Title: Migration of inflammatory cells into the brain in Parkinson’s
disease
Project period: 3. Semester
Project group: 10gr307
Participants:
_____________________________
Cecilie W. Skovholm
_____________________________
Heidi D. R. Jensen
_____________________________
Malene Cording Christensen
_____________________________
Mette Pedersen
_____________________________
Sanne Andersen
_____________________________
Sofie Pagter
Supervisor: Torben Moos
Copies: 8
Page count: 43
The specific causes to Parkinson’s disease
are still unknown. There are many theories
regarding the causes, some of them being
environmental and others genetic. The
leading theory is, that it is a mixture
between environmental and genetic factors.
Parkinson’s disease is characterized by an
inflammatory condition in the dopaminergic
neurons in substantia nigra pars compacta,
which affects the nigrostriatal pathway.
When the dopaminergic neurons in
substantia nigra pars compacta undergo
degeneration the active microglia recruits
the immune cells of the periphery immune
system, by secreting different chemokines,
which helps make the blood brain barrier
more permeable.
It is still unknown, why the dopaminergic
neurons undergo a constant degeneration,
but it is believed that the environmental and
genetic factors play a crucial part.
It is difficult to determine in which order the
different pathological changes occur,
because the brain of patients with
Parkinson’s disease can only be examined
post mortem.
Because the pathogenesis of Parkinson’s
disease is still unknown, it is difficult to find
the optimal treatment.
Number of appendix: 0
Completed: 20. December 2010
3
Preface
This project is created by group 307, on the third semester of the education ”Medicine
with Industrial Specialization” at the University of Aalborg.
The project is written in the period November 16th to December 20th 2010.
The target group for this project is students from Medicine with Industrial
Specialization and students on related educations.
The project is created with guidance from Torben Moos who the group would like to
thank for supervision.
4
Table of contents
Migration of inflammatory cells into the brain in Parkinson’s disease .......... Error!
Bookmark not defined.
Preface ..................................................................................................................................... 4
Table of contents ................................................................................................................... 5
Reading guide ........................................................................................................................ 6
Introduction ........................................................................................................................... 7
Background ............................................................................................................................ 9
Parkinson’s disease in general ............................................................................................... 9
The motor circuit .......................................................................................................................10
Statement of problems: .................................................................................................... 16
Problem solving .................................................................................................................. 17
Causes of Parkinson’s disease ...............................................................................................17
Oxidative stress.......................................................................................................................................17
Lewy bodies and alpha-synuclein .....................................................................................................18
Parkinson’s disease induced by virus ..............................................................................................19
Parkinson’s disease induced by pesticides .....................................................................................20
Familial Parkinson’s disease ..............................................................................................................23
The innate immune systems response in general .........................................................................24
The inflammatory response in general ............................................................................................25
Cells of the central nervous system ..................................................................................................26
Microglia...................................................................................................................................................26
Activation of the microglia .................................................................................................................27
Pathological potential of activated microglia ................................................................................28
The blood brain barrier .........................................................................................................................28
Migration of cells into the brain ........................................................................................................29
Histopathology in Parkinson’s disease ..............................................................................31
Discussion ............................................................................................................................ 34
Conclusion ........................................................................................................................... 36
Outlook ................................................................................................................................. 38
References ............................................................................................................................ 40
5
Reading guide
Composition
Throughout this project we have used both English and Latin names for anatomical
terms. We have been consistent in using the same name through the text, and the
places we have used an abbreviation, we have indicated the meaning of the
abbreviation the first time it is used, by writing the whole name first and the shortened
term in parenthesis, for example the central nervous system (CNS).
Method
The Aalborg Model is used as framework for the project. It has been a theoretic
project based on already known knowledge and therefore no experiment has been
made, thus we do not have any results to discuss.
To ensure a high level of evidence in the project we have only used scientific
databases such as PubMed, Google Schoolar and The library of Aalborg University.
We have furthermore used educational books with relevance for the projects subject.
References
As indication of the references we have used the Vancouver system, which is the
system normally used in physical science. It is also called the “author-number”
system because the references are continuously numbered down though the text in
order of their appearance.
6
Introduction
The dictionary defines neuro- as “relating to nerves or nervous system”. The
definition of degeneration is “deteriorative and loss of function in the cell of a tissue
or organ”. A neurodegenerative disease must therefore appear as a disorder in which
neurons in the brain loses structure and function, which results in cell death. (1)
Neurodegenerative diseases can have a negative influence on balance, speech,
breathing, heart function and movement. The neurodegenerative diseases are often
genetic, but alcoholism, tumours, strokes, toxins, chemicals and viruses can also cause
degeneration of neurons. Neurodegenerative diseases include Alzheimer’s disease,
Amyotrophic Lateral Sclerosis, Friedreich’s Ataxia, Huntington’s disease, Lewy body
disease, Spinal Muscular Atrophy and Parkinson’s disease. Most of the
neurodegenerative diseases have no cure, therefore the treatment mostly involves
relieving of symptoms and pain and increase of mobility to make the life easier for the
patients. (2)
Because these diseases are affecting both the society as well as the individual, in
terms of lost manpower and reduced quality of life, there is a great interest in
improving the treatment of neurodegenerative diseases. Therefore research is very
important in this area. (3)
One of the neurodegenerative diseases that currently is being heavily researched is
Parkinson’s disease, but the causes are still unknown. In Denmark 6000-8000 people
suffer from this disease. (4)
Some research suggests that the degeneration, that takes place in the brain, may be a
combination of genetic predisposition and external toxic influence, such as pesticides,
viruses and other chemicals.
In spite of the fact that there is a relatively low prevalence of Parkinson's disease in
Denmark, the prevalence is increased by age. From the age of 65 the prevalence is
1.6 % and rises to 3.6 % at the age of 80. (5)
Furthermore, there is a ratio of 3-2, between men and women in developing
Parkinson’s disease. (6)
7
The lack of knowledge in causes and treatment of Parkinson’s disease and the huge
social and physical costs for the patients, leads us to the initiating problem:
What are the causes of Parkinson’s disease?
8
Background
Parkinson’s disease in general
Parkinson’s disease is a progressive neurological disorder. TRAP covers the four
cardinal features of the disease; Tremor at rest, Rigidity, Akinesia (bradykinesia) and
Postural instability. (7)
Tremor at rest is the first symptom the patients experience often when they do their
everyday activities such as buttoning their shirt. (8)
Rigidity is characterised by an increased resistance against passive movements, and is
often associated with postural deformity. The patient appears to have a flexed neck
and trunk posture and flexed elbows and knees are often seen too.
