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TOP
The prefrontal cortex of the Göttingen minipig brain defined by neural
projection criteria and cytoarchitecture
Jacob Jelsing
Research Laboratory for Stereology and Neuroscience,
Copenhagen University Hospital, Bispebjerg, Copenhagen,
Denmark
Recently, the debate about the nature and characteristics of the prefrontal
cortex (PFC) in non-primate species has resumed. If specific subregions of
the PFC are unique to primates, then its role in cognition
and behaviour,
and its contribution to disease, can be studied only in these species.
Considering the dimensions and large nuclei of the gyrencephalic pig brain,
which is comparable to that of humans in gross anatomy, growth and
development, we believe pigs may potentially prove useful as an animal model
of human frontal lobe function and dysfunction. However, there is to date no
available data regarding the localisation of
the PFC in the pig brain.
In an attempt to delineate the PFC in the Göttingen minipig brain the
distribution of reciprocal thalamocortical projections was investigated
using anterograde and retrograde axonal tracing techniques and evaluated in
relation to the specific cytoarchitectonic organization. Tracers were
visualized using standard immunohistochemistry or evaluated in vivo using
manganese (Mn2+) as an MRI paramagnetic tracer. Injections directed towards
the mediodorsal thalamus labelled the medial and rostral pole of the frontal
lobe as well as anterior cingulate, anterior insular and dorsomedial frontal
cortices. Subsequently,
the reciprocity and specificity of these projections
was confirmed from injections into the outlined cortices.
Although, the granular layer IV characteristic of primate PFC was not
evident our data suggests that the
pig have a structurally divided
prefrontal cortex that includes not only features of the medial and orbital
areas in primates, but also some features of the primate dorsolateral
prefrontal cortex.
TOP Neuron volume in
the human neocortex
Anette.K. Stark1, Dorte P. Pelvig1, Henning Pakkenberg1, Morten Møller2
and Bente Pakkenberg1
1 Research Laboratory for Stereology and Neuroscience, Bispebjerg
University Hospital, Copenhagen, DK
2 Institute of Anatomy, Panum, University of Copenhagen, DK
The average life expectancy has risen dramatically since the turn of the
last century. Lengthening of lifespan has little merit if the quality of
life is not preserved, and in the elderly the decline in memory and
cognitive abilities is of great concern. With aging we see a small decrease
in the total number of neocortical neurons of 9.8% from age 20 to age 90
years, but a gender difference, where men have 16% more neocortical neurons
than women.
The aim of this study was to determine the mean volume of neuronal
perikaryon and nucleus in human neocortex according to age and gender.
We used a stereological method, the optical rotator, to get an estimation of
the three-dimensional size of the neurons in tissue that was isotropically
rotated.
The post-mortem material consisted of brains from 19 women average age 63.9
years (18-93 years) and
21 men average age 51.9 years (19-87 years).
We have preliminary results of this study; women have a mean neocortical
neuron perikaryon volume of
1364 mm3 and men have a mean neocortical neuron
perikaryon volume of 1574 mm3 a significant gender difference of 15%
(p=0.02). Women have a mean neocortical neuron nucleus volume of 264 mm3 and
men have a mean neocortical neuron nucleus volume of 273 mm3. Women have a
decrease in neocortical neuron perikaryon volume by 355 mm3 from the age of
20 to the age of 80 years. Men have a decrease in neocortical neuron
perikaryon volume by 331 mm3 from the age of 20 to the age of 80 years.
Women have
a decrease in neocortical neuron nuclear volume by 29 mm3 from
the age of 20 to the age of 80 years.
Men have a decrease in neocortical
neuron nuclear volume by 48 mm3 from the age of 20 to the age of
80 years.
None of the age differences were statistically significant. These data are
not corrected for in-between gender age and bodysize differences.
In conclusion our preliminary data have shown no difference in neuronal
volume decrease with aging and
no gender difference in neuronal nuclear
volume, but for perikaryon volume the gender difference shown
in studies of
total neocortical neuron number is replicated, men have a 15% larger
neocortical perikaryon volume than women.
TOP
Neocortical cell number in the common minke whale (Balenoptera
acutorostrata) brain
Nina Eriksen & Bente Pakkenberg
Research Laboratory for Stereology and Neuroscience, Bispebjerg Hospital,
Bispebjerg Bakke 23,
DK-2400 Copenhagen NV
Cetacean brains are among the least studied mammalian brains. Most research
has been carried out on odontocete physiology and anatomi, while the only
research on mysticete brains has been done on general morphology. Cetaceans
have the largest brain in the Animal Kingdom, but a low Encephalon Quotient
(EQ). Encephalisation is a phenomenon that occurred only in the mammalian
evolution, where brain size is supposed to increase with increasing body
mass. The cetacean brain is very different from the terrestrial mammalian
brain in many ways. Like the rest of the whale body, it posses several
traits that show adaptation to water. The migration of blowhole from frontal
to parietal has changed the shape of the brain and the distribution of the
cranial nerves.
This is the first attempt to estimate the neocortical cell number on any
species of cetacean - here we use the common minke whale as a model of all
mysticetes. Five brains from mature whales were used. Each hemisphere is cut
in 2.5 cm slabs using Cavalieri’s method, which is based on unbiased
principles for estimating volume of an object. Using a random start the
object is cut into parallel sections of equal thickness. The product of the
distance between the sections and the total cut area of the same side of
all
sampled sections provide an estimate of volume. Neuron and glial numbers are
counted using the optical disector, which is a stereological probe for
sampling objects according to their number.
A mean number of approximately 14x109 neurons and 10x1010 glial cells were
found in neocortex.
This is 2/3 of the human neuron number (19-23x109), but
14 times that of rhesus monkeys (1x109) and
900 times that of mice
(1.5x106).
