear to have a synergistic effect, in fact they seem to inhibit each other’s expected
normal behaviour.
3. ii
II
Acknowledgements
I would like to express my sincere gratitude to Professor Shane O’Mara for all his time and
guidance over the course of this project.
I would like to convey my special thanks to Dr. Charlotte Callaghan and Boon Wan Wang for
their continuous support, patience and technical assistance. I extend my thanks to all
members of the SOM lab for making it a great place to carry out my project work and a
thoroughly enjoyable experience.
4. iii
III
Abbreviations
ACh Acetylcholine
AChE Acetylcholinesterase
AD Alzheimer’s Disease
ADAS-cog Alzheimer’s Disease Assessment Scale cognitive subscale
ANOVA Analysis of Variance
APP Amyloid Precursor Protein
Aβ Amyloid-β
BADGE Bisphenol A diglycidyl ether
BuChE Butirylcholinesterase
CDR Clinical Dementia Rating
ChAT Choline Acetyltransferase
ChE Cholinesterase
CNS Central Nervous System
CoA Coenzyme A
DNA Deoxyribonucleic Acid
DON Donepezil
ELISA Insulin Enzyme-Linked Immunosorbent Assay
GFAP Glial fibrillary acidic protein
GLUT3 Glucose Transporter-3
5. iv
ICAM1 Inter cellular adhesion molecule 1
KO Knockout
LPS Lipopolysaccharide
LTP Long Term Potentiation
MA Middle Aged
MMSE Mini-Mental State Examination
NFT Neurofibrillary Tangles
NMDA N-Methyl-D-aspartate
NO Nitric Oxide
PMNs Polymorphonuclear neutrophils
PNS Peripheral Nervous System
PPARγ Peroxisome Proliferator Activated Receptor γ
RANTES Regulated upon Activation, Normal T-cell Expressed, and Secreted
RNA Ribonucleic acid
ROSI Rosiglitazone
T2DM Type 2 Diabetes Mellitus
TNFα Tumour Necrosis Factor α
TZD Thiazolidinedione
YA Young Adult
6. v
IV
List of Figures and Tables
Table 1: Adiponectin Standard Preparation
Figure 1: Treatment of donepezil and rosiglitazone decreases age related deficit of learning
and memory in the novel object recognition task
Figure 2: Combination of donepezil and rosiglitazone in YA rats does not improve memory
Figure 3: YACTL explore objects constantly throughout a given length of time
Figure 4: Variability between time points in each treatment group
Figure 5: Rosiglitazone and donepezil increase exploration time in the object recognition task
Figure 6: Treatment of Donepezil and Rosiglitazone does not significantly alter plasma
insulin levels in YA or MA rats
Figure 7: Donepezil significantly reduces stress levels in MA rats
Figure 8: Rosiglitazone Increases Plasma Adiponectin levels in YA and MA rats
Figure 9: Donepezil Increases ACh levels in YA rats
Figure 10: Treatment with rosiglitazone and donepezil does not significantly alter the levels
of mRNA expression in the AChE, α-7, GLUT3, PPARγ, IL-1β and IL-4 primers
Figure 11: A positive correlation is seen with donepezil and insulin levels in YA rats
Figure 12: A negative correlation in AChE improves discrimination ratio in MA rats
7. vi
V
Table of Contents
I Abstract i
II Acknowledgements ii
III Abbreviations iii
IV List of figures and tables v
V Table of contents vi
1. Introduction 1
1.1. Background 1
1.2. Alzheimer’s Disease 3
1.3. Cholinergic transmission and donepezil 5
1.4. PPARs and rosiglitazone 7
1.5. Object recognition task 10
1.6. Study aim 11
2. Materials and Methods
2.1. Behavioural experiments 12
2.1.1. Animals 12
2.1.2. Drugs and dosage 12
2.1.3. Novel object recognition 13
2.1.4. Analysis of recordings 13
2.1.5. Statistical analysis of behaviour 13
2.2. Molecular analysis
2.2.1. Blood samples 14
2.2.2. Tissue samples 14
2.2.3. Insulin Enzyme-Linked Immunosorbent Assay (ELISA) 14
2.2.4. Adiponectin ELISA 15
8. vii
2.2.5. Corticosterone ELISA 17
2.2.6. ACh ELISA 17
2.2.7. RNA purification and Quantification 18
2.2.8. Reverse transcription of cDNA and real time PCR 19
3. Results
3.1. Behavioural experiments
3.1.1. MA rats exploration 20
3.1.2. YA rats exploration 22
3.1.3. YA and MA total exploration time 24
3.1.4. YA and MA variability 25
3.1.5. Novel object exploration between time points and treatment groups 25
3.2. Molecular experiments
3.2.1. Insulin ELISA results 28
3.2.2. Corticosterone ELISA results 29
3.2.3. Adiponectin ELISA results 30
3.2.4. ACh ELISA results 31
3.2.5. RT-PCR results 32
3.2.6. Correlations 34
4. Discussion 37
5. Conclusion 43
5.1. Future studies 43
6. References 44
7. Appendix 47
9. 1
1. Introduction
Dementia is a loss of cognitive ability in a person that was previously unimpaired. It is
considered as being beyond what might be expected from the normal aging process.
Dementia is more common in the aged population but can occur in any stage of adulthood.
Dementia affects memory, thinking, language, judgement and behaviour. The main
pharmacological treatment groups for dementia are cholinergic neurotransmitter modifying
agents, such as acetylcholinesterase (ACh) inhibitors an example of which is donepezil.
Donepezil, marketed as Aricept, stops the breakdown of acetylcholine in the brain. It is used
to treat mild to moderate Alzheimer’s disease and can produce cognitive improvements in
people with dementia with lewy bodies (O'Brien and Burns, 2010). Rosiglitazone is an anti-
diabetic drug marketed as Avandia in the US. It is part of the thiazolidinedione class of drugs.
It binds and activates peroxisome proliferator activated receptor gamma (PPARγ) and alters
the expression of genes involved in glucose uptake and disposal and lipid metabolism
(Lebovitz et al., 2001). Rosiglitazone is also associated with cognitive enhancement.
Pedersen et al, showed Tg2576 mice administered rosiglitazone exhibited better spatial
learning and memory abilities than untreated Tg2576 mice (Pedersen et al., 2006). These
drugs have not yet been used in conjunction with one another. This project will investigate
how these drugs interact with one another.
1.1 Background
PPARs belong to the nuclear hormone receptor family. There are three isotypes of PPARs.
PPAR alpha was the first to be discovered and was described as a receptor that is activated by
peroxisome proliferators, thus giving them their name. The two other types are PPAR beta
and gamma. Each of these isotypes is coded for by a different gene. They have quite broad,
but specific, expression patterns (Michalik et al., 2006).
Their function surpasses peroxisome proliferation such as being involved in many cellular
and systemic roles. The major role of PPAR alpha is the regulation of energy homeostasis. It
activates fatty acid catabolism, stimulates gluconeogenesis and is also involved in the control
of lipoprotein assembly. The second isotype, PPAR beta, is needed for the development of
10. 2
the placenta and the gut. It controls energy homeostasis by stimulating genes involved in fatty
acid catabolism (Michalik et al., 2006). PPAR gamma is essential in adipose tissue
differentiation. Its expression is induced early when pre-adipocyte cell lines are being
differentiated (Lehmann et al., 1995). It is also important for the maintenance of adipocyte
specific functions such as, lipid storage in white adipose tissue and energy dissipation in
brown adipose tissue, and is needed to help differentiated adipocytes to survive (Michalik et
al., 2006). It is clear that PPARs are strongly involved in lipid existence and this is why
targeting them could aid in ways to combat type 2 diabetes mellitus (T2DM). PPAR gamma
is also implicated to have a role in reducing inflammation by being upregulated in
macrophages and inhibiting nitric oxide synthase among other things (Ricote et al., 1998).
The widespread view today is that PPARs act as lipid sensors that can translate changes in
lipid/fatty acid levels into metabolic activity which leads to either the catabolism of lipids or
to their storage (Michalik et al., 2006).
Cholinergic neurotransmission is the release of acetylcholine from the presynaptic neuron
into the synaptic cleft so it can interact with its receptors on the post synaptic neuron.
Acetylcholine is synthesized from acetyl CoA and choline through the catalytic activity of the
enzyme choline acetyltransferase (ChAT). The acetyl CoA is made in mitochondria that are
present in the nerve ending and the choline is transported from the extracellular fluid into the
terminal by a sodium dependant membrane carrier. Choline is derived from the diet and
delivered through the blood stream, thus it is the rate limiting step in the process (Amenta and
Tayebati, 2008). Acetylcholine synthesis is a rapid process which can support a high rate of
transmitter release. Recent evidence links changes in the choline transporter capacity with the
ability to perform tasks that tax attentional processes and capacities (Sarter and Parikh, 2005).
There are two types of cholinergic receptor: the nicotinic receptor which forms a ligand gated
ion channel, and the muscarinic receptor which is G protein coupled (Voss et al., 2010).
Learning, memory, attention and processing speed are regulated by acetylcholine. Enhancing
cholinergic transmission at nicotinic receptors by giving nicotine improves the performance
on learning, memory and attention tasks. Conversely, giving mecamylamine, an antagonist of
the nicotinic receptor, results in the impairment of attention and declarative memory (Voss et
al., 2010). Blocking muscarinic transmission using an antagonist such as scopolamine results
in impaired cognitive functions such as learning, memory, verbal fluency and attention (Voss
et al., 2010).
11. 3
There are differences between young and aging brains as would be expected. As we get older
our brain shrinks, they have larger sulcal widths and smaller gyri. Connections between
neurons are lost and neurogenesis is not as frequent. DNA damage occurs but results
regarding the age-dependent decline in DNA-repair capacity are conflicting and divided
(Subba Rao, 1993). Microglia are usually kept in an inactive state but in healthy aged brains
there are reports of increased microglial activity. There are also more pro-inflammatory and
less anti-inflammatory cytokines found which suggests an abnormal immune state of
microglia in the aged brain (Luo et al., 2010).