Akinesia is the most common clinical feature for patients with Parkinson’s disease. In
general, it is characterised by slowness in activities of the everyday tasks and also
slow movement and reduced reaction time. (6)
Besides the bodily disorders, the patients cannot show facial expressions; instead they
have a slightly open mouth and a staring look.
Postural instability results in a disturbance of the balance reflex, which makes the
patient fall easily. (9)
Besides the four cardinal motor symptoms, there are also other motor symptoms and
several non-motor symptoms. In table 1 these symptoms are shown. (7)
Table 1 shows the motor symptoms and non-motor symptoms in Pakinson’s Disease. (7)
9
The motor circuit
The basal ganglia (nuclei basales) are located in the ventral part of cerebrum, and is a
common name for some deep laying nerve cells in the grey substance of cerebrum.
The expression “basal ganglia” is actually a misleading name, because “ganglia”
refers to a bundle of nerves, located outside the Central Nervous System (CNS), and
the basal ganglia are nuclei in the CNS. “Basal brain nuclei” would therefore be a
better and more accurate name, than basal ganglia.(10)
The basal ganglia have influence on our motor skills and contribute, among other
things, to the speed of the movements, both when it comes to implementing and to
completing them. Diseases of the basal ganglia are causing movement disorders and
changes in the rest tonus1, like seen in Parkinson’s disease. (11)
The basal ganglia are divided into nucleus caudatus, nucleus lentiformes, claustrum
and amygdala. Figure 1 only shows the functional part of the basal ganglia in regards
to Parkinson’s disease.
Figur 1 dipicts a drawing of the basal ganglia inside the left hemisphere. b; photograph of a
frontal section of the brain showing the basal ganglia and other structures. c; the same
photograph as in (b) with nuclei highlighted .(13)
1
Tonus is the constant low-level activity of a body tissue, especially muscle tone.(1)
10
Striatum is the generic name for nucleus caudatus and nucleus lentiformis. Nucleus
lentiformis is divided into globus pallidus and putamen. Furthermore substantia nigra
is functionally connected to the basal ganglia, by sending dopamine from substantia
nigra pars compacta to striatum. This root is called the nigrostriatal pathway and is
one of the dopaminergic pathways.
Substantia nigra reaches out from the top of pons to the bottom of diencephalon, and
runs thereby all the way trough mesencephalon. It is located dorsal to the cerebral
penduncles, and is the largest nucleus in mesencephalon. Substantia nigra is
anatomically divided in two areas by the midline of the brain. Due to differences in
structure and function another division of substantia nigra is used. In this division
substantia nigra is divided into substantia nigra pars compacta, which is the dorsal
area of substantia nigra, and substantia nigra pars reticularis, which is the ventral
part. (10)
Substantia nigra pars reticularis is separated from globus pallidus interna by capsula
interna2. Both in structure and function pars reticularis and globus pallidus is alike and
they both consist mainly of GABAergic3 neurons. In pars reticularis the neurons lies
further from each other and the neurons are thinner than the dopaminergic neurons 4 in
pars compacta.
Substantia nigra pars compacta consist of dopaminergic neurons with a high content
of melanin5, which makes this part of substantia nigra appear dark. The neurons of
pars compacta are long and thick and are packed more densely than the neurons in
pars reticularis. Pars compacta is subdivided in to a ventral and a dorsal part. (12)
The basal ganglia and substantia nigra are a part of the motor circuit. A movement
originate as motivations in the prefrontal area of the cortex cerebri. Signals from the
prefrontal area are sent to another area of cortex cerebri, which is called the premotor
area. Here it is determent which muscles must be contracted, in which order and to
what degree. This information is send to the motor circuit, which includes both
inhibitory and excitatory pathways, and is explained in the following. At last, signals
2
Capsula interna is an area of white substance, which separate two structures. It contains both
ascending and descending axons. (10)
3 Neurons that uses GABA as neurotransmitter. (10)
4 Neurons that uses dopamin as transmitterstof. (10)
5
Melanin is a dark pigment, which makes the colour brown or black. (1)
11
from the motor circuit are send to the primary motor area of cortex cerebri, from
where the signal travel through descending pathways in medulla spinalis to the
peripheral nervous system, which innervate the skeletal muscles. (13)The mentioned
areas in cortex cerebri are shown in figure 2.
Figure 2: The figure shows the functional regions of the lateral side of cerebral cortex. (46)
The motor circuit is divided into a direct and indirect pathway. Figure 3 shows these
two pathways and supports the description below.
The direct pathway results in an excitatory stimulation of the skeletal muscles.


Cortex cerebri initiates this pathway by sending projections6 to striatum. These
projections are neurons containing glutamate7, which has an excitatory effect
on the neurons in striatum.
o The excitatory glutamate activate the GABAergic neurons in striatum.
Neurons then projects from striatum to globus pallidus interna8 and contain the
inhibitory amino acid GABA9. Thereby the GABA neurons in globus pallidus
interna are switched off.
Projections are connections between the cerebral cortex and other parts of CNS or sensory
organs. (44)
7 Glutamate is an aminoacid. (10)
8 Globus pallidus interna is the inner part of nucleus lentiformis. (14)
6
12


Because of the lack of the inhibitory GABA from globus pallidus interna to
thalamus, thalamus is continuously active.
Nucleis of thalamus sends glutamergic projections to cortex cerebri. This last
step initiates the movement of the skeletal muscles.
Figur 3: This figure shows the motor circuit, and the dopaminergic projections from substantia nigra pars
compacta to striatum. The blue lines show the direct pathway, and the red lines show the indirect
pathway. If the line is dotted it means that the projections is GABAergic, and if the line is full, it means
that the projections is glutamergic. The dotted black lines are present when cortex cerebri is initiated. The
full black line is at all time present – except when excitatory stimulated form the GABAergic neurons in
the indirect pathway. Furthermore is dopamine sent from substantia nigra pars compacta to striatum, and
thereby the indirect pathway becomes dominant. (10)
The indirect pathway inhibits the initiation of a movement, and skeletal muscles are
not stimulated.


9
The first step in this pathway is a glutamergic projection from cortex to
striatum.
o The GABAergic neurons in striatum are then turned on.