TOP
Early induced stress and the consequences on the total neuron number
in the
hippocampus of mus musculus domesticus
B.S. Katrine Fabricius, Faculty of Science, August Krogh Institute,
University of Copenhagen1,
Research Laboratory for Stereology & Neuroscience2,
Bispebjerg Hospital & Department of Veterinary Pathobiology, laboratory animal science3,
the royal
veterinary and agricultural university., 2: Professor dr.med. Bente Pakkenberg 3:
Assistant Professor Thomas C. Krohn.
The aim of the project is to investigate whether experimentally
early-induced stress by maternal separation in critical developmental stages
in the house mouse, mus musculus, causes behavioural differences and/or
neuron cell loss in the mouse hippocampus.
Recently, researchers have found that maternal separation or other pre- or
postnatal stress related experiences can change the feeding behaviour, cause
high anxiety levels, provoke fear responses and show reduced learning
impairments in both the rat and the mouse. Furthermore, a gender difference
has been found in both rodent types, where maternal separation causes males
to show a higher anxiety level than (diestrous) female.
Two different maternal separation models were used; in the first model the
pups were removed from the mother for a minimum of 3 hours per day from
postnatal day (PND) 1-14. In the second model the pups
were removed from the
mother at PND 9 for 24 hours. In the control litters, the pups went with the
dam
at all time undisturbed. The models were performed in both males and
females. When the pups reached adulthood, the level of stress was measured
with two behavioural tests; an open field test and an elevated plus maze
test. The Barnes elevated platform maze were used to test for any damage in
the hippocampus.
The maze is equivalent to the Morris water maze, and thus
tests for spatial learning and memory.
The mice were sacrificed and the total number, regional volume and density
of neurons will be estimated
in the hippocampus using the neuronal optical
fractionator design.
TOP
Linking Personality to Brain Neurotransmission in Animal Models
Nanna Marie Lind, DVM.
Department of Experimental Medicine, Panum Intitute, University of
Copenhagen. nml@cnsr.dk
This talk starts with definition of personality in humans, and addresses the
possibility of modelling personality in experimental animals. In particular,
the attempt to model the Novelty Seeking (NS) temperament trait (Cloninger
et al. 1993) in Göttingen minipigs will be presented.
1) The behavioural response to a novel object was recorded in 12 Göttingen
minipigs (test described in Lind et al. 2005a). Subsequently, the same
individuals underwent PET scans with the dopamine D2/3 specific radiotracer
[11C]raclopride, before and after injection of 1 mg/kg amphetamine. We
hypothesised that changes in striatal [11C]raclopride
binding in the amphetamine-challenge paradigm (as described in Lind et al.
2005b) would correlate with the behavioural response to novelty, and that
the correlation would be highest in the ventral striatum, a region linked to
the rewarding properties of amphetamine and other psychostimulants. The
propensity to explore an unfamiliar object (i.e. duration of object contact)
was elevated in pigs with high amphetamine-induced dopamine release,
primarily in the male pigs.
The results confirm a prior identical study in healthy men (Leyton et al.
2002), thereby supporting the behavioural response of minipigs to a novel
object as a possible animal model of the human novelty seeking temperament
trait (Lind et al. 2005c).
2) In the same cohort of pigs, learning rate was measured in a Go/No-Go test
(test described in Moustgaard et al. 2005). Informal observation of the pigs
suggested that fearful animals learned the GNG task faster than less fearful
animals. We therefore hypothesised that learning of the GNG task in minipigs
could be predicted from their response to novelty, an index of
“temperament”. The results showed that pigs with
low behavioral response to
novelty learned the GNG task faster than did pigs with a strong behavioural
response to novelty, indicated by the tendency to approach novel objects (i.e.
number of contacts).
We hypothesise that the critical factor in this
relationship is difference in emotional reactivity rather than difference in
motivation for exploration (Lind & Moustgaard 2005).
The duration of object contact during the Novel Object test has properties
similar to the NS temperament trait in humans. Future
investigations should address whether the number of contacts with a novel
object could be an animal homologue of other human temperament traits, such
as the Harm Avoidance (HA) trait, and if correlations exists between HA and
other neurotransmitter systems.
In summary, The Novel Object Test for Göttingen minipigs provides the means
to model the human NS temperament trait. This pig model could prove valuable
in future attempts to establish and model behavioural correlates to brain
neurotransmission. This would be of particular relevance to research of the
pathophysiology of neuropsychiatric disorders.
References:
Cloninger CR et al. (1993). Arch Gen Psychiatry, 50:975-990
Leyton M et al. (2002). Neuropsychopharmacology, 27:1027-1035
Lind NM et al. (2005a). Scand J Lab Anim Sci , in press.
Lind NM et al. (2005b) Brain Res Bull, 65:1-9
Lind NM et al. (2005c) Behavioural Brain Research, accepted
Lind NM & Moustgaard A (2005) Neural Plasticity, accepted
Moustgaard A (2005). Behavioural Processes 2005; 68: 97-102
TOP
Functional changes of synaptic memory in a double
transgenic mouse model
of Alzheimer's disease
Morten Skovgaard Jensen
The Synapse Project,
Institute of Anatomy, University of Aarhus
The â-amyloid hypotheses suggest that an overproduction of â-amyloid and its
aggregation in senile plaques is the root cause of Alzheimer’s disease.
Neurotoxic effects of â-amyloid and the consequent loss of nerve cells could
explain the memory deficits and the cognitive decline that characterize the
disease. Newer data indicate that APP processing and the production of
â-amyloid is involved in regulation of excitatory synaptic transmission.
â-amyloid may therefore interfere with synaptic information processing and
memory storage in the brain long before the loss of neurons is significant.
I order to test the latter we investigated synaptic transmission and the
induction and the storage of synaptic memory at the age of tree month in
normal mice and transgenic mice producing human â-amyloid. The transgenic
mice express and produce a mutated form of human APP (APPswe) and the
ã-secretase presenilin 1 (PS1) known to course in an early onset of Familiar
Alzheimer’s disease.