1.2 Alzheimer’s Disease
Alzheimer’s disease (AD) is the most common form of dementia. There are currently more
than 44,000 people with dementia in Ireland, with the majority of cases being AD. With an
aging population there is estimated to be an increase of 303% of dementia cases by 2036 but
with only a 40% increase in the population (Ireland, 2010). AD was discovered in 1906 by a
German doctor, Alois Alzheimer. He described the symptoms of his patient Auguste Deter
and these are the same basic symptoms described by clinicians today. There are two forms of
AD, early onset and sporadic. In early onset AD there are genetic links found. If a person has
a mutant Presenilin 1 or 2 gene or APP (amyloid precursor protein) gene they are more likely
to develop AD. Presenilin 1 can decrease the age of onset to as early as 25 years old in some
cases. However, most early onset cases would only be seen at 40 years and older. The
sporadic form of AD is the most common form and it also has a genetic risk factor, ApoE4.
Everyone has Apolipoproteins but in people that inherit ApoE4 the risk of developing AD is
much greater (Olarte et al., 2006).
The early signs of AD are usually mistaken as being related to aging or stress. These early
symptoms present as difficulty in remembering things that have just recently occurred and the
inability to form new memories. Symptoms are unique for each patient but in general as the
disease progresses more symptoms appear including confusion, mood swings, irritability and
aggression, language breakdown, long term memory loss and a general withdrawal of the
patient as they decline (Massoud and Gauthier, 2010).
12. 4
There are two main competing hypotheses suggested to explain the events that lead to AD.
These are the amyloid cascade hypothesis and the hyperphosphorylation of protein tau
hypothesis. Amyloid plaques are found in the entorhinal cortex, the hippocampus and the
neocortical areas but these plaques are also found in brains of normal individuals and do not
correlate with dementia. Neurofibrillary tangles (NFT) correlate with cognitive decline as
they are deposited in an ordered fashion. NFTs start in the entorhinal cortex and gradually
spread to the hippocampus, the rest of the temporal lobe, the association areas of the
prefrontal and parietal cortices, eventually covering all areas of the neocortex (Massoud and
Gauthier, 2010).
The amyloid hypothesis is the more predominant hypothesis and formulates that the amyloid
beta (Aβ) leads to secondary events that include the hyperphosphorylation of tau and
generation of NFT, inflammation, oxidation and excitotoxicity. An apoptotic cascade is
activated as a result with neuronal cell loss and a decrease in neurotransmitters. The reduction
in acetylcholine and to a lesser degree serotonin and noradrenaline is thought to be
responsible for the clinical manifestations of AD. Amyloid precursor protein (APP) is the
precursor to the Aβ peptide. It is cleaved by α-secretase, β-secretase and γ-secretase. When
APP is cleaved by α-secretase followed by γ-secretase it produces p3. This is a small non-
toxic soluble peptide and is neurotrophic. However when it is cleaved by β-secretase
followed by γ-secretase amyloid-beta protein is produced that deposits into plaques. This is
neurotoxic. More than 25 mutations in APP have been identified that are causative of the
hereditary form of familial AD (Thinakaran and Koo, 2008). APP gene duplication alone
causes early onset AD which is consistent with the finding of AD in Down syndrome patients
who carry 3 copies of chromosome 21, which holds the APP gene (Thinakaran and Koo,
2008).
Recent data suggest that cerebrovascular disease has a role in the pathophysiology of AD.
Regional brain hypoperfusion can lead to degenerative changes and cognitive impairments
that are described in the early stages of AD. Clinicopathological studies show that most cases
of dementia are due to a contribution of degenerative and cerebrovascular lesions (Massoud
and Gauthier, 2010).
13. 5
1.3 Cholinergic transmission and donepezil
Donepezil is a new class of acetylcholinesterase (AChE) inhibitor that has an N-
benzylpiperidine and an indanone moiety which gives it longer and more selective action
(Sugimoto, 2001). It is reversible and non-competitive. It was found by chance by Sugimoto
and his team in Eisai labs and they worked on it for four years to make the anti-AD drug. It is
the best AChE developed so far (Sugimoto, 2001). Donepezil is approved for use in mild to
moderate AD and is currently under review for the treatment of vascular dementia.
Cholinesterase (ChE) inhibitors improve cognitive activities and produce recognisable
clinical changes in AD and they have been associated with an improvement in functional
activities and behavioural symptoms. Donepezil reaches its peak plasma concentration 3 – 4
hours after oral administration and is well absorbed with a relative oral bioavailability of
100%. The absorption is not affected by time of administration or food. It has a long terminal
elimination half-life of about 70 hours and therefore can have the convenience of once daily
administration. It has a therapeutic effect at a 5 mg dose but 10 mg tablets are also available.
After several doses, donepezil will accumulate in the plasma up to sevenfold and reaches a
steady state level after 15 days of oral administration. Donepezil is metabolised mainly by the
cytochrome P450 system and has extensive first pass metabolism i.e. a lot is lost during the
absorption process. In comparison with placebo tests donepezil has been found to be safe and
well tolerated in clinical trials up to a 10mg daily dose. The most common side effects seen in
the patients included nausea, diarrhoea, insomnia, vomiting, muscle cramps, fatigue and
anorexia. This is a reflection of increased cholinergic activity induced by cholinesterase
inhibition (Sugimoto, 2001).
AChE is the primary ChE of the nervous system and muscle. The function of AChE is the
termination of cholinergic transmission by rapid catalytic hydrolysis of ACh. This releases
choline and acetic acid. Butirylcholinesterase (BuChE) has similar effects but it is less
specific and is inhibited by many agents. AChE is present as a membrane bound tetramer
(G4) in the human brain. There are a variety of different forms of AChE and this allows it to
be present intracellularly, intra-axonally, bound to the extracellular basal membrane and as a
soluble protein (Roman and Rogers, 2004). ACh is an important neurotransmitter in the
central nervous system (CNS) as well as the peripheral nervous system (PNS). In the PNS,
ACh can be found at the neuromuscular junction, in autonomic ganglia and at
parasympathetic effector junctions. Cholinergic transmission mediates most of the autonomic
14. 6
effects produced by vagus nerve stimulation. In the CNS the cholinergic neurons are found in
the brain and the spinal cord. They are important in mediating attention, learning, memory,
speech and emotion. Choline acetyltransferase is the synthetic enzyme for ACh and is used in
immunohistochemistry to identify cholinergic neurons in nervous tissue. They can also be
identified by the presence of positive in situ hybridization for AChE mRNA. AChE-rich glial
cells are found in all cortical layers. Neurons that contain BuChE are also found in the brain
but to a lesser extent. Most amyloid beta plaques and NFTs show intense AChE and BuChE
activity (Roman and Rogers, 2004).
There are many different clinical rating scales for AD taking in different views of the disease.
Mild to moderate dementia is usually defined by a score on the Mini-Mental State
Examination (MMSE) test (Roman and Rogers, 2004). This is a questionnaire to screen for
cognitive impairment. The Clinical Dementia Rating (CDR) scale provides a global rating of
dementia from 0 (normal, no impairment) to 3 (severe impairment) (Roman and Rogers,
2004). It evaluates memory, orientation, judgement and problem solving and in functional
domains it assesses community affairs, home and hobbies, and personal care. The ADAS-cog
(Alzheimer’s disease Assessment Scale cognitive subscale) is the accepted standard
measurement of cognitive abilities in patients with AD (Roman and Rogers, 2004). Decline
in untreated patients’ shows a score increase of 4 – 6 points per year. Donepezil trials that
were double blinded and placebo controlled for a duration of 24 weeks showed statistically
significant improvements in cognition in the ADAS-cog and MMSE, as well as in global
function by CDR (Roman and Rogers, 2004). Donepezil treatment can in some cases delay
the need for nursing home placement by up to two years, it improves disruptive behaviours
and has shown significant preservation of function versus placebo in two major 1-year phase
III studies (Roman and Rogers, 2004).
The current known mechanism underlying the neuroprotection of donepezil is the up-
regulation of the PI3K/Akt cascade. PI3K is a lipid kinase involved in the regulation of a
number of processes such as glucose metabolism, cell growth, proliferation etc. It is activated
by hormones such as insulin and growth factors e.g.NGF. Shen et al. show that there could be
another possible pathway that donepezil takes. This is by a decrease in glutamate toxicity
through the down-regulation of NMDA receptors, following stimulation of alpha7 nAChRs.
(Shen et al, 2010)
15. 7
1.4 PPARs and rosiglitazone
Rosiglitazone is part of the family of Thiazolidinediones (TZDs). TZDs are a new class of
oral anti-diabetic drug that selectively enhances or partially mimics certain actions of insulin
on carbohydrate and lipid metabolism, causing a slowly generated anti-hyperglycaemic effect
in T2DM. This is usually accompanied by a reduction in circulating concentrations of insulin,
triglycerides and non-esterified fatty acids. Rosiglitazone is a selective agonist at the PPAR
gamma nuclear receptor. It reduces glycaemia by reducing insulin resistance at adipose
tissue, skeletal muscle and liver. Ciglitazone was the first described TZD in the 1980’s and
many followed after that such as troglitazone, pioglitazone, and rosiglitazone (Annex, 2010).
Rosiglitazone is marketed under the name Avandia for use in T2DM, but was recently
removed from the European market. It is still available in the US. It is contraindicated in
patients that; are known to be hypersensitive to TZDs, have cardiac failure, have an acute
coronary syndrome, hepatic impairment or diabetic ketoacidosis. Rosiglitazone can cause
dose-dependent fluid retention. When it is used with insulin there is an increase in risk of
oedema, as both are associated with fluid retention. This could increase the risk of ischemic
heart disease. In clinical trials there was evidence of dose related weight gain with
rosiglitazone especially when being used in conjunction with insulin. It is also related to a
reduction of haemoglobin levels so in patients with already low levels there is a risk of
anaemia. Long term use of rosiglitazone showed an increase in the incidence of bone
fractures in patients, in particular females (Annex, 2010).