Inhibitory GABA neurons from striatum project to globus pallidus externa.
o Normally globus pallidus externa sends inhibitory GABA to nucleus
subthalamicus, which thereby is turned off.
o When globus pallidus externa receives GABA from striatum, globus
pallidus externa is turned off and nucleus subthalamicus is active
because of the glutamate it constantly receives from cortex cerebri.
Gamma-aminobutyric acid (19)
13




Nucleus subthalamicus sends excitatory glutamate to globus pallidus interna,
which is turned on.
The activated GABAergic neurons of globus pallidus interna send inhibitory
GABA to thalamus.
Thalamus is turned off and does thereby not stimulate the cortex cerebri to
initiate movement.
When movement is not initiated, skeletal muscles are not stimulated.
When dopamine is sent from substantia nigra pars compacta to striatum, the direct
pathway becomes dominant, which results in an initiation of movement.
Dopamine(14) is send from substantia nigra pars compacta, when it is stimulated by
cortex cerebri. In Parkinson’s disease the lack of dopamine from substantia nigra pars
compacta to striatum results in a dominance of the indirect pathway, and thereby an
absence of movement initiation. (10)
Because of the degeneration of the pigmented neurons, substantia nigra pars compacta
gradually loses its characteristic
dark colour. This is seen in figure 4.
Furthermore
this
degeneration
results in trouble initiating and
maintaining
movements,
for
patients with Parkinson’s disease.
Rigidity, tremor, akinesia and the
missing facial expressions are a
result of the lack of dopamine
neurons termination in striatum.
However these symptoms does not
show until 80 % of the neurons are
degenerated.
(10)
To diagnose Parkinson’s disease,
Figur 4 shows a horizontal view of the brain in which
substantia nigra is seen. In the right upper corner is a
healthy substantia nigra where pars compacta appears as
dark. In the right lower corner is substantia nigra from a
patient with Parkinson’s disease shown. The degeneration
of pigmented dopaminergic neurons leaves pars compacta
appears as pale like the rest of substantia nigra (23)
akinesia and at least one of the
other TRAP symptoms must be present. If Levodopa, which currently is the most
effective treatment of Parkinson’s disease, has a positive effect on the patient, it will
14
confirm the diagnosis.(9) However, this treatment is not the most ideal because 50 %
of the patients experience a progressive reduction of the effect after 5-7 years.
Levodopa is decarboxylated 10 to dopamine in the bowels and the liver. Dopamine
does not pass the blood-brain-barrier (BBB). By adding a decarboxylase inhibitor, a
greater amount of Levodopa will stay unchanged and thereby, pass the BBB.
Decarboxylase inhibitors do not pass the BBB, and that is why Levodopa is
decarboxylated to dopamine in the brain. The transport of Levodopa over the bowel
and the BBB occurs by using a carrier mechanism.
Levodopa is only a symptomatic treatment of Parkinson’s disease, with the goal to
delay the development of the disease, ease the symptoms, and improve quality of life.
Currently, there is no cure for Parkinson’s disease and therefore, research is necessary.
(15)Furthermore a lot of research is concentrated on finding the cause to Parkinson’s
disease, since this is not enlightened. In this project we will focus on oxidative stress,
genetic factors, viruses and pesticides, which are some of the possible causes to
Parkinson’s disease.
By autopsy of patients with Parkinson’s disease, an inflammation, which is a result of
neurodegeneration, of substantia nigra is seen. It is still unknown what causes the
inflammatory state, but there are a lot of theories about this. Because of the fact that it
is an inflammatory condition our focus will, in relation to this, be on the innate
immune system.
The abovementioned indistinctness leads us to our statement of problems.
10
Release of CO2. (45)
15
Statement of problems:

What causes Parkinson’s disease?

How does the immune system react to neurodegeneration in the brain?

What causes the periphery immune system to migrate into the brain in
neurodegenerative diseases?
16
Problem solving
Causes of Parkinson’s disease
The specific cause of Parkinson’s disease is still unknown, but many theories about
the causes exist. One of them is, that it might be a mix between the genetic mutations
and the environmental factors. In many resent experiments Parkinson’s disease is
induces by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a narcotic used by
substance abusers, which is known to cause the same neurodegeneration as in
Parkinson’s disease. (16)
In the following we will enlighten some of the suggested causes. First mentioned is
oxidative stress, which is the strongest theory of degeneration of neurons in relation to
Parkinson’s disease.
Oxidative stress
Hypothesis; Oxidative stress can induce Parkinson’s disease.
Oxidative stress is a cellular process in which free radicals cause damage to vital
structures and functions of the cells.(17) Free radicals are atoms, molecules or ions
with unpaired electrons. They react with other atoms or molecules by donating the
unpaired electron or by abstracting a hydrogen atom, whereby a new free radical is
formed and a chain reaction can begin. A free radical can also react with another free
radical, by which the reactions will stop.(18)
If a free radical removes hydrogen from a lipid in the cell membrane, a chain reaction
will start, and several new radicals are formed. These radicals can change the cell
membrane and cause disruption of the cell’s function and may lead to degeneration of
the cell. The chain reaction will continue until no more lipids are available, unless
stopped by antioxidants.(19) Free radicals can also oxidize the sulfur-containing
groups in proteins and thereby destroy the structure and thus the function of the
protein. If free radicals react with DNA bases, it may lead to mutations.(18)
It is crucial to maintain a natural balance between free radicals and antioxidants to
avoid oxidative stress. (17)
17
In Parkinson’s disease there have been demonstrated increased levels of iron in the
substantia nigra pars compacta. The increased level of free iron produces free radicals,
which, as mentioned above, could be the cause of oxidative stress.
Iron exists naturally in the brain and is stored as ferritin, why there is almost no free
or reactive iron present in the brain. Therefore an elevated level of iron in the brain
will normally lead to an increased ferritin synthesis.
An elevated level of free iron in the brain tissue, caused by abnormal metabolism of
iron to ferritin, is believed to be one of the courses of oxidative stress. If the iron
exists as free iron it can react with hydrogen peroxide (H2O2) and create cytotoxic
oxygen free radicals, such as OH and Fe3+. As mentioned above these free radicals
induce lipid peroxidation in the cell membrane and change the internal homeostasis in
the cell. The change of homeostasis leads death of the cell. It is yet unknown whether
oxidative stress is the cause of neurodegeneration or if degeneration of neurons is the
cause of oxidative stress, but they probably have an influence on one another. (20)
Lewy bodies and alpha-synuclein
In patients with Parkinson’s disease there have been detected Lewy bodies in the
cytoplasm of neurons.