The production of â-amyloid in the transgenic mice is
elevated from the birth and accumulates over time. The
aggregation of â-amyloid into placks occurs at about the 9 month of age.
Synaptic transmission was evaluated by extracellular recording from the CA1
area in the in vitro hippocampus slices preparation. Synaptic potentials
were evoked by stimulation of the schaffer-collaterals and population field
responses were recorded in the stratum radiatum. Basic synaptic transmission
was evaluated by its input-output relationship and was not different when
comparing the control mice and the transgenic group of mice.
High frequency stimulation was used to induce synaptic long-term
potentiation (LTP) in the synapses.
In normal mice this consist of a
transient phase of potentiation which decayed in about half an hour and
a sustained phase lasting more than tree hours. The amplitude of the transient
LTP was reduced whereas
the amplitude and saturation level of the sustained
form of LTP were not different between the groups.
Post induction test
stimulation, using a paired pulse protocol known to destabilize the
sustained phase,
was found to be more efficient in reducing the sustained
LTP in slices from the transgenic animals.
The transgenic mice show therefore defects in both the induction and the
storage of synaptic potentiation month before â-amyloid is aggregating as
placks. The data suggest that an overproduction of â-amyloid directly
affects the mechanisms underlying potentiation of excitatory synaptic
transmission, and consequently may interfere with acquisition and storage of
memory.
TOP
Structural Changes in Synapses in the Hippocampus of a
Transgenic Model
of Alzheimer’s Disease
Mark J. West and Georg Bach Jensen
The Synapse Project,
Anatomy, University of Aarhus
We are studying potential changes in the numbers, sizes and
types of synapses in the striatum radiatum
of the hippocampal CA1 region of
a transgenic mouse model of beta-amyloidosis that produces amyloid plaques
that are similar to the type observed in Alzheimer’s Disease (AD). The study
is designed to test
the extended amyloid hypothesis of AD, which suggests
that one of the initial events, in the cascade of pathological events that
leads to AD, is a change in synapses caused by the beta Amyloid protein (Abeta)
that is found in abundance in AD brains. We have earlier reported that the
stratum radiatum of the CA1 region is particularly vulnerable to AD
pathology in humans. We are presently in the process of developing the
methodology and equipment required for this analysis. Preliminary data on
changes in the number of
CA1 neurons, which will form the background upon
which potential changes in synapses are interpreted, indicate that there are
no significant differences in the number of CA1 neurons at 12 months of age.
This observation indicates that 12 month old transgenic mice are optimal for
our studies in that significant amyloid pathology is present at this time,
but there is no sign of the more advanced pathology, i.e. neuron loss change
in volume.
TOP
Of Mice and Men: MicroPET imaging of beta amyloid plaque in
transgenic mice
Dirk Bender
PET Center, Århus Sygehus
A cardinal pathological characteristic for AD is an increased
formation and deposition of plaques in distinguished brain regions. Recently
several PET tracers imaging plaques were developed. A new development in PET
is scanning of mice and rats in dedicated small sized PET scanners (MicroPET).
It is therefore possible to scan transgenic mice (“AD mice”), which develop
plaques like human AD patients. The study will combine the three tools
consisting of a pre-clinical animal model, the PET “in vivo autoradiography”
and an imaging reagent for AD related plaque to study the suitability of a
small animal
PET scanner (MicroPET) for detection of plaques in transgenic
“AD mice”, to follow the age dependent formation of these plaques in the
transgenetic mice and to evaluate the suitability of a PET tracer for
imaging plaque deposition. The outcome of the study will eventually result
in valuable information on the potential of this diagnosis approach in human
AD patients.
Preliminary results obtained with C-11 labeled PIB in mice are presented.
TOP
Perfusion Weighted Magnetic Resonance Imaging in Alzheimer’s
disease
and Mild Cognitive Impairment: Preliminary Results
Louise Gyldensted, MD
Neuroradiologic Research Unit, Aarhus University Hospital
Alzheimer’s disease (AD) is associated with several vascular
risk factors pointing to a microvascular insufficiency of gray and white
matter in AD pathogenesis [1, 2]. Cerebral perfusion measurements in AD with
PET and SPECT show hypoperfusion in: Hippocampus, temporo-parietal and
frontal cortices [3, 4].
We hypothesize that AD patients have microvascular
disturbances of subcortical white matter and that perfusion weighted (PW)
MRI with subsequent coregistration can serve as a tool for delineating
microvascular perfusion abnormalities across AD patients and thus better
characterize disease progression. We studied a group of AD and MCI patients
(n=9) and healthy age matched controls (n=18) with contrast bolus PWI MRI.
Our initial findings support our hypothesis.
[1] de la Torre,JC: Neurosci.Behav.Rev.
18:397-401, 1994; [2] Brown,WR: Ann.NYAcad.Sci. 903:39-45, 2000; [3]
Friedland,RP: JCAT 7:590-98, 1983; [4] Waldemar,G:
Cerebrovasc.BrainMetab.Rev. 7:89-130, 1995;
TOP
The ferret and the microPET
Aage Kristian Olsen, DVM, Ph.D.
PET-center, Aarhus University Hospital, Noerrebrogade 44, 10c, Dk-8000
Aarhus
We use mainly rodents (mice and rats) in our microPET
scanner. However, we like to use the ferret as
a supplement in some brain
studies. The weight of the rat brain weighs approximately two grams, and
therefore only small amounts of blood can be sampled. The ferret brain
weighs 7-10 grams, and the body weight is approximately 1-1½ kg. Therefore,
we can get more details when scanning the brain and we can sample more blood
in ferrets compared to rats. Furthermore, the cerebral cortex is rough in
the ferret brain as in humans. However, there are also some disadvantages in
using the ferret as a laboratory animal: Compared to other rodents, ferrets
are only rarely used, and therefore much information about ferret
biology is
not available. Furthermore, we have some practical problems with the
anesthesia of ferrets
and the surgical implantation of catheters. Also the
blood glucose seems to vary during the anesthesia.