The bioavailability of rosiglitazone after an oral dose is approximately 99% and its plasma
concentrations peak around 1 hour after dosing. There are no significant effects seen in the
overall exposure of the drug when administered with food and its absorption is not affected
by increases in gastric pH. It has high plasma protein binding that is not influenced by
concentration or age. The major routes of metabolism are N-demethylation and
hydroxylation, followed by conjugation with sulphate and glucuronic acid. The major
metabolite, para-hydroxy-sulphate, cannot be ruled out as a contributing factor to the activity
of rosiglitazone as its effects are not fully understood yet. The terminal elimination half-life
of rosiglitazone is approximately 3 to 4 hours and its major route of excretion is urine
(Annex, 2010).
16. 8
Uemura et al. studied the effect of insulin on neuronal glucose uptake by assaying glucose
uptake, translocation of GLUT3 to the plasma membrane (PM) and fusion of GLUT3 vesicles
with the plasma membrane. They found that insulin stimulated the translocation of GLUT3 to
the PM but this is not sufficient to increase glucose uptake. The increase in glucose uptake is
due to increased fusion of GLUT3 vesicles with the PM which results in more GLUT3 being
exposed to the extra cellular surface. This paper suggested that glucose uptake is regulated by
at least two separate factors, the promotion of GLUT3 to the PM, and a membrane
depolarization that will induce fusion of GLUT3 vesicles with the PM (Uemura and
Greenlee, 2006). Another study researching glucose transporters showed that rosiglitazone
substantially improves peripheral insulin sensitivity in vivo by the normalisation of GLUT4
protein in fat and an increase of GLUT1 protein levels in fat and skeletal muscle. They also
found a direct effect of rosiglitazone to increase GLUT1 expression in vitro in adipocytes
suggesting the drug was exhibiting a direct insulin sensitising effect (Kramer et al., 2001).
García-Bueno et al. investigated the effects of rosiglitazone in the brain after stress in rats.
They found that stress decreased the cortical synaptosomal glucose uptake but that this effect
was prevented with treatment of rosiglitazone by restoring protein expression of GLUT3
(García-Bueno et al., 2006).
Cowley et al. investigated changes in LTP and astrocytosis. They found that microglia
activation was increased with age but that it was not due to rosiglitazone. CD11b is a cell
surface marker for microglia and the increase in its expression was unaffected by
rosiglitazone treatment so from this they concluded that microglia are not a target for
rosiglitazone. GFAP is a marker for astrocytes in the brain. There is an age related increase in
GFAP mRNA and GFAP protein in the thalamus, hypothalamus and hippocampus. With the
treatment of rosiglitazone in aged rats, GFAP immunoreactivity was inhibited which suggests
that rosiglitazone specifically targets astrocytes. Astrocytes and endothelial cells are a major
source of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) in
the brain. Its expression is stimulated by inflammatory cytokines such as TNFα. Treatment
with rosiglitazone noticeably inhibits RANTES mRNA which is consistent with the finding
that the promoter region of its gene has a PPARγ response element. The evidence from this
project suggests that activated astrocytes in aged rat brains release TNFα which causes a
negative impact on LTP, while rosiglitazone down-regulates astrocyte activation, thus
decreasing TNFα production and consequently leads to restoration of LTP. From this study it
was concluded that rosiglitazone mediates its effects on endothelial cells and that its
17. 9
immunomodulatory and anti-inflammatory effects are as a result of interactions between
endothelial cells and astrocytes (Cowley et al., 2010).
Loane et al. provide data that shows rosiglitazone attenuated the age related increases in IL-
1β mRNA and protein and the associated nitric oxide (NO) production in the hippocampus.
This suggests that rosiglitazone down-regulates microglial activation as they are the main cell
source of IL-1β and NO. However there was no reduction in MHCII which is a cell surface
marker for microglia. This proposes that the action of rosiglitazone is to target the
transcription of inflammatory genes. They found that increases in IL-1β mRNA and protein
induced by LPS, and also the increase in iNOS expression in glia was attenuated by
rosiglitazone. The data showed that neither age nor rosiglitazone affected PPARγ mRNA
expression or age related protein expression but they did find that rosiglitazone increased
PPARγ in hippocampal tissue prepared from aged rats. There is also evidence that
rosiglitazone might exert its anti-inflammatory effects by up-regulating IL-4 for example in
tissue prepared from aged, rosiglitazone treated rats, IL-4 was increased and secondly, the IL-
4 concentration decrease in the hippocampus that occurs in aged rats was reversed in tissue
prepared from rosiglitazone treated rats. To reinforce the finding that the action of
rosiglitazone is mediated by IL-4 they investigated the effects of rosiglitazone on LPS-
induced changes in glia prepared from wild type and IL-4 knockout (KO) mice. They showed
that in the wild type mice rosiglitazone attenuated the LPS-induced increases in MHCII
mRNA and IL-1β concentration in cells, but this effect was not seen in the KO mice. Overall
Loane et al. suggest that there is strong evidence for a central role for IL-4 in mediating the
effects of rosiglitazone (Loane et al., 2009).
Cuzzocrea et al. investigated a role for rosiglitazone in reducing acute inflammation. From
recent studies it was shown that both PPARα and PPARγ have a role in regulating the
inflammatory response. PPARγ was found to be expressed prominently in vivo in activated
monocytes and tissue macrophages. They tried to establish a mechanism for rosiglitazone by
using bisphenol A diglycidyl ether (BADGE) which is a PPARγ antagonist. The study found
that rosiglitazone attenuated the development of carrageenan-induced paw oedema and
pleurisy; it weakened the infiltration of the lung with polymorphonuclear neutrophils (PMNs)
and also weakened the expression of ICAM-1 and P-selectin, among other things. When the
animals were pre-treated with BADGE it attenuated the protective effects of rosiglitazone.
From this they proposed that the activation of PPARγ reduces the development of acute
inflammation and that the activation of PPARγ contributes to the anti-inflammatory effects of
18. 10
rosiglitazone. They also found that rosiglitazone attenuated the production of TNFα and IL-
1β similar to the findings of Loane et al. This attenuation in the cytokines was then reversed
if rats were pre-treated with BADGE. From this they concluded that acute inflammation
results in the activation and subsequent expression of pro-inflammatory cytokines, and that
rosiglitazone activates the PPARγ receptor resulting in the reduction of the release of these
pro-inflammatory cytokines (Cuzzocrea et al., 2004).
It is well known that a high fat diet will lead to glucose intolerance and insulin resistance.
Pathan et al. studied the effects of rosiglitazone in rats fed a high fat diet to assess any effects
on cognitive function. They used rosiglitazone as it does not cross the intact blood brain
barrier (BBB) so by these means it would be possible to see if the effects of the drug were
indirectly mediated through its peripheral actions. They found in the high fat diet rats there
was a significant increase in plasma glucose, plasma triglyceride, cholesterol and basal
insulin. In the rosiglitazone rats all these factors were lowered, suggesting an improvement in
insulin sensitivity. To test the spatial memory of the rats a Morris water maze was used. In
normal diet fed rats their latency to reach the platform declined over 5 days training
indicating they could use the spatial cues to direct them to the platform. In high fat diet fed
rats their latency did not decrease as much as the controls, suggesting their spatial memory
was slightly impaired due to the high fat diet. This was backed up by the fact that when
rosiglitazone was given, their escape latency was reduced. Insulin resistance and chronic
peripheral hyper-insulinemia causes a down regulation of insulin into the brain, which leads
to a brain insulin-deficient state (Pathan et al., 2008). Insulin in the brain is needed for
modulating glucose utility in regions such as the hippocampus and hypothalamus. Also for
the modulation of neurotransmitters like ACh, noradrenaline and dopamine, and it is also
required for activation of signalling pathways like the PKC pathway which is involved in
memory processing and synaptic plasticity (Pathan et al., 2008). Thus rosiglitazone, from this
study, is proposed to reverse the learning and memory deficits of a high fat diet in rats by
correcting peripheral insulin resistance.
1.5 Object recognition task
The aim of a non-matching-to-sample task is to study recognition memory due to the fact that
rats and mice have the tendency to interact with a novel object more than with one previously
seen. The object recognition is perceived by the amount of time spent investigating the novel
object. This task generally consists of two phases. The animal is kept in a housing cage that is
19. 11
distinct from the environment that the experiment will take place in. For the first phase the
animal is put into the new environment with ‘identical to be familiarised’ objects and given
time to investigate them. The animal is placed with its nose pointing away from the objects so
that it doesn’t have any bias towards them. Afterwards the animal is put back into its home
cage. For the second phase the animal is put into the training environment with one of the
objects being the previously investigated one, but the other novel. An interval of 1 hour can
be used to test for short term memory and an interval of 24 hours for long term memory. For
our purposes the training environment will be circular to prevent the rats from possibly hiding
in a corner. It forces them to explore the objects. The results can be viewed in different ways,
for example, a difference score, which involves subtracting familiar object interaction time
from novel object interaction time, or by a discrimination ratio, which is the novel object
interaction time divided by the total interaction time for both objects. A ratio of 0.5 indicates
more time was spent with the novel object (Bevins and Besheer, 2006).
1.6 Study aim
Donepezil is the gold standard drug in treating Alzheimer’s disease and is used world-wide in
patients presenting with dementia and AD. Rosiglitazone has shown its cognitive benefits in
animal trials from the papers discussed above. In this experiment the drugs are combined and
administered to young adult (YA) and middle aged (MA) male Wistar rats for 5 days. This
combination has not previously been researched. The aim is to investigate whether combining
these two drugs will work synergistically to further enhance their cognitive ability.
20. 12
2. Materials and Methods
2.1 Behavioural Experiments
2.1.1Animals
The rats used were male Wistar rats and obtained from Bioresources on campus in Trinity
College. Middle aged (MA) rats were aged between 13-14 months and young adult (YA) rats
were between 3-4 months old. The MA rats weighed 635±20g and the YA rats weighed
331±10g. The rats were housed in groups of two in a controlled environment (temperature:
20-22ºC, 12/12 h light/dark cycle) with food and water ad libitum. Animals were then
randomly assigned to donepezil-treated (DON), rosiglitazone-treated (ROSI), donepezil- and
rosiglitazone-treated (ROSI+DON) and control (CTL). There were 64 animals in total, 32
MA and 32 YA split further into four groups of eight for each age group giving a total of
eight subgroups (MACTL, MADON, MAROSI, MAROSI+DON, YACTL, YADON,
YAROSI, YAROSI+DON). Rats were handled 3 days prior to experimentation to familiarise
them with the surroundings and handling in general. All experiments were carried out under a
license from the Department of Health and Children (Ireland) and with ethical approval from
Trinity College Dublin Animal Users Ethical Committee.