Lewy bodies occur, when a protein, such as alpha-synuclein, accumulates, thereby
forming insoluble fibres, called fibrils.
When alpha- synuclein accumulates it is believed to result in toxicity by forming
reactive oxygen species (ROS), which can lead to oxidative stress, thereby making the
accumulated alpha-synuclein able to change the storage of vascular dopamine. The
accumulation is a result of a mutation in the gene coding for alpha-synuclein. The
reason for this mutation can be oxidative stress.(6)
The pathological characteristics of lewy bodies are that they are intraneuronal
eosinophile11 aggregates, which consist of a number of different proteins, of which
alpha-synuclein seems to be the most important. Study of Lewy bodies revealed
masses of aggregated filaments with a diameter of 7-25 nm, but the precise molecular
11
Cell or cell component stained by eosin, which is a red dye anion(14)
18
composition, the reason why they are neurotoxic and the role they might play in the
degeneration of neurons remains non-clarified.(21) (9) (22)
Parkinson’s disease induced by virus
Hypothesis; A virus can induce Parkinson’s disease.
A rapport from The Rockefeller University shows that it is possible to induce
symptoms and pathological changes, like those found in Parkinson’s disease, by
inoculating rats with Japanese encephalitis virus (JEV).
In this experiment two groups of rats were examined. One group were inoculated with
JEV and the other group was an age similar control group.
The brains susceptibility to JEV depends on the age of the rat at the time of the
inoculation.
In table 2 the effect of inoculation depending on the age of the rat is shown.
These results indicate that if the rat is infected with JEV at the age of 14 days, it may
results in pathological changes like those found in Parkinson’s disease.
Furthermore it shows that the most affected area in substantia nigra was the region of
pars compacta.
Age:
1-3 days
10 days
14 days
Effect:
Developed widespread infection in the whole brain.
No JEV-antigens were found in the cortex cerebri, but in
other parts of the brain.
The JEV-antigens were only found in the basal ganglia and
the substantia nigra.
Table 2 shows the effect of JEV according to the age of the rats. (23)
This experiment also showed that there were fewer TH-positive 12 neurons in
substantia nigra of the inoculated rats than in the rats of the control group. Some of
the inoculated rats, who lived a whole year, had almost no TH-positive neurons in
substantia nigra. This indicates that the neurons in substantia nigra are especially
vulnerable to viruses.
The inoculated rats showed distinct akinesia, which were significantly improved,
when treated with Levodopa.
12
TH – tyrosine hydroxylase – an enzyme that is responsible for the conversion of L-tyrosine to
DOPA (dihydroxyphenulalanine), which is a precursor for dopamine. (19)
19
Furthermore the two groups of rats had
their motor skills tested. The infected
rats
were
much
slower
than
the
uninfected rats. Later on the infected
rats were treated with Levodopa and
tested again. Their motor skills had
improved, but were still slower than the
control group, see figure 5.
All of these neurological changes were
present in more than 80 % of the rats
inoculated with JEV. (23)
These results indicate that viruses can
induce Parkinson’s disease.
Parkinson’s disease induced by
pesticides
Hypothesis; If a person is exposed to
Figure 5 shows the motor skills of tested rats.
The vertical axis shows the time to perform the
task in seconds and the horizontal axis shows
the group of rats (the control group, the
inoculated group and the inoculated group
treated with Levodopa).(26)
small doses of pesticides for a long period of time, their risk of developing
Parkinson’s disease will increase.
Many different experiments have shown that pesticides can induce the same
neurological damages as seen in Parkinson’s disease.
In a rapport from 201013 rats were exposed to Dichlorvos (DDVP), a pesticide widely
used in greenhouses to protection against vermin. (24)
DDVP is an organophosphate, which is a common name for a group of esters or
phosphate acids. These organophosphates irreversible inactivate acetylcholine
esterase, which leads to an inactivation of the whole neuron. (25)
Pesticides and therefore also DDVP are extremely hydrophobic, why they easily can
pass membranes, such as the BBB.
In the experiment from 2010 rats were exposed to 2,5 mg DDVP pr. kg. every day in
12 weeks.
13
”Nigrostriatal neuronal death following chronic dichlorvos exposure” (24)
20
DDVP was pulsating administrated to make a resemblance to the real world, where
those who work with pesticides inhales it, get it on their skin and digests it.
At this low dose a distinct degeneration of the dopaminergic neurons in substantia
nigra pars compacta was seen. Furthermore there was a formation of Lewy bodies in
the neurons and a prominent deterioration of the mitochondria.
If the rats were exposed to a higher dose of DDVP there was a systemic poisoning and
non-specific brain lesions.
In relation to this experiment several different compounds were measured.
The results of the measuring are seen in figure 6. (24)
Figure 6 shows effect of a chronic dichlorvos exposure on mitochondrial electron transport chain. (A)
NADH dehydrogenase activity. (B) cytochrome oxidase activity. (C) ROS levels. (D) lipid peroxidation.
(24)
21
A. Figure 6.A shows that the rats, which were exposed to DDVP, have a distinct
decrease in numbers of NADH dehydrogenase enzymes (complex I) in the
mitochondria in substantia nigra and in striatum. Complex I is the first step in
the electron transport chain in mitochondria, which contributes to the
formation of ATP – the cells energy.
B. Figure 6.B shows an increased activity of the enzyme Cytochrome oxidase
(complex IV). This enzyme is the last step in the electron transport chain, in
mitochondria, where it reduces the molecular oxygen, which is linked to the
ATP-production. The complex IV activity was increased by 56,7 % in
substantia nigra and by 55,4 % in striatum. This increase in enzymatic activity
may lead to the formation of free oxidative radicals, which in worst case can
lead to oxidative stress.
C. In figure 6.C the ROS levels were measured, which indicates the quantity of
free radicals. As shown the levels were considerable higher in the rats exposed
to DDVP, both in substantia nigra and in striatum, than the level in the control
rats.
D. In figure 6.D the lipid peroxidation levels were measured. This level indicates
the destruction of lipid membranes of the neurons, which will lead to the
degeneration of the cell. The lipid peroxidation level was increased by
50,29 % in substantia nigra and by 38 % in striatum.