We are at the moment
trying to solve these problems, and furthermore, we are creating a ferret
brain atlas. The uptake of 11C-raclopride in the ferret basal ganglia will
be shown during the presentation.
TOP
GABAA receptor partial agonists: mechanism of receptor activation
Uffe Kristiansen1, Carolina Cannillo1,2, Martin
Mortensen1, and Flemming F. Johansen2
1 Dept. of Pharmacology, Danish University of Pharmaceutical Sciences
2 Molecular Neuropathology Group, Institute of Molecular Pathology,
University of Copenhagen
The GABAA receptors are ligand gated chloride channels.
Activation of the receptors in most cases results
in inhibition of neuronal
activity. Partial agonists are defined by their limited ability to activate
a receptor (limited efficacy), in this case implying a reduced maximum
chloride flux. We have studied activation of GABAA receptors by a number of
partial agonist on the single-channel level in order to extract information
on their mechanism of receptor activation. Briefly, the ability to activate
the receptor correlates with the ability to stabilize the receptor in the
open conformation, while the flux of chloride ions through the open
conformation is similar regardless of the efficacy.
Partial agonists compete with full agonists for the receptor binding sites.
When a full agonist is displaced
by a partial agonist, the probability of
finding the channel open is reduced to a lower level determined by
the
partial agonist. In this way, the partial agonist exhibits antagonist
properties. It is normally assumed that the potencies of a partial agonist
with regard to its agonist and antagonist properties are equal, because both
are mediated through binding to the same sites. Our experiments suggest that
this is not always the case. Using a generally accepted kinetic model for
the receptor together with rate constants extracted from our single-channel
experiments, we have simulated the interaction of full and partial agonists
and found that the antagonist potency of a partial agonist would be expected
to be higher than its agonist potency.
In other words, due to the inherent activation mechanism of the GABAA
receptor, the antagonist properties of partial agonists would be effectuated
at lower concentrations than the agonist properties.
This may also be true
for other ligand gated ion channels with similar activation mechanisms.
TOP
Protein tyrosine phosphatase signaling in the nervous system
Jan Sap
Cell regulation Laboratory, Institute of Molecular
Pathology, University of Copenhagen
Specific and reversible phosphorylation on tyrosine residues
is a major mechanism of intracellular signal transduction. The number of
protein tyrosine phosphatases (PTPs) in the genome rivals that of tyrosine
kinases, indicating important regulatory roles for this enzyme family.
Medically, PTPs are relevant as tumor suppressors and as potential
pharmacological targets, yet these possibilities are only beginning to be
studied. There is increasing evidence that PTPs play important functions in
brain development and synaptic regulation. We will illustrate the possible
biological functions of these enzymes based mostly on studies with a
ubiquitous PTP, RPTPá. Studies in PC12 cells show that RPTPá can regulate
neurite outgrowth in response to NGF, FGF, and EGF in complex ways. Genetic
studies in mice reveal unexpected roles in radial neuronal migration, and
synaptic plasticity. We will also briefly discuss plans to search for PTPs
that regulate neurotrophin sensitivity and neuronal survival
TOP
Agonists and Antagonists: do they bind to the same site?
Autoradiographic study of the abundance of agonist/antagonist sites
for histamine H3 and dopamine D2/3 receptors.
Luciano Minuzzi
PET Center, Århus Sygehus
Introduction:
G-protein coupled receptors are a large family of receptors that mediate
transmembrane signal transduction in response to the binding of many
neurotransmitters, peptides, hormones and autacoids. Agonist binding changes
the G-protein complex, increasing the activity of enzymes which in turn
regulate second messengers. When there is no dissociation of the G-protein
complex, agonists bind with high affinity to the receptor. Upon agonist
binding, the G-protein is activated and the receptor converts to a low
affinity state for agonists. Antagonist binding is unaffected by G-proteins.
Since only the agonist fraction is functional, endogenous neurotransmitter
and drug efficacies are determined by agonist potency on the receptors in
the high affinity state.
On the basis of various studies in vitro and in vivo, the
fraction of dopamine receptors in the high affinity state has been estimated
to be 40%. However the function of the receptors in the low affinity state
is uncertain. In order to obtain a better understanding of dopamine D2/3
agonist binding, we carried a series
of experiments to test the sensitivity
of [3H]NPA (D2 agonist) and [3H]raclopride
(D2/3 antagonist) in rat brain cryostat section to LSD
competition, and their saturation binding parameters. In another study, we
used quantitative autoradiography to measure the saturation binding
parameters for [3H]N-M-HA (H3 agonist) and [125]IPP
(H3 antagonist) in several regions of cryostat sections from
brain of Landrace pigs.
Methods: Twenty m m-thick cryostat
sections from rats (Lewis/Mol strain) and four adult female
Landrace-Yorkshire pigs were used in the experiments. Rat brain sections
were incubated with 1 nM [3H]NPA or 2 nM [3H]raclopride
along with LSD at final concentrations ranging from 10-13 M to 10-4
M. Addition of 100µM guanylyl-5'-imidodiphosphate (Gpp(NH)p) induced
dissociation of the G-protein complex changing the dopamine receptor to a
state with low affinity for agonists. To assess comparable processes in the
case
of histamine H3 receptors, saturation binding experiments
were carried out in pig brain sections incubated with [3H]N-M-HA
or [125]IPP. One-site and two-site models were fitted to the
displacement series and
the saturation binding experiments for the
calculation of the IC50s, Bmax and Kds
(Sigma Plot 8.02).
Results and Discussion: Preliminary results will be presented and
discussed in the context of PET competition models.