2.1.2 Drug and dosage
Rosiglitazone is a thiazolidinedione (5-[4-(2-[methyl(pyridin-2-
yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione) and is marketed by GlaxoSmithKline as
Avandia. Donepezil is an acetylcholinesterase marketed by Pfizer as Aricept. Maple syrup
was used as a control (Pure Canadian Maple Syrup, Newforge®). All drugs were
administered orally mixed in a solution of maple syrup via a 1ml syringe. Each rosiglitazone
rat received 0.2ml of a 6mg/kg/day rosiglitazone and maple syrup suspension. Each donepezil
rat received 0.2ml of a 0.3mg/kg/day of donepezil and maple syrup suspension. Rats
receiving a combination of the two drugs got 0.2ml of a mixed 6mg/kg/day rosiglitazone and
0.3mg/kg/day donepezil with maple syrup suspension. Dosing was for 5 days. Animals were
not dosed on the last day of testing i.e. on day of sacrifice.
21. 13
2.1.3 Novel Object Recognition
The arena was circular, 45cm high x 50cm wide, made from black cardboard. Sawdust was
used as a base. A recording camera was placed on the ceiling giving an aerial view of the
arena, and was linked to a computer behind the curtain. CyberLink® Powerdirector software
was used to record the animals. The arena was located in a quiet room surrounded by black
curtains with two 60 watt lamps during experimentation. Each rat was habituated with the
arena for a period of 10 minutes in the 2 days preceding testing. On the first testing day two
identical objects were placed in the arena (sample object A). Each rat was placed in the centre
of the arena, with its nose pointing away from the objects, to avoid bias. Sample exposure
was for 10 minutes. 1 hour later each rat was placed in the arena with sample object A and an
unseen i.e. novel object B, for 3 minutes. 24 hours later the same was done but with novel
object C. Objects used were made from Lego and cleaned with 70% alcohol spray between
each animal.
2.1.4 Analysis of Recordings
A timer was assigned to each object. The appropriate timer was started when the animal came
in contact with the object and stopped when animal ceased contact. This was continued for
the duration of the recording. Times were converted to milliseconds and discrimination ratio,
total exploration time and difference scores were found using Microsoft Excel. Graphs of
discrimination ratio and total exploration time were plotted and analysed (GraphPad Prism 5,
USA).
2.1.5 Statistical Analysis for Behaviour
One- and two-way ANOVA’s (Analysis of Variance) were used to analyse data. A one-way
ANOVA compares means of two or more samples, a two-way ANOVA takes into
consideration two variables i.e. treatment and time. A Bonferroni multiple comparisons post-
hoc test was also used to determine which time and treatment group were significantly
different from each other. Data was deemed statistically significant when p<0.05.
22. 14
2.2 Molecular Analysis
2.2.1 Blood Samples
Animals were sacrificed by decapitation between 1 and 2 hours after the last behavioural
experiment. Trunk blood was run through a funnel rinsed with 15µl
ethylenediaminetetraacetate (EDTA) and collected into a 15ml Falcon tube which contained
15µl EDTA. EDTA was used as an anticoagulant. Blood was stored on ice until all samples
were collected then centrifuged at 2000rpm for 10 minutes at 4ºC. Blood plasma was taken
off and frozen at -80ºC.
2.2.2 Tissue Samples
Rat brain was cut in half, left hemisphere was dissected and right hemisphere was snap frozen
in dry ice for storage. The hippocampus, pre-frontal cortex, parietal cortex and cerebellum
were dissected and each divided into 3 parts for use in ELISA (Insulin Enzyme-Linked
Immunosorbent Assay), western blot and PCR (polymerase chain reaction) analysis. ELISA
tissue samples were homogenised in Krebs buffer (see appendix), western blot tissue samples
were homogenised in Lysis buffer (see appendix) and PCR brain tissue was snap frozen on
dry ice. All were then kept in a -80ºC freezer.
2.2.3 Insulin Enzyme-Linked Immunosorbent Assay (ELISA)
Blood plasma samples prepared as above were used. All blood specimens used were
generated under non-fasting conditions. Endogenous plasma insulin levels were determined
with a rat/mouse insulin ELISA kit (EMD Millipore (NYSE: MIL), Billerica, Massachusetts,
USA) following the manufacturer’s instructions. Assay procedure was carried out as in the
following steps. 10X wash buffer concentrate provided was diluted 10 fold with 450ml
deionised water. Each well was washed 3 times with 300µl of diluted wash buffer per wash.
All washes were carried out using the labtech LT-3000 microplate washer. Residual wash
buffer was removed by inverting the plate and tapping it onto absorbent tissue. All wells were
done in duplicate. 10µl assay buffer was added to the blank and sample wells. 10µl matrix
solution was added to the blank, standard and quality control wells. 10µl rat insulin standards
were added to the standard wells in descending order (10ng/mL, 5ng/mL, 2ng/mL, 1ng/mL,
0.5ng/mL and 0.2ng/mL). 10µl of quality control 1 was added to its wells on plate 1 and 10µl
23. 15
of quality control 2 to plate 2. To sample wells 10µl of the plasma sample to be tested was
added. 80µl detection antibody was added to all wells. The plate was covered with a plate
sealer and incubated at room temperature for 2 hours on a plate shaker (Stuart Scientific, UK)
that rotated at 400rpm. The plate sealers were removed and solutions decanted. Plates were
tapped as before to remove residual solution. Wells were washed 3 times with 300 µl diluted
wash buffer per well per wash. 100µl enzyme solution was added to each well. Plate was
sealed and incubated at room temperature for 30 minutes on the plate shaker. The sealer was
removed and solutions decanted. Wells were washed 6 times with 300µl diluted wash buffer
per well per wash. Plates were tapped and residual solution removed as before. 100µl
substrate solution was added to each well and the plate was sealed and shaken by hand to
gently mix solution in wells. When appropriate blue colour was seen in most wells 100µl stop
solution was added. The plate was shaken to ensure complete mixing of stop solution which
turned blue colour to yellow (acidification). Any bubbles were burst with a needle. The
absorbance was read at 450nm and 595nm in a plate reader (ELx800 Universal Microplate
Reader, Bio-Tek Instruments Inc. U.S.A).
2.2.4 Adiponectin ELISA
Plasma samples were diluted 1:500 with 1X assay buffer provided in the Millipore rat
adiponectin ELISA kit (EMD Millipore (NYSE: MIL), Billerica, Massachusetts, USA). The
dilution was made by adding 10µl plasma sample to 990µl 1X assay buffer, then 100µl of
this diluted sample was added to 400µl 1X assay buffer to give a 1:500 dilution. Rat
adiponectin standards were prepared by adding 0.5 ml distilled water into the glass vial of
standard provided to reconstitute a 200ng/mL concentration of adiponectin standard. The vial
was inverted, allowed sit for 5 minutes then vortexed (GV Lab, Gilson) gently. The following
table gives the process of serial dilution that was used to give various rat adiponectin
standards:
24. 16
Table 1: Adiponectin Standard Preparation
Standard Concentration
ng/ml
Volume of Deionized Water
to Add
Volume of Standard
to Add
200 0.5 ml 0
Standard Concentration
ng/ml
Volume of 1X diluted
Assay Buffer (Sample
Diluent) to add
Volume of Standard
to Add
100 0.25 ml 0.25 ml of 200 ng/mL
50 0.25 ml 0.25 ml of 100 ng/mL
25 0.25 ml 0.25 ml of 50 ng/mL
12.5 0.25 ml 0.25 ml of 25ng/mL
6.25 0.25 ml 0.25 ml of 12.5ng/mL
3.125 0.25 ml 0.25 ml of 6.25ng/mL
Rat adiponectin quality controls 1 and 2 were also reconstituted with 0.5 ml distilled water.
They were inverted, allowed sit for 5 minutes then mixed well. All reagents were pre warmed
to room temperature prior to assay setup. 10X wash buffer was diluted 10 fold by mixing its
contents with 900 ml distilled water. Plates were assembled and labelled accordingly then
washed 3 times with 300µl diluted wash buffer. A plate washer (labtech LT-3000 microplate
washer) was used for all wash steps. 80µl assay running buffer was added to all wells. An
additional 20µl assay running buffer was added to blank wells. 20µl rat adiponectin standard
was added in descending order to the standard wells. 20µl QC1 was added to plate 1 and 20µl
QC2 was added to plate 2. 20µl of each sample of rat plasma was added to the remaining
wells. The plate was covered with the plate sealer provided and incubated at room
temperature for 2 hours on a plate shaker (Stuart Scientific, UK). The plate sealer was
removed, solutions decanted and tapped on absorbent tissue to remove residual solution. Each
well was washed 3 times as before. 100µl detection antibody was added to all wells. The
plate was covered with a plate sealer and incubated at room temperature for 1 hour on a plate
shaker (Stuart Scientific, UK). The plate sealer was removed, solutions decanted and plates
tapped as before. Each well was washed 3 times as before. 100µl enzyme solution was added
25. 17
to each well, plate was covered and incubated at room temperature for 30 minutes on a plate
shaker. The plate sealer was removed, the solutions decanted and tapped on tissue to remove
any residual solution. Each well was washed 3 times with diluted wash buffer as above.
100µl substrate solution was added to each well. The plate was covered and shaken by hand.
A blue colour formed in the standard wells with intensity proportional to increasing
concentrations. When a blue colour was seen in most wells the reaction was stopped with
100µl stop solution. Air bubbles were burst with a needle and plates were read at 450nm and
590nm (control) in a plate reader (ELx800 Universal Microplate Reader, Bio-Tek
Instruments Inc.).