The experiments shows that risk of developing Parkinson’s disease will increase if a
person is exposed to small doses of pesticides for a long period of time. (24) This kind
of results are not only seen in experiments with DDVP, but also with other pesticides
as rotenone. (23)
All above-mentioned causes lead to degeneration of neurons, especially in substantia
nigra. Occurrence of degeneration of neurons in CNS activates the immune cells of
the brain, initiating inflammatory processes, which is described in the following.
22
Familial Parkinson’s disease
Hypothesis; Gene mutations can cause Parkinson’s disease.
95% of the cases of Parkinson’s disease occur spontaneously. The last 5% is a result
of a genetic mutation. (26)
Familial Parkinson’s disease is a result of a gene mutation, but sporadic Parkinson’s
disease can also originate from a genetic mutation, along with many other
environmental factors as mentioned above.
In familial Parkinson’s disease mutations in at least 11 gene-loci have been shown,
and six gene products have been proved to have a pathogenic mechanism (6). We will
focus on the gene-loci PARK8, which have the gene product leucine rich repeat
kinase 2 (LRRK2). Among individuals with familial Parkinson’s disease, mutations
in the LRRK2 gene are shown in 10 % of the cases and are the most frequent.
PARK8 is an autosomal dominant familial mutation that causes Parkinson’s disease
of varying phenotype 14 . LRRK2 is a very complex cytosolic protein, which has a
kinase domain15. The mutation takes place in the kinase domain of the LRRK2 protein.
Expression of the LRRK2 mutations in the cells leads to degeneration with aggregated
formation. Study shows that by inactivating the kinase domains in the LRRK2,
delayed degeneration and prevention of aggregated formation will occur. This
indicates the importance of this mutation regarding to the pathogeneses of Parkinson’s
disease.(6)
14
15
The result of the interplay between individual genes (genotypes) and the environment. (44)
Domain with enzymatic functions. (44)
23
The immune system
The innate immune systems response in general
The body’s primary protection of any invading foreign organisms is the skin and
mucous membranes. Even though, foreign organisms can pass these barriers, mostly
through the airway and the digestive tract, and thereby cause infection.
The body’s internal environment is a perfect place for bacteria and virus to proliferate.
If this proliferation is allowed to continue it will in a short period of time kill the host.
Fortunately a range of defence systems against foreign substances, such as the innate
immune system, exists. The innate immune system is also activated if a cell appears
different from what is normal e.g. because of damage to the cell membrane, since the
cell then is detected as foreign to the body.
Detection of a foreign substance immediately activates the innate immune system,
which defence mechanisms are nonspecific, in other words it is not limited. (27)
Table 3 shows the different types of cells in the innate immune system and their
primary function.
Cell
Neutrophils
Primary function
Phagocytosis and inflammation; usually the first cell to leave the blood
and enter infected tissues.
Monocyte
Leaves the blood and enters tissues to become a macrophage.
Macrophage Most effective phagocyte; important in later stages of infection and in
tissue repair; located throughout the body to ”intercept” foreign
substances; processes antigens; involved in the activation of B- and Tcells.
Basophils
Motile cell that leaves the blood, enters tissues, and releases chemicals
that promote inflammation.
Mast cell
Non-motor cell in connective tissues that promote inflammation
through the release of chemicals.
Eosinophils Enters tissues from the blood and releases chemicals that inhibit
inflammation.
NK-cell
Lyses tumor and virus-infected cells.
Table 3 shows the cells of the innate immune system and their primary function. (23)
There are three main components of innate immunity. First there is a mechanical
mechanism, such as the skin and mucous membranes, which maintain barriers to
prevent the entry of foreign substances into the body. Chemical mediators are the
24
second component, which covers the many molecules, e.g. complements proteins,
responsible for the many functions of innate immunity. The last component is the
cells involved in phagocytosis, seen in Table 3, and the production of chemicals that
participate in the immune system’s response.(13)
When the body becomes infected or damage to cells occurs, the innate immune
system firstly recruits macrophages and neutrophilic granulocytes (neutrophils),
which will try to phagocyte foreign substance. Furthermore specific microorganisms
activate complement proteins16, and the activation will increase the binding of the
abovementioned phagocytes cells to the invading organisms. (27) Complement
proteins are in the circulating blood and in normally conditions, they are in an inactive,
non-functional state. When a foreign substance is detected, they become activated in a
cascade, in which each component activates the next.(13) Once activated, the
complement proteins induce inflammation by attracting phagocytising cells. (28)
The inflammatory response in general
Inflammatory processes are a part of the innate immune system and are the body’s
reaction against infection and cell damage. If the cells of the immune system detect
any foreign objects, such as bacteria, parasites, viruses or damaged cells,
inflammation is the body’s frontline defence and enables it to fight the unwanted
substances.
Inflammation can occur in two ways, locally or systemically, and have different
processes and symptoms. Local inflammation is confined to a certain area and the
arising symptoms are redness (rubor), heat (calor), swelling (tumor), pain (dolor) and
loss of function (functio lesa). The systematic inflammation can occur at different
sites in the body and three additional features can be present. One of them is
production of neutrophils that promote phagocytosis. Pyrogens are other molecules
that get released by microorganisms, macrophages and neutrophils, and induce fewer.
The last feature, which only happens in severe cases, increased vascular permeability,
which is so widespread, that large amounts of fluid are lost from the blood to the
tissues. This results in a decreased blood volume and can lead to shock and ultimately
death.
16
Includes around 40 plasma proteins and cell surface receptors. (13)
25
The actual inflammatory response is a sequence of events performed by chemical
mediators and cells of the innate immune system. When tissue is damaged a release of
chemical mediators will occur. These chemical mediators have three important effects
regarding the inflammatory response;
Vasodilatation will increase the blood flow and thereby bring a large amount of
phagocytes and white blood cells to the site of injury.
There will be a chemotactic attraction of phagocytes, which leaves the blood and
enter the damaged tissues.