TOP
2,3-Dihydroxybenzoic Acid Attenuates Kanamycin-induced Volume
Reduction
in Mouse Utricular Type I Hair Cells
Stig Å. Severinsen1, Mette Kirkegaard2, Jens R. Nyengaard1
1 Stereology and Electron Microscopy Research Laboratory, Institute of
Clinical Medicine, University of Aarhus, Denmark
2 Center for Hearing and
Communication Research, Karolinska University Hospital, Stockholm, Sweden
Sensory hair cells are delicate structures which can be
damaged by exposure to aminoglycosides.
The aim of this study was to
estimate cellular quantitative changes in the utricular balance organ of
mice
to assess a possible damage to cells after treatment with the
aminoglycoside kanamycin. Antioxidant therapy with 2,3-dihydroxybenzoic acid
(DHB) was also introduced to attenuate the cell damaging effect
of kanamycin.
Mice were injected twice daily with either saline, kanamycin
or kanamycin + DHB for 15 days and perfusion fixed three weeks after last
injection. Total volume of the utricle as well as number of hair and
supporting cells was estimated using stereological methods. Volume of hair
cells (HCI & HCII), supporting cells (SC)
and neural elements were estimated
on electron micrographs.
Mean volume of the utricle was 3.65 · 10-3 mm3 (0.07) in control animals and
3.50 · 10-3 mm3 (0.06) in kanamycin + DHB-treated animals. In animals
treated with kanamycin the volume was reduced to 3.14 ·
10-3 mm3 (0.04) which was significantly different from the two other groups.
Although hair and supporting cell numbers remained unchanged regardless of
treatment, cell volume was significantly affected. Mean volume of HCI was
270 microm3 in control animals. Kanamycin-treated animals had a HCI mean
volume of 209 microm3 and were significantly different from kanamycin +
DHB-treated animals (P=0.04), which had a HCI mean volume of 286 microm3,
but only tended to be different from controls (0.09). Mean volume of HCII
and SC did not differ significantly between the groups (P0.05).
We did not
find any apoptotic cells (no caspase-3 reactivity).
This experiment demonstrates that two kanamycin-injections of 900 mg/kg/day
do not alter the number
of sensory hair cells in the utricular macula of
mice. However, a significant decrease in volume of the utricular sensory
epithelia indicates that the tissue was affected by the kanamycin treatment.
Mean volume of HCI and total volume of the utricular macula did not decrease
in animals co-treated with
the antioxidant DHB, indicating that antioxidant
therapy could be a successful way of otoprotection against
aminoglycoside-induced inner ear damage.
TOP
EAE, Cannabinoids & Cytokines
Henrik Hasseldam
Molecular Neuropathology Group, University of Copenhagen, Copenhagen,
Denmark
Molecular Neuropathology Group, Institute of Molecular
Pathology, University of Copenhagen
Cannabinoids are increasingly in focus as a possible symptomatic treatment
for Multiple Sclerosis (MS). MS patients describe a relief of pain, tremor
and paralysis when smoking marihuana. These effects are supported by
clinical studies showing minor overall improvements in the general
well-being of MS patients.
With this in mind we have established an animal model, Experimental
Autoimmune Encephalomyelitis (EAE), which shares substantial clinical and
pathological characteristics with MS. In order to explore the effects
of
symptomatic cannabinoid treatment on the development and severity of EAE, we
set up a treatment regimen which included daily i.p. injections (2.5 & 5.0
mg/kg/24h) with the synthetic cannabinoid analogue WIN-55,212-2, initiated
at symptom debut.
The results clearly demonstrated an effect on the neuropathological level,
with regards to a reduction in
the amount of inflammatory cells and degree
of demyelination. This was especially true for the high WIN-55,212-2
concentration. Furthermore, we wanted to test whether the cannabinoid
treatment, had an influence on the concentration of cytokines in the CNS. We
focused on the pro-inflammatory cytokines TNF-á and IFN-ã, and actually
observed a significant increase following cannabinoid treatment. This is
somewhat surprising in the sense that both cytokines have been shown to be
toxic to neurones and oligodendrocytes, and to augment the inflammatory
activity in the CNS. On the other hand, the presence of these cytokines at
later time points has been shown to induce programmed cell death in T-cells
and promote remyelination, thus reducing the disease burden in animal models
of MS.
TOP
Protein nitration in organotypic culture models of Parkinson's disease
Trine R. Larsen1,2, Ann-Sofi Peterson3, Kenneth Caidahl3,
Peter Roepstorff2, Jan Bert Gramsbergen1.
1 Anatomy and Neurobiology, Institute of Medical Biology, University of
Southern Denmark.
2 Biochemistry and Molecular Biology,
Faculty of Science and Engineering, University of Southern Denmark.
3 Clinical Physiology, Sahlgrenska University Hospital,
University of Göteborg, Sweden.
Objectives Nitrated proteins
have been detected in nigral Lewy bodies of Parkinson’s disease (PD)
patients, but it is unclear whether protein nitration is a critical event in
the degeneration of dopaminergic neurons. Here we quantified protein-bound
3-nitrotyrosine (3-NT) in toxin-treated organotypic, mouse ventral
mesencephalic (VM) cultures and correlated this with dose-dependent dopamine
depletions.
Materials and Methods Organotypic VM cultures of embryonic day 12
mice (CBL57/bl6) were grown for 3 weeks and then treated (acute: 24 or 48
hours, chronic: 3 weeks) with the peroxynitrite donor 3-morpholino
sydnonimine (SIN-1), 1-methyl-4-phenylpyridinium (MPP+) or rotenone, or
co-treated with MPP+ and the nitric oxide synthase inhibitor N-nitro
L-arginine methyl ester (L-NAME). Protein-bound 3-NT was quantified using a
novel gas chromatograpy/negative chemical ionisation tandem mass
spectrometry technique.
Tissue dopamine and medium 3,4-dihydroxyphenylacetic
acid (DOPAC) levels were assessed by HPLC with electrochemical detection.