2.2.5 Corticosterone ELISA
Plasma samples were obtained as above and diluted 1:22 by adding 10µl plasma sample to
210µl sample diluent provided by the kit. An IDS Corticosterone ELISA kit was used
(Immunodiagnostic Systems Limited, UK). All samples were vortexed (GV Lab, Gilson) to
mix thoroughly. To the antibody coated plate 100µl each of the standards, quality control and
samples were added to the appropriate wells in duplicate. 100µl of enzyme conjugate solution
was added to all wells using a multichannel pipette. The plate was covered with a plate sealer
and incubated for 18 hours at 5ºC. All wells were washed 3 times with 300µl wash solution
as above with an automated plate washer (labtech LT-3000 microplate washer). The plate
was tapped on absorbent tissue to remove excess wash buffer. 200µl of TMB substrate was
added to all wells and the plate was incubated at room temperature for 30 minutes. 100µl of
stop solution was added to all wells. The absorbance was measured at 450nm and 650nm
(control) using a plate reader (ELx800 Universal Microplate Reader, Bio-Tek Instruments
Inc.).
2.2.6 ACH ELISA
Hippocampal tissue was prepared as above. Before use a Bradford protein assay was carried
out to assess the level of protein in each sample. The samples were then brought to the same
protein concentration by diluting the tissue sample in the appropriate volume of krebs buffer.
A Wuhan EIAab Science kit was used (Wuhan EIAab Science Co., Ltd, China). All reagents
were brought to room temperature before use. Wash buffer was made up by diluting 30ml of
wash buffer concentrate in 750ml distilled water. The standard was reconstituted with 1ml of
sample diluent giving a stock standard of 200nmol/L. The remaining standards are made
26. 18
using a serial dilution and the sample diluent acts as a zero standard. 100µl of either standard,
blank or sample was added to the appropriate wells. The plate was covered with a plate sealer
and incubated for 2 hours at 37ºC. The liquid was removed from the wells but the wells were
not washed. 100µl of detection reagent A was added to each well. The plate was covered with
a plate sealer and incubated at 37ºC for 1 hour. Each well was washed 3 times with 400µl
wash buffer in an automated plate washer (labtech LT-3000 microplate washer). Excess
liquid was removed by tapping the plate on absorbent tissue. 100µl detection reagent B was
added to every well, the plate was covered with a plate sealer and incubated at 37ºC for 1
hour. The plate was washed 5 times and tapped dry. 90µl of substrate solution was added to
every well. The plate was sealed with a plate sealer and incubated at 37ºC for 15-30 minutes.
The plate protected from light by placing paper over it. 50µl stop solution was added to every
well and the plate tapped to ensure uniform mixing. The plate was read at 450nm in a plate
reader (ELx800 Universal Microplate Reader, Bio-Tek Instruments Inc.).
2.2.7 RNA Purification and Quantification
RNA was purified from hippocampal samples using the Macherery-Nagel Nucleospin® Rna
II isolation kit (Machery-Nagel GmbH & Co. KG, Germany). Buffer RA3 and rDNase were
prepared according to manual instructions. Tissue was homogenised in 350µl buffer RA1 and
3.5µl β-mercaptoethanol. This step was carried out under the fume hood as β-
mercaptoethanol is highly toxic. The lysate was then pipetted onto a NucleoSpin® Filter and
centrifuged at 14,000 rpm for 1 minute. This reduced viscosity and cleared the lysate. 350µl
ethanol (70%) was added to the lysate, pipetted up and down 8 times and put on a
NucleoSpin® RNA II Column and centrifuged for 30 seconds. The elute was disposed of as
the RNA was attached to the membrane. 350µl membrane desalting buffer was added and
centrifuged for 1 minute and elute disposed. DNase reaction mixture was prepared by adding
10µl reconstituted rDNase to 90µl reaction buffer for rDNase. 95µl of this reaction mixture
was pipetted directly onto the centre of the silica membrane and incubated for 15 minutes.
200µl buffer RA2 was added to the NucleoSpin® RNA II Column and centrifuged for 30
seconds and elute disposed. Buffer RA2 inactivates rDNase. 600µl buffer RA3 was added to
the NucleoSpin® RNA II Column, centrifuged for 30 seconds and elute disposed. 250µl
buffer RA3 was added to the NucleoSpin® RNA II Column and centrifuged for 2 minutes to
fully dry the membrane. The column was placed into an RNase free tube and 60µl RNase-
free H2O was added to the column and centrifuged for 1 minute to elute the RNA. The RNA
27. 19
was put back onto the membrane and centrifuged again to ensure all RNA was obtained. The
amount of RNA in each sample was found using a spectrophotometer (Thermo-Scientific,
Nanodrop Spectrophotometer ND-1000, UK). 1µl of RNA was placed on the bottom pedestal
and the top arm closed. Between every 4 samples the machine was blanked using 1µl RNase
free H2O. From these results 20µl RNA sample was equalised with the appropriate amount of
RNase free H2O.
2.2.8 Reverse Transcription of cDNA and Real Time PCR
10µl of the equalised RNA was placed in PCR tubes and 10µl master mix (Applied
Biosystems, US) was added. Samples were put in a thermocycler (Peltier Thermal Cycler
PTC-200, MJ Research, US) for 2 hours to make cDNA. For use in the PCR machine, cDNA
was diluted 1:4 by adding 60µl RNase free H2O. Each sample of cDNA was placed in a well
on a 96 well MicroAmp™ (Applied Biosystems, US) plate. To all wells a mixture of
TaqMan, β-actin (as a control) and a primer (IL-1β, IL-4, GLUT3, PPARγ, α-7 or AChE) was
added (15µl) (Applied Biosystems, US). The plate was sealed well to ensure none of the
sample evaporates during PCR, then the plate was centrifuged for 1 minute at 1100rpm to
ensure all solution is at the bottom of the well. The plate was placed in the real time PCR
machine (Applied Biosystems, US) and run using the 7300 system software.
28. 20
3. Results
3.1 Behavioural Experiments
3.1.1 MA Rats Exploration
Analysis of performance scores in the object recognition task were done using non-
parametric analysis of variance (ANOVA). A one-way ANOVA was used for within group
analysis followed by Bonferroni’s Multiple Comparison Test as a post hoc measure. In the
MACTL (F(2,21)= 3.986, p<0.05) group the rats did not learn the task meaning they were
unable to identify the novel object. At 1 hr they spent more time with object B than chance
levels but this was not significant. However they spent significantly less time exploring
object C than object A at 24 hrs further proving the task was not learned. Exploration of the
novel object was above chance levels at both time points for MADON but they did not
successfully identify the novel object as there was no significance (F(2,21)=2.313, p>0.05)
between the time points. MAROSI rats were able to identify the novel object at the 1 hr time
point (F(2,21)=5.433, p<0.05) showing they learned the task as did MAROSI+DON
(F(2,21)=4.115, p<0.05), but at 24 hrs neither group significantly explored object C longer than
object A.
30. 22
3.1.2 YA Rats Exploration
YACTL, YADON and YAROSI were able to identify the novel object at all time points.
Using a one-way ANOVA with a Bonferroni’s multiple comparison post hoc test,
significance levels showed donepezil treated rats (F(2,21)= 33.82, p<0.0001) and rosiglitazone
treated rats (F(2,21)=22.75, p<0.0001) learned the task and identified the novel object. Control
animals (F(2,21)=9.348, p<0.05) also identified the novel object. Rats that received a
combination of rosiglitazone and donepezil (F(2,21)=8.485, p<0.05) were unable to identify the
novel object although after 1 hr they did explore object B more so than object A. At 24 hrs
YAROSI+DON rats had a lower discrimination ratio than YACTL at the same time point.
YAROSI+DON rats only explored object C to the same extent as object A showing they were
unable to learn the task. It is clear from figure 2D that when these drugs are given in
combination their cognitive enhancing ability is lost.
32. 24
3.1.3 YA and MA Total Exploration Time
Comparing YACTL and MACTL exploration times will show if MA rats explored the
objects as much as YA rats. This does not take into account the difference between objects
i.e. A and B. It is a measure of the overall time spent exploring the objects. A two-way
ANOVA with a Bonferroni post hoc test was used. At 0hr YACTL spend significantly more
time exploring than MACTL (**, p<0.001). At this first time point the rats were given 10
minutes to explore objects A and A to familiarise themselves with object A which they will
see again and also to investigate the surroundings. At 1 hr and 24 hrs there is no significant
difference between exploration times suggesting that MACTL and YACTL are exploring the
objects to a similar scale. These time points however are only 3 minutes long. A possible
reason for MACTL rats not spending as much time exploring at 0 hr is perhaps 10 minutes is
too long. They lose interest whereas YACTL have more expendible energy and are
stimulated to keep exploring.
0
hr
1
hr
24
hr
0
50000
100000
150000
yactl
mactl
*
TotalExplorationTime(ms)
Figure 3: YACTL explore objects constantly throughout a given length of time. At 1hr and
24 hrs YACTL and MACTL are on par whereas at 0 hr MACTL have a lower exploration
time compared to YACTL. (n=8±SEM, *p<0.05).
33. 25
3.1.4 YA and MA variability
Variability plots show the spread between the data. A one-way ANOVA was used with
Bonferroni’s multiple comparisons as a post hoc measure. YACTL and MACTL at both the 1
hr and 24 hr time points vary significantly (F(5,42)=3.733,*p<0.01) (figure 4A). YACTL have
a tighter discrimination ratio compared to MACTL which are more spread. At 24 hrs
MACTL rats become more spread compared to what they were at 1 hr. This shows the
difference in performance levels between individual rats in the same group. In MADON
(F(5,42)=9.027,***p<0.0001) at 1 hr and 24 hrs there is again a large spread (figure 4B). At the
0 hr time point MAROSI are far more spread compared to YAROSI
(F(5,42)=6.636,***p<0.0001) (figure 4C). Individual YA and MA rats receiving rosiglitazone
and donepezil show a lot of variability at all time points (F(5,42)=3.515,*p<0.01) (figure 4D).