An increased vascular permeability will facilitate the diffusion of complement
proteins from the blood to the tissues. This defence mechanism is primarily performed
by mononuclear phagocytes (MP)17, among the MPs are the macrophages. In CNS
another kind of MPs are present. These so-called brain MPs18 have the same function
as MPs in the rest of the body. Among the brain MPs are microglia.(29)
Cells of the central nervous system
Tissue of the central nervous system consists of neurons and non-neural cells. Some
of the non-neural cells are called glia cells and are further divided into three subtypes;
astrocytes, oligodendrocytes and microglia. The non-neural cells primary function is
to protect and support the neurons. Furthermore the function of the astrocytes is,
among others, to form the scar tissue in connection to decomposition and removal of
damaged cells, the oligodendrocytes produce the myelin sheets of the neurons and the
microglia phagocyte foreign substances or damaged cells.
In the following we mainly describe the microglia, which will be considered in
relation to Parkinson’s disease. (13)
Microglia
10 % of the glia cells are microglia and are mainly located in hippocampus, olfactory
telencephalon, basal ganglia and substantia nigra.(30)
Microglia is in the embryonic state derived from circulating monocytes, which are the
progenitors of microglia.
17
18
MP: monocytes, tissue macrophages and dendritic cells.(29)
Brain MP: perivascular and parenchymal macrophages.(29)
26
The morphology of microglia is branchy and thereby microglia have a lot of
peripheral processes.(31)
The microglia exists in three stages; resting, activated and phagocytic. In the healthy
brain only resting microglia are present. When the brain is injured microglia then
perform as a part of the immune system of the CNS.(30)
Furthermore microglia recruits cells from the immune system circulating in the blood
by releasing proinflammatory cytokines. Further information about this process is
found later in this section.
Activation of the microglia
Microglia have classical immunological receptors (e.g. cytokine) and classical neuroligand receptors (e.g. glutamate and GABA).
Numerous receptors and immune molecules recognition sites make the microglia a
good detector for any damage to the tissue of the CNS. When microglia detects
foreign substances it is activated and may even become phagocytising. Both microglia
in the phagocytic and active state are a part of the active brain defence system.
Furthermore, the resting microglia cells have their own territory in which they
randomly move, and thereby the brain can be completely scanned several times a
day.(30)
In the first stage of activation of the microglia, resting microglia retract their
processors, which become thicker and fewer. Moreover the cell body increases in size
and there is a change in expression of different enzymes and receptors. All this result
in a production of immune response molecules19 and the microglia cell is now in the
active state.
At the activated state the microglia gets in a proliferate mode and multiplies around
the infected area. Finally the microglia becomes motile and moves to the site of the
tissue damage.(30)
By producing different substances, activated microglia is involved in inflammatory
processes linked to neurodegeneration.(29)
If the cells in CNS die or is damaged, the microglia undergoes further transformation
and becomes phagocytising. In addition to the phagocytic state, microglia produce
19
E.g. cytokines and growth factor. (30)
27
and secrete potential neurotoxins, which may cause neural damage and thereby
promote neurodegeneration. (47) In relation to Parkinson’s disease, this happens
especially in substantia nigra pars compacta. (30)
The starting signal, to activate the microglia cells, consists of a wide range of
molecules either associated with cell damage or with foreign matter invading the brain.
Especially damage of the neurons can release high amounts of ATP, nucleotides,
growth factors etc. All this can be detected by microglia, which thereby gets activated.
Besides the cell damage and the foreign substances, the astrocytes are as well capable
of activating the microglia by releasing molecules. Alternatively activated sister cells
of the microglia can send signals that in turn activates other microglia cells.(30,32)
Astrocytes are “star-like” cells with many cytoplasmic processors. Some of the
processors are in contact with blood vessels, which forms the BBB. Astrocytes
separate the neurons and their processors from each other.
When injury occurs, astrocytes will migrates to the site of injury and develop a
hypertrophic morphology. They are now reactive astrocytes. They do not attack the
pathological target but wall it off.
In all, microglia and astrocytes can affect the developing stages of neurodegeneration
as explained below.(29)
Pathological potential of activated microglia
Microglia are capable of contributing to both protection and destruction of the CNS.
In neurodegenerative diseases, such as Parkinson’s disease, the long-lasting
phagocytic state of microglia may lead to neuronal death or the death of
astrocytes.(30)
To protect the brain against intrusion of harmful substances, an outer barrier between
the blood and the brain consists, which is called the BBB.
The blood brain barrier
There are two kinds of barriers in the brain, the blood cerebrospinal fluid barrier
(Blood-CSF-barrier) and the BBB. The BCB surrounds the choroid plexus in the
ventricles of the brain, where the cerebrospinal fluid is produced, and the BBB is
between the capillaries in the brain and the brain tissue. There are some areas in the
28
brain, that are not covered by the BBB, called circumventricular organs, e.g. near the
hypothalamus and pineal gland. Here the diffusion between blood and tissue is easier
and products are sent directly into the systemic circulation.
The BBB and BCB are very permeable to water, O2, CO2, glucose and most
liposoluble molecules (alcohol, anaesthetics etc.), but are almost impermeable to
water soluble- and large molecules.
The barriers impermeable effect is caused by the way the endothelial cells are
connected to each other by tight junctions, which make the space between the cells
incredibly small. Furthermore there is a thick basement membrane around the
capillaries in the brain. Astrocytes processors are pressed against the capillaries where
they secrete specific molecules, which help maintain the permeability. (33) (34)
When areas in the brain are inflamed, leucocytes are recruited from the blood to the
inflammatory area, and among others this recruitment changes the structure of the
BBB, which becomes more permeable. A higher permeability of the BBB makes it
easier for the cells to migrate into the brain. (35,36)
Migration of cells into the brain
When neurodegeneration occurs in the brain it is necessary to phagocyte the damaged
cells and repair the tissue. The presence of dead and damaged cells activates the
microglia, which releases proinflammatory cytokines, e.g. CCL2 that binds to CCR2,
which is a receptor located on the endothelium of the BBB. This binding, among
others, up regulates the expression of different adhesion molecules on the surface of
the endothelium cells. The effect of the binding of CCL2 to CCR2 is further described
in the section “Histopathology in Parkinson’s disease”. The cells of the immune
system bind to the adhesion molecules, which promote the migration of these cells
through the wall of the blood vessel, in this case the BBB, into the brain.
Two of the important adhesion molecules are Inter Cellular Adhesion Molecule
(ICAM) and Vascular Cellular Adhesion Molecule (VCAM). Both ICAM and VCAM
are located in the membrane of the endothelium in the blood vessels, and furthermore
ICAM is expressed on some of the cells of the immune system. When cells of the
immune system bind to the endothelium’s adhesion molecules, the speed of which the
29
immune cells pass the inflamed area is reduced, and migration of these cells into the
brain is promoted.