Results Acute SIN-1 or MPP+ treatments caused dose-dependent protein
nitration reaching a maximum
of 8-fold by 10 mM SIN-1 or 2-fold by 10 µM
MPP+. Chronic MPP+ or rotenone treatments caused maximum protein nitration
by 1 µM (2-fold) or 50 nM (4-fold) respectively. Co-treatment with L-NAME
(300 µM) prevented protein nitration by acute MPP+.
Conclusions Protein nitration and dopamine depletion by MPP+ or
rotenone treatments are closely related, but inhibition of protein nitration
did not prevent dopamine depletion in the acute MPP+ model. Whether protein
nitration plays a role in dopamine
depletion and neuronal cell loss under chronic conditions remains
to be
investigated.
TOP
Does neuroglobin protect neurons after transient cerebral ischemia?
C. Hundahl1+; J. Kelsen2+; L.C.B. Rønn3; and J.R.
Nyengaard4
1 Department of Zoophysiology, Institute of Biology; 2Water and Salt Research
Centre; 4Stereology Research Lab, University of Aarhus; and 3NeuroSearch
A/S, DK-2750 Ballerup, Denmark
+These authors contributed equally to this work.
Introduction: Brain ischemia due
to stroke is a major cause of brain injury leading to disability and death.
Neuroglobin (Ngb) is a novel characterized heme globin primarily expressed
in retinal nerve cells and at very low levels in endocrine active brain
regions in vertebrates. When artificially over-expressed, Ngb can reduce the
infarct size in a transient model of Middle Cerebral Artery occlusion (MCAo)
in rats. Hence modulation
of Ngb expression may have a significant potential
in new treatment strategies of stroke. However, it is not known whether a
protective effect is present at physiological levels of Ngb.
Materials and methods: Thirteen Spontaneously Hypertensive Rats (SHRs)
subjected to either ninety minutes of transient MCAo or sham surgery on the
right side were euthanized on day eight after surgery.
We used free-floating
immunohistochemistry with polyclonal and monoclonal antibodies directed
against
Ngb and a neuronal marker (NeuN), respectively. Design-based
stereologic quantification of Ngb and NeuN positive cells in the striatum of
all animals was performed using the optical fractionator.
Results: The ischemic damage affected primarily striatal structures
and constituted 16.6 mm3 in the MCAo rats and was not present in the sham
rats (p<0.005) . We a significant lower number of Ngb positive neurons in
MCAo (n=7) rats compared to sham (n=6) ( p>0.005). These findings suggest
that Ngb does not have a protective effect at physiological levels.
Furthermore, Ngb expression is mainly confined to the hypothalamus and
amygdale, which is not directly affected by the ischemic lesion. The level
of expression was also lower in the ischemic hemisphere when compared to the
non-ischemic hemisphere of MCAo rat brains.
Conclusion: Ngb, expressed at
physiological levels after transient MCAo in SHRs, does not provide
protection against ischemic injury.
Support by: EU (grant QLG3-CT-2002-01548). No conflict of
interests.
TOP
Temporary cerebral ischaemia upregulates the 5-HT(1) receptor
in the middle cerebral artery
Jacob Hansen-Schwartz1,2, Flemming Fryd Johansen2, Lars
Edvinsson1,3
1 Department of Experimental Research, Glostrup University Hospital,
Glostrup, Denmark
2 Institute of Molecular Pathology, University of Copenhagen, Copenhagen,
Denmark
3 Department of Internal Medicine, University of Lund, Lund, Sweden
Stroke gives rise to an immediately infarcted area surrounded
by an area of critically perfused neural tissue ('area at risk'); the fate
of this tissue being dependent on increased perfusion subacutely.
Using
experimental temporary occlusion of the middle cerebral artery (MCA) in the
rat we studied the possible upregulation of the 5-HT(1) receptor in the MCA.
Experimental stroke: The MCA of an anaesthetised rat was exposed and
temporarily occluded with a hook for 120 mins. Afterwards the rat was
revitalised.
In vitro pharmacological examination: After two days the rat
was killed and the MCA's were harvested and suspended on wires for
measurement of contractile properties. Selective 5-HT(1) agonists and
antagonists were used to study contractile responses. Contractile responses
to 5-HT(1) stimulation were clearly enhanced after stroke (compared to the
contralateral MCA). This receptor upregulation implies an increased
sensitivity towards
the endogenous agonist 5-hydroxytryptamine. It has
earlier been shown that temporary occlusion leads to upregulation of a
contractile ET(B) receptor. The combined upregulation of contractile
receptors may further reduce the blood supply to the area at risk, thus,
increasing the infarct size with subsequent increased neurological
disability.
TOP
D-Amphetamine promotes cognitive recovery and physical therapy
improves
fine motor rehabilitation in a rat embolic stroke model
Rune Skovgaard Rasmussen, MA; Karsten Overgaard, MD;
Elisabeth S. Hildebrandt-Eriksen, PhD; Gudrun Boysen, MD, DMSc.
Neurovascular Research Laboratory, University Hospital of
Copenhagen, Teilum Building
Background and Purpose: The purpose of this study was to examine
effects of d-amphetamine and physical therapy alone or combined in terms of
fine motor performance, gross motor performance and cognition after middle
cerebral artery thromboembolization in rats.
Methods: Seventy-four rats underwent surgery and 59 were embolized in
the right carotid territory with
an embolus and randomly assigned to: 1)
SHAM (non-embolized, saline), 2) CONTROLS (embolized, saline), 3) D-AMPH (embolized,
d-amphetamine), 4) THERAPY (embolized, saline + physical therapy) and 5) D-
AMPH+THERAPY (embolized, d-amphetamine + physical therapy). Groups 3-5
received d-amphetamine treatment and/or physical therapy on days 1, 3, 5 and
7 after surgery. Animals from all groups were re-tested during days 21 to 28
after surgery.