The variability plots show some MA rats are performing like YA rats and some of the MA
rats are underperforming in the task in general.
3.1.5 Novel object exploration between time points and treatment groups
A two-way ANOVA along with Bonferroni’s multiple comparison post hoc test were used to
test significance. At the 0 hr time point control and drug treated groups of both YA and MA
rats have a similar level of exploration. YACTL and MACTL appear to have very similar
discrimination ratios. This shows all animals explored objects A and A to the same extent
(figure5A). At 1 hr YADON identify the novel object and explore it for a longer period than
all other groups and to a significant level between MACTL and MADON (* p<0.05). At 24
hrs MACTL were unable to identify the novel object and spent equal amounts of time with
each object. Again YADON explored the novel object more so than other groups.
Interestingly YADON had a significantly (*p<0.05) higher discrimination ratio than
YAROSI+DON suggesting rosiglitazone was hindering donepezil and taking away from its
full effect. In figure 5B, YADON and YAROSI spent significantly more time exploring the
novel object at both time points (***p<0.0001). MAROSI and MAROSI+DON spent more
time at with the novel object at 1 hr (*p<0.05).
36. 28
3.2 Molecular Experiment Results
3.2.1 Insulin ELISA Results
Plasma insulin levels in rat groups were measured using an ELISA. A one-way ANOVA
showed the means are significant (F(7,56)=2.655, p<0.05). However Bonferroni’s multiple
comparisons post hoc test showed no significance between treatment groups but they are
trending in that direction. MACTL rats have a lower level of plasma insulin compared to
YACTL which is then brought to a similar level when given donepezil. MADON rats have a
much higher level of plasma insulin than MACTL and MAROSI which is still seen even
when donepezil is given in conjunction with rosiglitazone. Rosiglitazone in YA rats gives a
small rise in insulin levels which is then decreased to below control levels when the drugs are
combined.
yactl
m
actl
yadon
m
adon
yarosim
arosi
yarosi+don
m
arosi+don
0.0
0.5
1.0
1.5
2.0
2.5
PlasmaInsulinng/ml
Figure 6: Treatment of Donepezil and Rosiglitazone does not significantly alter plasma
insulin levels in YA or MA rats. Plasma insulin levels were obtained from trunk blood
samples. MA rats have lower levels of insulin than YA. (n=8±SEM).
37. 29
3.2.2 Corticosterone ELISA Results
Plasma corticosterone levels were tested on trunk blood using an ELISA. A one-way
ANOVA gave significance of means (F(7,55)=4.347, p<0.001). Bonferroni’s multiple
comparison post hoc test showed MACTL rats had significantly higher corticosterone levels
compared to YACTL as would be expected. In MA rats donepezil significantly reduced
corticosterone levels compared to controls (** p<0.001). Rosiglitazone and the combination
of rosiglitazone and donepezil reduced corticosterone levels in MA rats but not to a
significant level. In YA rats donepezil increased corticosterone levels but not to a significant
level. Rosiglitazone decreased corticosterone levels in both YA and MA rats. The
combination of rosiglitazone and donepezil appears to be averaging the effects of the drugs
compared to their levels when used alone.
yactl
m
actl
yadon
m
adon
yarosim
arosi
yarosi+don
m
arosi+don
0
100
200
300
400
500
***
PlasmaCorticosterone
ng/mL
Figure 7: Donepezil significantly reduces stress levels in MA rats. Donepezil decreases
corticosterone levels in MA rats but rosiglitazone interferes with this when the drugs are
given in combination. (n=8±SEM, *p<0.05).
38. 30
3.2.3 Adiponectin ELISA Results
An adiponectin ELISA was performed on trunk blood to test its levels in YA and MA rats. A
one-way ANOVA gave means to be significantly different (F(7,56)=15.94, p<0.0001).
Donepezil did not change adiponectin levels in YA or MA rats but a Bonferroni post hoc test
shows that rosiglitazone significantly increases adiponectin levels (***p<0.0001) in both YA
and MA rats compared to control groups and to donepezil treated groups. Rosiglitazone and
donepezil combined also increase adiponectin levels in YA and MA rats. However there is a
small decrease in adiponectin when rosiglitazone and donepezil are given together in MA rats
compared to when it is given alone, suggesting donepezil is taking away from its full effect,
but this is not significant.
yactl
yadon
yarosi
yarosi+don
m
actlm
adon
m
arosi
m
arosi+don
0
10000
20000
30000
40000
50000
***
***
***
***
PlasmaAdiponectin
ng/mL
Figure 8: Rosiglitazone Increases Plasma Adiponectin levels in YA and MA rats.
Adiponectin levels are similar for YACTL and MACTL rats. Rosiglitazone increases these
levels but in MA rats when rosiglitazone is combined with donepezil the adiponectin levels
are not significantly increased. (n=8±SEM, *p<0.05).
39. 31
3.2.4 ACh ELISA Results
Acetylcholine was measured from rat hippocampal samples. A one-way ANOVA found
means to be significantly different (F(7,54)=3.533, p<0.05). A Bonferroni post hoc test showed
that in YA rats, donepezil significantly increased ACh levels (* p<0.05) compared to the
control rats but this was not seen in MA rats. The levels of ACh in MA rats does not
significantly change between treatment groups but there is a small reduction in ACh when
rosiglitazone is given in MA rats. Donepezil is impaired when given in conjunction with
rosiglitazone as ACh levels in YAROSI+DON rats are brought back to nearly the same as
YACTL.
yactl
m
actl
yadon
m
adon
yarosim
arosi
yarosi+don
m
arosi+don
0
5
10
15 *
**
*** *
AChnmol/L
Figure 9: Donepezil Increases ACh levels in YA rats. Levels of ACh raised by donepezil are
decreased when it is given in combination with rosiglitazone. (n=8±SEM, *p<0.05).
40. 32
3.2.5 RT-PCR Results
mRNA expression of AChE, α-7, PPARγ, GLUT-3, IL-1β and IL-4 were found using real
time PCR from hippocampal tissue. There are no significant treatment related changes in any
of the primers in mRNA expression. AChE mRNA expression (F(7,55)=1.1246, p>0.05) is
increased in MADON rats whereas in YADON it is decreased but not to a significant level.
YACTL and MACTL α-7 mRNA expression (F(7,56)=0.8725, p>0.05) levels are not altered
with age. In YA rats drug treatment causes a small decrease in α-7 which can be seen in
MADON and MAROSI, while MAROSI+DON brings levels back to that of controls (not
significant). GLUT3 mRNA expression (F(7,56)=0.7174, p>0.05) increases in YADON rats
then it decreases when rosiglitazone and donepezil are combined but not to a significant
level. In PPARγ mRNA expression (F(7,55)=1.246, p>0.05) all MA rats have higher levels
than YA rats, although not significant. IL-1β mRNA expression (F(7,53)=0.3780, p>0.05)
appears to be reduced in MA rats with the treatment of donepezil and rosiglitazone both
separately and in combination but again this result is not significant. IL-4 mRNA expression
(F(7,53)=0.9985, p>0.05) shows variability between age and treatment group but not to a
significant level. IL-4 is trending towards a decrease when treated with rosiglitazone.
42. 34
3.2.6 Correlations
Correlations were performed with the discrimination ratios of YA and MA rats at the 1 hr and
24 hr time point against ELISA and PCR results. This was done by plotting an XY scatter
graph with linear regression and a Pearson correlation test to test for significance. Figure 11
shows a correlation between discrimination ratio and plasma insulin levels. There is a
significant correlation between discrimination ratio and YADON rats (r = 0.7125,* p<0.05),
showing a higher insulin level gives a better discrimination ratio i.e. a longer amount of time
spent with the novel object. At 24 hrs there is a positive correlation between discrimination
ratio and YAROSI+DON (r = 0.7712, *p<0.05). Figure 12 shows the correlations between
discrimination ratio and AChE mRNA expression. There is a positive correlation seen in
YADON at 24 hrs with AChE (r = 0.739, *p<0.05) (figure 12B). Donepezil prevents the
action of AChE possibly causing its up-regulation. There is a negative correlation seen at the
24 hr time point in AChE mRNA expression with MACTL (r = -0.8907, *p0.05) and
MAROSI (r = -0.9144, *p<0.01) (figure 12D). This suggests lower levels in AChE results in
a better discrimination ratio.
43. 35
0 1 2 3
0.5
0.6
0.7
0.8
0.9
yactl
yadon
ya rosi
yarosi+don
r = 0.7125
P<0.05
11A
Plasma Insulin ng/ml
DiscriminationRatio
1hrTimepoint
0 1 2 3
0.0
0.2
0.4
0.6
0.8
1.0
yactl
yadon
ya rosi
yarosi+don
r = 0.7712
P<0.05
11B
Plasma Insulin ng/ml
DiscriminationRatio
24hrTimepoint
0 1 2 3 4
0.0
0.2
0.4
0.6
0.8
1.0
mactl
madon
marosi
marosi+don
11C
Plasma Insulin ng/ml
DiscriminationRatio
1hrTimepoint
0 1 2 3 4
0.0
0.2
0.4
0.6
0.8
mactl
madon
marosi
marosi+don
11D
Plasma Insulin ng/ml
DiscriminationRatio
24hrTimepoint
Figure 11: A positive correlation is seen with donepezil and insulin levels in YA rats. 11A
represents the correlation at 1hr between DR and insulin in YA, 11B represents the
correlation at 24 hr between DR and insulin in YA rats, 11C represents the correlation at 1
hr between DR and insulin in MA and 11D represents the correlation at 24 hrs between DR
and insulin in MA rats. (n=8±SEM, *p<0.05).