Migrations of immune cells into the brain can occur in two ways transcellular
diapedesis or paracellular diapedesis 20 . By transcellular diapedesis immune cells
migrate directly through the body of the endothelium cell, whereas they by
paracellular diapedesis migrate through the space between the endothelium cells.
Release of different substances, such as cytokines and enzymes also promotes the
migration. For instance macrophages releases cytokines, which initiate the expression
of adhesion molecules on the endothelium, and migrating leucocytes secrete the
enzyme proteases, which degenerate the basement membrane of the blood vessel and
thereby ease the migration. (37)
20
Diapedesis is the passage of blood cells through the intact wall of the capillaries , typically
accompanying inflammation. (1)
30
Histopathology in Parkinson’s disease
There are many hypotheses about why the chronic inflammation of the brain in
Parkinson’s disease occurs.
Neuroinflammation in the patients with Parkinson’s disease have been observed in
postmortem studies. By autopsy of patients with Parkinson’s disease cells, as active
microglia, reactive astrocytes and periphery immune cells have been discovered in
substantia nigra pars compacta. In fact a six-fold increase in numbers of phagocytic
microglia, which phagocytize dopaminergic neurons, have been shown. Furthermore
these microglia secrete many pro-inflammatory cytokines and a dysfunction of the
BBB has been revealed. (29)
Figure 7 shows a proposed theory describing the neuroinflammatory processes
in Parkinson’s disease. (31)
Figure 7 explains a proposed relation between the immune cells, the disrupted BBB
and the dopaminergic neuronal death in the neuroinflammatory process. The figure
31
illustrate that the death of the dopamineric cells in substantia nigra pars compacta
either leads to microglia activation, reactive astrocytes, BBB dysfunction and
migration of periphery immune cells into the brain, or that these four features leads to
the death of dopaminergic neurons. This cascade continues and thereby the
neuroinflammation becomes chronic. (31)
In chronic neuroinflammation glia cells secrete inflammatory chemokines, which are
chemotactic cytokines i.e. cytokines that attract other cells to specific areas of the
body. Furthermore chemokines activate the inflammatory cells, such as monocytes
and glia cells. One theory is that there is a connection between specific chemokines
and the inflammatory state that occur in Parkinson’s disease. Even though many
chemokines are considered to contribute to loss of dopaminergic neurons in substantia
nigra pars compacta there are many reasons to concentrate on the specific chemokine
CCL2. It is believed, that because of the fact that the ligand receptor pair CCL2 and
CCR2 are expressed on the dopaminergic neurons in substantia nigra, these
chemokines must have an influence on the representative neurons.
CCL2 binds to CCR2, which is a chemokine-receptor located on endothelial cells,
astrocytes, microglia and neurons. Upon binding, monocytes, T-cells and NK-cells
will migrate through the BBB to the inflammatory site and perform their
inflammatory response. Furthermore the chemokine is able to activate other microglia
and recruit them to the damaged area. Furthermore it has been shown that a deletion
of the gene coding for CCR2 will lead to a reduced migration of monocytes, T-cells
and NK-cells to the brain. (38) (39)
In the normal brain microglia will be in a resting state and will only become activated,
when the microglia detects any change in the tissue of the brain, for example when a
cell dies, the microglia will start an inflammatory response.
If the inflammation becomes chronic, because of constant changes in the environment
in the brain, the microglia will become phagocytic, which is the case in Parkinson’s
disease.
A study has tried to clarify the interaction of microglia and neurons in Parkinson’s
disease. The result of the study showed that phagocytic microglia kills the damaged
neurons. Because of these observations, it has been shown that if the microglia are
removed and Parkinson’s disease then is induced, the disease will not develop.
32
However this is not a suitable solution, since the absence of glia cells in the brain will
result in many other diseases. (40)
33
Discussion
It is debatable of how great importance the genetic factors plays in relation to the
environmental factors, as genetic mutations only appear in 5% of the cases of
Parkinson’s disease. Even though mutations are seen in 5% of the cases, is it not
tantamount with genetic as the only cause to the outbreak of Parkinson’s disease in
those cases. Besides genetics there are, as mentioned before, many environmental
factors that may cause Parkinson’s disease.
All the environmental factors initiate oxidative stress, which is connected to the
degeneration of the dopaminergic neurons in substantia nigra pars compacta. It is still
unknown whether oxidative stress occurs before or after the degeneration of these
neurons, or both. If oxidative stress occurs before the degeneration, one can argue that
oxidative stress is the reason for degeneration of neurons in substantia nigra. On the
other hand the degenerated cells, which are seen in cases of Parkinson’s disease, may
lead to oxidative stress presumably by causing damage to the cell membrane. It is
difficult to determine the order of the oxidative stress and degeneration, because the
brain cannot be examined before the patient has passed away. Further are patients
with Parkinson’s disease diagnosed very late in the pathogenesis, which makes it
impossible to examine whether the high level of oxidative stress appears before the
neurons are degenerated. Because of this, only theories about the connection between
oxidative stress and neurodegeneration are present.
In patients with Parkinson’s disease oxidative stress, which is seen as an increased
level of free radicals, has been observed post mortem. One theory is that increased
levels of free iron generate free radicals that result in oxidative stress, which is
assumed to lead to neurodegeneration. The level of iron in the brain increases with
age, which enhances the risk of oxidative stress. Age can’t be the cause of Parkinson’s
disease because the prevalence of the disease is relatively low, better reasons must be
clarified, which then can lead to a cure and not just a symptomatically treatment,
which is the only existing treatment at this point of time. Furthermore it is believed
that patients with Parkinson’s disease have an abnormality in the synthesis of ferritin,
34
which thereby increases the level of free iron in the brain. This supports the
arguments that there must be a more precise cause of Parkinson’s disease than age.
It is known that damage to the neurons activates microglia, which become
phagocytising and by secreting chemokines recruit peripheral immune cells, which
migrate into the brain. Of unknown causes the degeneration of neurons persists and
therefore the microglia remain phagocytising, by which the inflammatory response
becomes chronic. It is unknown whether it is microglia or the migrated immune cells
that have the biggest influence on the phagocytosis of the neurons in substantia nigra
pars compacta. Experiments shows, that by removing or inactivating the microglia
and then induce Parkinson’s disease, the development of the disease will fail to appear.