Results: No differences in infarct volumes were observed between
groups of embolized animals. When evaluating performances on days 21 to 28
after surgery, SHAM and THERAPY had better fine motor performance than
CONTROLS (P < 0.05), while SHAM and D-AMPH achieved better cognitive
performance than CONTROLS (P < 0.05). No significant differences were
observed between any groups regarding gross motor performance.
Conclusions: After embolization physical therapy improved fine motor
performance and d-amphetamine accelerated rehabilitation of cognitive
performance.
TOP
Hypothermia accounts for a significant part of the neuroprotection
provided
by cannabinoids following cerebral ischemia.
Peter K. Bonfils, Jakob Reith, Henrik Hasseldam, and
Flemming Fryd Johansen*
Molecular Neuropathology Group, University of Copenhagen, Copenhagen,
Denmark
Cannabinoids have neuroprotective potentials, and the
expression of endocannabinoids as well as cannabinoid receptors are induced
after cerebral ischemia. They also induce hypothermia by lowering the
hypothalamic set point. We have investigated the significance of such
hypothermia in ischemic neuroprotection following systemic administration of
WIN 55,212-2, a synthetic cannabinoid receptor agonist. Results showed that
WIN 55,212-2 significantly reduced infarct volumes of rats subjected to
focal cerebral ischemia (middle cerebral artery occlusion) and significantly
decreased ischemic CA1 damage in rats subjected to global cerebral ischemia
(two-vessel occlusion). A significant (approximately 50%) part of this
neuroprotection was provided by WIN 55,212-2 induced hypothermia (33.7 1.1C
/ 34.9 1.6C), because prevention of hypothermia by maintaining body core
temperatures between 37.0-38.0C dissolved the neuroprotective effect into a
hypothermic component and an unidentified component. Finally, the ability of
WIN 55,212-2 to reduce levels of the proinflammatory cytokine IFN in the
infarcted hemispheres of rats subjected to focal cerebral ischemia required
hypothermia.
We conclude that cannabinoids are reliable candidates for drug induced
hypothermia and neuroprotection. These neuroprotective effects could provide
the basis for potential therapeutic uses of cannabinoids and/or
endocannabinoids in stroke.
TOP
GFP-transgenic mice to quantify hippocampal Inhibitory Interneuron
Loss
by stereology after fluid percussion Brain Injury.
B. M. Witgen1,2, J. Lifshitz3, J. R. Nyengaard1, M. S.
Grady2.
1 Stereology and Electron Microscopy Research Laboratory, Aarhus Denmark;
2 Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA;
3 Department of Anatomy and Neurobiology, Virginia Commonwealth University,
Richmond,VA.
Susceptibility of hippocampal neurons to human brain injury
can also be identified in experimental models. After lateral fluid
percussion brain injury in the mouse, design-based stereology confirmed a
uniform loss of 25-35% of all neurons in the four major subregions by one
week post-injury, which does not progress by
one month. Since excitatory and
inhibitory networks combine to
establish hippocampal function, the neuronal loss could be all excitatory
neurons, all inhibitory neurons, or a combination of the two populations.
To begin to address the nature of hippocampal neuronal loss after TBI, we have
taken advantage of GFP-GAD ( FVB-TgN(GadGFP)45704Swn) transgenic mice to
quantify the remaining population of somatostatin-positive inhibitory
interneurons in the hippocampus using combined
transgenic-fluorescent-stereology.
At one week, the brains of brain-injured
and uninjured control mice were processed for stereological quantification
of GFP-positive somatostatin-positive inhibitory interneurons in the hilus.
Uninjured control mice had 1140 (CV=0.06) somatostatin-positive interneurons,
whereas brain-injured mice 900 (CV=0.05).
In the hilus at one week after
injury, 21% of the interneurons are lost, but previous data shows a 34%
total neuronal loss. Consequently, the proportion of somatostatin-positive
inhibitory interneurons in the hilus increases after brain injury (9.9% to
11.9%), with potential functional implications. Further studies address the
remaining hippocampal subregions and other inhibitory interneuron
subpopulations.
TOP
Dynamic changes of the corticospinal tract after ischemic stroke
detected by
MR-fibertracking
Mette Møller1,2, Jesper Frandsen1, Grethe Andersen3, Dora
Zeidler1, Albert Gjedde1,2,
Peter Vestergaard-Poulsen
1 Center of Functionally Integrative Neuroscience1, Aarhus University,
PET-Center, Aarhus University Hospital
2 Department of Neurology3, Aarhus
University Hospital, Denmark
Introduction
The integrity and connectivity of nerve fiber tracts in the brain are
important in assessing plastic changes related to
functional recovery and to obtain prognostic information and formulate
future therapeutic strategies for stroke patients.
Previous imaging studies have shown that the adult brain after ischemia can
regenerate and compensate for motor deficits by plastic changes, though
demonstration of wallerian degeneration is associated with more permanent
neurological deficts.
To our knowledge, the integrity of the corticospinal tract in a longitudinal
study of stroke from the onset of the acute stroke and throughout
rehabilitation has never previously been documented in humans.
Using diffusion measurements (DTI) and computer based 3D axonal fiber
tracking algorithms in correlation with neurological motor scale we
hypothesized that changes in axonal integrity along the corticospinal tract
after stroke can be detected as reduction in fractional anisotropy (FA-index)
and that a sustained low FA-index is indicative of axonal loss and
correlates with poor motor outcome. Finally we speculated whether a
reduction of FA-index is associated with a low density ratio (ipsi/contra-lesional)
of the tracked corticospinal fibers.
Methods
Five right-handed patients with first-time subcortical ischemic infarcts and
motor deficits were tested with Medical Research Council (MRC) and
MR-scanned on day 1-4, 30 and 90 post-stroke. Serial DTI was performed using
a birdcage head-coil and double spin echo single shot EPI at 1.5 T,
employing 17 isotropically-distributed directions, and a b-factor of 1000
s/mm2, with additional b-factor=0 s/mm2 images.