44. 36
0.0 0.5 1.0 1.5 2.0
0.5
0.6
0.7
0.8
0.9
yactl
yadon
ya rosi
yarosi+don
12A
AChE mRNA
Fold Change
DiscriminationRatio
1hrTimepoint
0.0 0.5 1.0 1.5 2.0
0.0
0.2
0.4
0.6
0.8
1.0
yactl
yadon
ya rosi
yarosi+don
r = 0.739
P<0.05
12B
AChE mRNA
Fold Change
DiscriminationRatio
24hrTimepoint
0.0 0.5 1.0 1.5 2.0
0.0
0.2
0.4
0.6
0.8
1.0
mactl
madon
marosi
marosi+don
12C
AChE mRNA
Fold Change
DiscriminationRatio
1hrTimepoint
0.0 0.5 1.0 1.5 2.0
0.0
0.2
0.4
0.6
0.8
mactl
madon
marosi
marosi+don
r = -0.8907
P<0.01
r = -0.9144
P<0.01
12D
AChE mRNA
Fold Change
DiscriminationRatio
24hrTimepoint
Figure 12: A negative correlation in AChE improves discrimination ratio in MA rats. 12A
represents the correlation between DR and AChE in YA rats at a 1 hr time point, 12B
represents the correlation between DR and AChE in YA rats at a 24 hr time point, 12C
represents the correlation between DR and AChE in MA rats at a 1 hr time point and 12D
represents the correlation between DR and AChE in A rats at a 24 hr time point. (n=8±SEM,
*p<0.05).
45. 37
4. Discussion
The main finding from this study was that donepezil given in combination with rosiglitazone
does not enhance either drug’s cognitive ability. In fact the opposite is true. They impede
each other and are not able to fully perform to their best ability. Donepezil alone increases
ACh availability in YA rats but it is unable to replicate this when given with rosiglitazone.
Similarly, donepezil reduces corticosterone levels in MA rats but when combined with
rosiglitazone it cannot perform to the same aptitude. Rosiglitazone alone is acting as a
metabolic drug and increases plasma adiponectin in MA rats but again this is decreased when
rosiglitazone and donepezil are given concomitantly.
The results from the behavioural experiments show that MACTL do not learn the object
recognition task. This particular task may prove too difficult for the MA rats. MA rats treated
with donepezil did not learn the task either, but rats treated with rosiglitazone and
rosiglitazone and donepezil combined did learn the task and were able to identify the novel
object at the 1hr time point. YA rats did learn the task and were able to identify the novel
object. All groups except the rosiglitazone and donepezil combined group continued to
distinguish between the novel and previously seen object at the 1 hr and 24 hr time point.
When each group and time point were compared against each other there was no significant
result between control and drug treated rats suggesting that the drugs did not greatly enhance
learning in YA rats. However when rosiglitazone and donepezil are given in combination,
there is a significant decrease in the animals ability to identify the novel object between this
drug group and donepezil given alone, at the 24 hr time point. This suggests that rosiglitazone
is taking away from donepezil’s full effect.
Tissue was harvested following behavioural experiments and used for molecular analysis.
Interleukin-1β (IL-1β) is a pro-inflammatory cytokine expressed highly in the hippocampus
and hypothalamus (Lynch et al., 2001). It is synthesised and released from neurons and glia
in response to stress, injury or insult (Murray et al., 1998). IL-1β is known to affect appetite,
sleep and the activity of the hypothalamic-pituatary-adrenal axis, as well as its interesting
ability to inhibit hippocampal LTP. IL-1β exerts its effects by binding to its receptor IL-1RI
(IL-1 receptor Type I). The ability of IL-1β to inhibit LTP in vitro is a mechanism not yet
known but one possibility is that IL-1 induces the formation of reactive oxygen species in the
brain (Murray et al., 1998). Lynch et al. show that the age related increase in IL-1β
46. 38
concentration was paralleled by an increase in IL-1RI expression. They also show that an
increase in hippocampal IL-1β concentration attenuated LTP levels in aged and stressed rats.
This follows other observations of an inverse relationship between IL-1β concentration and
LTP (Lynch et al., 2001). In aged rats an increase in hippocampal IL-1β concentration is
accompanied by a decrease in IL-4 (anti-inflammatory). IL-4 under normal circumstances
decreases IL-1β mRNA synthesis and IL-1β release from glia (Loane et al., 2009). An
increase in inflammatory cytokines in the brain profoundly affects behaviour. Oitzl et al. first
showed that rats injected intracerebroventricularly with IL-1β showed poor performance in
the Morris water maze task. Hippocampal-dependant learning such as contextual fear
conditioning and behaviour in the Morris water maze were affected by persistent
overexpression of IL-1β in rat hippocampus (Lynch, M. 2010). However it is important to
note that a low concentration of IL-1β and other pro-inflammatory cytokines such as IL-6 are
likely to play a role in the maintenance of LTP (Lynch, M. 2010). IL-4 is secreted by T
helper type 2 cells. Nolan et al. for the first time show that glia are capable of secreting IL-4
and this activates IL-4 receptors on neurons thereby exerting its anti-inflammatory effects.
IL-4 has the ability to inhibit the release of pro-inflammatory cytokines and to upregulate the
synthesis of IL-1 receptor antagonist. Hippocampal IL-4 concentration is decreased and IL-4
stimulated signalling is down-regulated with age. These changes together with an increase in
IL-1β concentration and IL-1β induced signalling are responsible for a deficit in LTP (Nolan
et al., 2004). Loane et al. have shown that rosiglitazone attenuated the increase in IL-1β in the
hippocampus by its ability to increase hippocampal concentrations of IL-4 in aged rats. The
results obtained from real time PCR on rat hippocampal tissue in this study were not
significant. This could be due to having used MA rats, not old rats, so levels of either IL-1β
or IL-4 would not be extremely high or low, respectively. Also the drug was given for a
period of 5 days which may not have been a sufficient treatment trial length. Although not
significant, rosiglitazone and the combination of rosiglitazone given with donepezil is
trending towards a reduction in IL-1β in MA rats. The levels of IL-1β and IL-4 did not vary
in the YA rats, as expected.
Rosiglitazone is associated with PPARγ and GLUT-3. The data from this study shows that
neither age nor treatment group has any effect on PPARγ mRNA expression. This is
consistent with the findings by Loane et al. This paper also states that rosiglitazone acts in a
manner that is independent of PPARγ activation and that rosiglitazone gains access to the
brain due to a reduction in the BBB allowing permeability in older rats (Loane et al., 2009).
47. 39
To confirm their finding they tested GW9662, a PPARγ antagonist, to see if it would abolish
the effect of rosiglitazone. The data indicated that it did not modulate the inhibitory effect of
rosiglitazone on LPS-induced IL-1β in glia (Loane et al., 2009). Conversely, Cuzzocrea et al.
demonstrate the mechanisms that underlie the protective effects of rosiglitazone are
dependent on the activation of PPARγ. They show that BADGE, a PPARγ antagonist,
reduces the protective effects of rosiglitazone in rats thus proposing activation of PPARγ
reduces the development of acute inflammation and contributes to the anti-inflammatory
effects of rosiglitazone (Cuzzocrea et al., 2004). The observation from this study of GLUT-3
mRNA expression showed no change between age or treatment group. GLUT-1 and GLUT-3
are responsible for glucose transport across the blood brain barrier. GLUT-3 is localised at
the neuronal cell membrane and possesses a higher affinity for glucose than GLUT-1. GLUT-
3 activity provides neuroprotection and subsequently when not functioning correctly it is
related with neuronal damage (García-Bueno et al., 2007). García-Bueno et al. also found
that stress reduced the expression of GLUT3 in Western blot analysis but that rosiglitazone
prevented this. Dello Russo et al. showed TZD’s can modify glucose metabolism in brain
glial cells. They showed TZD’s increased astrocyte glucose uptake from the media. In
agreement with Loane et al. Dello Russo et al. suggest the effects of TZD’s are independent
of PPARγ activation, and instead it works by rapid activation of an intracellular signalling
system (Russo et al., 2002). Kramer et al, showed GLUT-1 levels increased in vivo and in
vitro by rosiglitazone (Kramer et al., 2001).
Donepezil is an AChE inhibitor. It works by blocking the breakdown of ACh by AChE
leaving more ACh free in the extracellular membrane. Zivin et al. showed that long term
treatment (28 days, 2mg/kg s.c) with donepezil increased hippocampal AChE mRNA levels
and its activity in the CNS compared to saline treated controls (Zivin et al., 2008). The results
from RT-PCR of AChE show no significant change at the mRNA expression level following
treatment for 5 days with 0.3 mg donepezil, which is most likely why there was no change.
Kume et al. studied the effect of donepezil and galanthamine, another AChE inhibitor, on the
function and expression of nicotinic receptors namely α-7 and α-4 in rat primary cortical
neurons. They found both drugs induced the upregulation of nicotinic receptors without a
change in the mRNA level after a 4 day treatment (10microM). The results from this study
are consistent with the above as no change in mRNA expression of α-7 was found. They
further state donepezil induced the up-regulation of α-7 and of α-4 nicotinic receptors which
caused an increase in the protein level while having no change to mRNA, suggesting
48. 40
donepezil modulates the up-regulation at a posttranscriptional level (Kume et al., 2005). To
get an indication of the ACh levels in the hippocampus and to see if these levels were
affected by treatment and/or age, an ACh ELISA was carried. The results showed donepezil
increased ACh in YA rats compared to YA and MA controls but there was no alteration of
ACh levels in MA rats treated with donepezil. Cai et al. measured ACh levels using an
ELISA following the treatment of young rats with donepezil for 6 months (0.65 mg/(kg·d)).
They found an increase in hippocampal ACh levels with treatment of the drug (Cai et al.,
2011) however they did not test on MA or old rats. The correlation plots between
discrimination ratio and AChE mRNA expression show increasing AChE levels with
YADON. The explanation being donepezil works as an AChE inhibitor thus not allowing it
to function. The increased correlation seen could be due to an upregulation of AChE because
its action is being inhibited. In MA rats there is a negative correlation seen with
discrimination ratio and AChE mRNA expression. Thus proving with reduced amounts of
AChE, performance in the object recognition task is improved.
Adiponectin is a fat-derived protein hormone that modulates metabolic processes such as
glucose regulation and fatty acid catabolism. Adiponectin levels are reduced in diabetic
patients. Rosiglitazone was previously used as an anti-diabetic drug with one of its actions
being to restore and increase adiponectin levels. An adiponectin ELISA was carried out in rat
plasma to ensure the drug was working correctly and to determine how its levels varied
between age and drug treatment group. Between YA and MA controls there was no
difference in adiponectin levels suggesting age is not a factor in changes of this hormone.