If both microglia and peripheral immune cells are kept, but they are deprived of their
ability to communicate and thus microglia’s ability to call in the peripheral immune
cells - for example by inhibition of the communication between CCR2 and its ligand
CCL2 - it is possible that the extent of neurodegeneration in the brain will be reduced.
This suggests that the microglia is the most crucial factor of the inflammatory state in
the brain.
A lot of research concerning Parkinson’s disease has been made. The research is
mainly based on experiments with mice and rats and therefore one wonders whether
the results can be transferred humans. There are several differences between the
human body and the body of a mouse or rat. For example when Parkinson’s disease is
induced in a rat the disease will develop in a short period of time whereas in humans
the disease will first appear around the age of 60. Furthermore it is not necessarily the
same way Parkinson’s disease develops in humans as in mice. In mice injecting
MPTP can induce the disease and even though the symptoms are the same the cause
to Parkinson’s disease in humans is rarely MPTP, since the drug is no longer a
widespread drug among substance abusers. Therefore it can be discussed whether the
results can be transferred to humans.
35
Conclusion
There are many different theories about what the cause to Parkinson’s disease is, but
the exact causes still remains unknown.
However we do know that many different factors are involved in the development of
Parkinson’s disease, genetic and environmental factors among others.
Among genetic factors different mutations in different genes have been shown, e.g. it
is known that a mutation in the PARK8 gene can lead to an increased disposition of
Parkinson’s disease. Among environmental factors pesticides and viruses are known
to cause the same neurological changes as seen in patients with Parkinson’s disease.
One theory is that Parkinson’s disease occurs as a result of a mixture of genetic
mutations and environmental influences.
In Parkinson’s disease, induced by both genetic mutations and environmental factors,
oxidative stress has been seen and is considered to be the most likely cause to
degeneration of the neurons in e.g. substantia nigra pars compacta. In some cases a
high level of free iron is also detected and is assumed to be one of the causes to the
development of oxidative stress. Furthermore Lewy bodies, in dopaminergic neurons,
have often been detected. Lewy bodies occur as a result of a mutation in the gene
coding for the protein alpha-synuclein, which then accumulates and forms insoluble
fibrils. It is uncertain how, but it is believed that the formation of Lewy bodies
contributes to the degeneration of the neurons.
Moreover it is still unknown whether oxidative stress leads to degeneration of the
neurons or if the degenerations of the neurons lead to oxidative stress, or both.
One of the reasons, why it is so difficult to clarify the precise cause to Parkinson’s
disease and in which order the neurological changes occur, is because it is only
possible to examine the brain post mortem.
The brains immune system consists of brain MP, which includes microglia. In
neurodegenerative diseases, like Parkinson’s disease, an inflammatory state occurs as
a result of degeneration of neurons. First microglia in the affected area receives
36
signals from the degenerating neurons and is hereby activated. Afterwards the
microglia secrete chemotactic cytokines e.g. CCL2 which is believed to play a great
part in Parkinson’s disease.
The inflammatory chemokine’s recruits immune cells from the periphery immune
system, which helps to phagocyte the degenerating neurons. The chemokines, among
others, contributes to making the BBB more permeable to the periphery immune cells,
which thereby are able to migrate into the brain.
Normally the microglia returns to an inactive state, when the substance causing the
inflammation is phagocytised, but in Parkinson’s disease a chronic state of
inflammation occurs as a result of continued activation of microglia.
It is unknown why a constant degeneration of neurons occurs, causing the on-going
activation of microglia, but it is believed that the above mentioned genetic and
environmental factors plays a crucial part.
Because the pathogenesis of Parkinson’s disease is still unknown it is difficult to find
the optimal treatment.
37
Outlook
Many aspects about Parkinson’s disease are yet unclear, and as explained above it is
not clarified, why the disease develop and no one has yet found the most ideal
treatment. Many theories exist and a lot of research is currently made in these areas. A
big step in finding the cause of Parkinson’s disease is to know what comes first; the
degeneration of the dopaminergic cells or oxidative stress, which is much like the
proverb about the chicken and the egg. The exact connection is still unknown, and the
same is obtained for a lot of other factors regarding Parkinson’s disease.
In the following we have selected some possible treatments and diagnostic, which are
currently researched in; stem cells, biomarkers and cytokines.
Biomarkers can revolutionize the screening of a disease in the wide population.
Biomarkers can e.g. be a high level of a certain protein connected to a specific disease.
A recent study has found four peripheral biomarkers, which are believed to be in
relation to Parkinson’s disease. These biomarkers opens the possibility for an earlier
diagnosis and thereby a sooner beginning of the treatment. (42) (43)
The inflammatory condition that is seen in patients with Parkinson’s disease can
possibly be reduced if the peripheral immune system does not respond to the
microglia’s ”call for help”.
One might imagine that by making an antagonist to CCL2 the peripheral immune
cells would not respond to the microglia’s ”call for help”, which would imply that the
peripheral immune system would not, in the same way, migrate into the brain and this
would result in a decreased inflammation compared to what normally is seen in
Parkinson’s disease. Because the binding of CCL2 to CCR2 is believed to have an
influence on the migration of peripheral immune cells, an antagonist would possible
reduce the number of migrating periphery immune cells into the brain and thereby
reduce the extent of phagocytosis of the dopaminergic neurons.
It is important to clarify the percentage role of the periphery immune cells in the
inflammatory condition that is seen in Parkinson’s disease. In relation to this it must
38
be considered if an oppression of these periphery immune cells has a useful effect. If
the peripheral immune cells only have a small role in the inflammatory condition in
the brain, it would not be advantageous to reduce the effect of these cells, since this
would result in a decreased effect of the peripheral immune system in general and
only have a small influence on the inflammatory condition in the brain.
Furthermore studies with animals suggest that a decreased activation of microglia can
be gained by using non-steroidal anti-inflammatory drugs, which thereby reduce the
neural damage. (47)
The abovementioned research areas are only a small fraction of the currently research.
Because of the obscurity within Parkinson’s disease researchers are interested in
clarifying the many aspects of the disease, finding the ideal treatment and maybe even
a cure. Within the last few decades several theories about Parkinson’s disease have
been made and the researchers have come significantly closer to an answer of the
many questions regarding the disease. However much is still unknown and therefore a
lot of research is still necessary in the future.
39
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