Rosiglitazone showed a significant increase in adiponectin levels in both age groups showing
that the drug is working as it should. Donepezil does not cause any change in adiponectin
levels. In fact, while not significant, donepezil is trending towards hindering rosiglitazone’s
efficacy in MA rats when the two drugs are combined. Maeda et al. show that TZD treatment
dramatically increased adiponectin plasma levels in young mice and that TZD’s enhanced the
expression of adiponectin mRNA in adipose cells (Maeda et al., 2001).
A resistance to insulin develops with age along with an overall decrease in insulin sensitivity.
Escrivá et al. conclude this is due to an accumulation of retroperitoneal and total body fat
which leads to an impairment of glucose uptake in the muscle (Escrivá et al., 2007). An
insulin ELISA was carried out to determine the effects between age and drug group on
insulin levels. There were no significant changes in the insulin levels between YA and MA
49. 41
rats and drug treatment didn’t make a significant difference. Insulin is needed in our bodies to
remove glucose from our blood. The occurrence of diabetes increases with age, with almost a
third of the elderly population being diagnosed with this metabolic disorder (Dunican et al.,
2011). Diabetic patients either do not make enough insulin to carry out this function, or they
are resistant to insulin. Rosiglitazone attaches to insulin receptors on cells throughout the
body and causes them to become more sensitive to insulin thus allowing removal of glucose
from the blood (Dunican et al., 2011). A correlation plot between discrimination ratio and
plasma insulin showed an increase in plasma insulin levels was correlated with an increase in
performance in the novel object recognition task.
Kizaki et al. showed that serum corticosterone concentration increased in old rats compared
to young (Kizaki et al., 2000). There are other papers stating otherwise but Sapolsky
concludes if the corticosterone measures in young rats are truly basal i.e. unstressed, there is a
considerable increase in basal glucocorticoid concentrations in aged rats. Sapolsky suggests
looking at measures such as the method and speed of obtaining the blood sample, the social
conditions of the rat housing, and/or the recency with which there was a disturbance in the
animal room, to ensure an accurate level of corticosterone is being measured (Sapolsky, RM.
1992). From this study corticosterone levels were found to be significantly increased in
MACTL rats compared to YACTL. There were no changes observed in the corticosterone
levels of YA rats with drug treatment but in MA rats, donepezil significantly decreased stress
levels and treatment with rosiglitazone and the combination of rosiglitazone with donepezil
are trending towards a decrease in corticosterone levels also. The fact that corticosterone
levels in the MA rats are 3 times as high as those seen in YA rats could possibly suggest a
reason as to why MA rats did not learn the task.
The aim of this experiment was to investigate the combined effect of rosiglitazone and
donepezil on learning and memory. It is conclusive from these experimental conditions that
taking these drugs concurrently does not have an added benefit. Firstly looking at YA rats in
the object recognition task, it is seen that the combination of drugs certainly does not improve
the amount of time spent with the novel object because at 24 hrs these rats were unable to
identify the novel object whereas the control rats and rats treated with each drug separately
were able to identify the novel object at this time point. Another example can be seen in the
corticosterone ELISA. In MA rats donepezil is decreasing levels significantly but when given
in conjunction with rosiglitazone there is no significant reduction in corticosterone levels.
50. 42
The drugs are appearing to work independently of each other. It is also clear that when they
are given together neither one can exert its full effect. It appears the combination of
rosiglitazone and donepezil diminish the metabolic effects rosiglitazone has alone, as
indicated by the adiponectin ELISA.
51. 43
5. Conclusion
In conclusion, I would not recommend the use of rosiglitazone and donepezil in conjunction
with one another based on the findings from this experiment. This study has shown that each
drug alone is enhancing cognition and appears to carry out normal function. In the case of
donepezil it is providing more ACh in the brain and rosiglitazone is increasing adiponectin
concentration in the blood. When combined these drugs do not exert their full potential. They
do not complement each other or have a synergistic effect.
5.1 Future Studies
This experiment provided rats with 5 days dosage of drug so perhaps if the dose of drug itself
was increased or given for a longer period of time, different results would be seen. I would
expect to see donepezil performing better in the MA rats. However, because the drugs
combined after 5 days didn’t have a synergistic effect, with longer treatment the same effect
or even a further decrease in each drugs cognitive ability would more than likely be expected.
Also recognition memory was tested here so in future experiments spatial memory e.g.
Morris water maze task or working memory could also be examined.
To improve the rats performance in the object recognition task I propose a second exposure
to the test objects i.e. objects A and A, should be given. This would reinforce in the rats
memory that this object has already been seen and explored and is therefore not of much
interest when a novel object is provided. An additional time point could be used to see at
what stage the rat is no longer able to identify the novel object.
52. 44
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7. Appendix
Lysis Buffer (100 ml):
0.158g of 10mM Tris HCL
0.292g of 50mM NaCl
0.446g of 10mM Na4P2O7+OH2O
0.210g of50mM NaF
pH to 7.4
1 ml of 1% IGEPAL
Just before use add (for 20ml):
200µl of 1mM Na3VO4 (1:100 dilution)
200µlof 1mM PMSF (1:100 dilution)
200µl of protease cocktail inhibitor (1:100 dilution)
Krebs Buffer (1000ml):
7.95g of NaCl
0.19g of KCl
0.16g of KH2PO4
0.27g of MgSO4
1.34g of NaHCO3
1.8g of glucose
pH to 7.3
Just before use add (for 20ml):
40 µl of CaCl2 (1:500 dilution) (1.47g in 10ml distilled water)
200µl protease inhibitor cocktail (1:100 dilution)
Reagents provided by Millipore Rat/Mouse Insulin ELISA Kit:
Rat/Mouse Insulin ELISA Plate
Coated with mouse monoclonal anti-rat insulin antibodies.
10X HRP Wash Buffer Concentrate
10X concentrate of 50 mM Tris Buffered Saline containing Tween-20.
Rat/Mouse Insulin Standards
Rat insulin in Assay Buffer: 0.2, 0.5, 1, 2, 5 and 10 ng/ml.
Rat/Mouse Insulin Quality Controls 1 and 2
Rat insulin in QC buffer.
Matrix Solution
Charcoal stripped pooled mouse serum
Assay Buffer
0.05 M phosphosaline, pH 7.4, containing 0.025 M EDTA, 0.08% sodium azide, and
1% BSA.
Rat/Mouse Insulin Detection Antibody
Pre-titered biotinylated anti-insulin antibody.
56. 48
Enzyme Solution
Pre-titered streptavidin-horseradish peroxidase conjugate in buffer.
Substrate (Light sensitive, avoid unnecessary exposure to light)
3, 3’, 5, 5’-tetramethylbenzidine in buffer.
Stop Solution
0.3 M HCl
Reagents provided by Millipore Rat Adiponectin ELISA kit:
Rat Adiponectin ELISA Plate
Coated with Monoclonal Anti-Adiponectin Antibodies
10X HRPWash Buffer Concentrate
10X concentrate of 50 mM Tris Buffered Saline containing Tween-20
Quantity: 2 bottles containing 50 ml each
Preparation: Dilute 1:10 with distilled or deionized water
Rat Adiponectin Standard
Adiponectin Calibrator lyophilized.
Quantity: 200 ng/ml upon hydration.
Preparation: Reconstitute with 0.5 ml distilled or deionized water to obtain 200 ng/ml.
Rat Adiponectin Quality Controls 1 and 2
One vial each, lyophilized, containing diluted serum at two different levels of
Adiponectin.
Quantity: 0.5ml/vial upon hydration
Preparation: Reconstitute each vial with 0.5ml distilled or deionized water
10X Assay Buffer (Sample Diluent)
Quantity: 50 ml
Preparation: Dilute 1:10 with distilled or deionized water to make 1X Assay Buffer
(0.05M Phosphosaline containing 0.025M EDTA, 0.08% Sodium Azide, 1% BSA)
Assay Running Buffer
0.05M Phosphosaline containing 0.025M EDTA, 0.08% Sodium Azide, 1% BSA, and
animal serum IgG
Rat Adiponectin Detection Antibody
Pre-titered Biotinylated Monoclonal anti-Adiponectin Antibody
Enzyme Solution
Pre-titered Streptavidin-Horseradish Peroxidase Conjugate in Buffer
Substrate (Light sensitive, avoid unnecessary exposure to light)
3, 3’, 5, 5’-tetramethylbenzidine in buffer
Stop Solution (Caution: Corrosive Solution)
0.3 M HCl
Reagents supplied by IDS for Corticosterone EIA:
Calibrators ( REF AC-1401A - AC-1401G):
Lyophilised phosphate buffered saline containing corticosterone, protein and
preservative. 1 mL per bottle, 7 bottles per kit.
Antibody Coated Plate ( REF AC-1402W):
Microplate with polyclonal rabbit anti-corticosterone antibody linked to the inner
surface of the polystyrene wells, 12 x 8-well strips in a foil pouch with desiccant.
57. 49
Enzyme Conjugate ( REF AC-1403):
Lyophilised phosphate buffered saline containing corticosterone labelled with
horseradish peroxidase, protein, enzyme stabilisers and preservative. 2 mL per bottle.
Buffer ( REF AC-1403B):
Phosphate buffered saline containing preservative, 12 mL per bottle.
Controls ( REF AC-1405A - AC-1405B):
Lyophilised mouse or rat serum pre-diluted in Sample Diluent, 1 mL per bottle, 2
bottles per kit.
TMB Substrate ( REF AC-SUBS):
A proprietary aqueous formulation of tetramethylbenzidine (TMB) and hydrogen
peroxide, 30 mL per bottle.
Stop Solution ( REF AC-STOP):
0.5M hydrochloric acid, 14 mL per bottle.
Sample Diluent ( REF AC-1400B):
Phosphate buffered saline containing horse serum, protein and preservative, 15 mL
per bottle.
Wash Concentrate ( REF AC-WASHL):
Phosphate buffered saline containing Tween, 50 mL per bottle.