Maximum gradient strength was 36 mT/m. 50 slices of 3.3 mm thickness were
acquired, covering a 24 cm FOV in a 128x128 matrix. TR/TE =17000/84 ms. The
sequence was repeated four times for a total 22 min acquisition time. The
eigensystem was calculated from the diffusion tensor in each voxel using
diagonalization. Fiber tracking was calculated with the FACT algorithm.
Results
The temporal evolution of the ratio (ipsi/contralesional) of FA-index was
unchanged (above 0.9) in the four patients with good outcome (MRC=4),
whereas a decline was seen in the patient with poor outcome (MRC=1.2) The
reduction of the FA-index was associated with a decreased intensity of the
affected cerebral peduncle in the FA-maps and a low density ratio of the
tracked fibers indicative of axonal loss.
Discussion
The novel findings seem to indicate an association between motor function
and reduction in both FA-index and density of the tracked fibers and that
these parameters appear to be sensitive of structural changes in the
corticospinal tract after stroke.
The data are preliminary based on five patients and inclusion of more
patients and a methological study on the connection between FA-index and the
fiber-tracking algorithm is planned.
TOP
In vivo studies of the biological activity of functionalised nanoparticles
designed
for drug delivery usage with focus on delivery to the CNS.
Sys Zoffman
Stereology and Electron Microscopy Research Laboratory,
Aarhus Denmark
Development and usage of nano-scale particles as carriers of
drugs have attracted considerable interest
as potential diagnostic and
therapeutic agents.
Positively charged polymeric molecules such as chitosan are able to
encapsulate negatively charged nucleic acid molecules. Furthermore, the core
can be encapsulated by a second type of polymer, which increases the
bloodstream circulation and potentially also the transfection activity.
Incorporation of small interfering RNAs (siRNA) into polymer-based
nanoparticles constitute a possible solution as a drug for treating
neurodegenerative disorders (Alzheimer’s disease and Pakinson’s diseases).
However, the blood-brain barrier (BBB) constitute a major obstacle in the
delivery of drugs to the central nervous system (CNS) as only small,
lipid-soluble molecules are allowed to pass the BBB.
It is therefore of
great interest to develop particles of nano-scale size, which can either
diffuse freely through the BBB or which are functionalised with ligands to
be transported with receptors present in the BBB. In this study we have used
an EGFP (Enhanced Green Fluorescent Protein) expressing mouse model
to test the delivery and activity of nasally administered chitosan-encapsulated
siRNA against the EGFP
mRNA to the mouse olfactory bulb and brain. We are
still facing technical problems and would like to know more about ligands
which are suitable candidates for crossing the BBB.
TOP
The decline of the Canadian
penny
Paul Cumming
Centre for Functionally Integrative Neuroscience, Aarhus
Introduction: Kinetics can mean the study of the effects of
forces on material objects, but in the context of chemistry and biochemistry
often refers to the laws governing rate of change in concentration of a
molecule. The mathematics underlying simple kinetic analysis is simple
indeed; One calculates the rate of change in the concentration of a chemical
product, or the rate of entry of a tracer into a metabolic compartment as a
function of the concentration of precursor and a first order rate constant.
While a biochemist can often keep the concentration of a precursor constant
in the course of the experiment, this
is not the case in PET studies; An
intravenous bolus of tracer results in a peak concentration occurring one
minute after injection, followed by a decline, which can be modeled as a
series of parallel first order processes, i.e. mixing in the blood,
distribution in body tissues, and metabolism or renal elimination.Plasma
pharmacokinetics is a well-established field for describing the fate of a
PET tracer or indeed any medication. The same approach can be used for the
study of diverse other systems, in which the concentration or abundance of
something (minke whales, coins in circulation) changes as a function of
time, according to some orderly law. Historians have used this approach to
attempt to reconstruct ancient economics, on the basis of large sample of
coins, collected as "hoards". Surprising, this can also be done at home, and
without a metal detector.
Methods: A sample of 1600 Canadian pennies was hoarded in a large
pickle jar during a period of two years. The sample was stratified by age,
and corrected for the annual output of pennies from the Royal Canadian Mint.
Population survival curves were calculated for the sample. In another study,
a hoard of 400 American coins in circulation in Canada was likewise examined.
Results: The survival curve of the Canadian penny revealed two phases;
an initial rapid phase, reflecting the intrinsic half-life of the coin (five
years). Some 40% of the coins disappeared with a much slower half-life,
close to 20 years. This may reveal the re-entry of hoarded coins into
circulation after a delay of two decade. The total hoard is calculated to
consist of 17,000,000,000 coins. The American penny survival curve revealed
an early growth function (Tau), indicating the delay for entry into
circulation. The population then declined with a half-life of 20 years,
suggesting that surviving pennies enter into the pool with slow turnover,
i.e. pickle jars.
TOP
Axonal lesion-induced reactive microglial proliferation and expression
of
ICAM-1
in IFN-Beta and vehicle-treated mice
Dissing-Olesen L., Dalmau I., Ladeby R., Nielsen H. H.,
Finsen B.
Medical Biotechnology Center, University of Southern Denmark,
Winsløwparken
25, DK-5000 Odense C, Denmark
Demyelination and axonal degeneration are prominent in
multiple sclerosis (MS). Interferon (IFN)- is known
to have a beneficial
therapeutic effect with relapsing remitting MS patients and is known to
reduce microglial proliferation in vitro and endothelial expression of the
intercellular adhesion molecule (ICAM)-1 in vivo in rodents.
A murine model for axonal degeneration was used to investigate if IFN-
reduces the axonal-lesion induced microglial reaction, which may contribute
to disease progression. Transection of the entorhino-dentate perforant
path-projection results in an anterograde axonal (Wallerian) and terminal
degeneration that induces a region-specific activation and proliferation of
microglia in the dentate gyrus. By applying double-label
immunohistochemistry for the macrophage antigen (Mac)-1 and the
proliferation marker bromodeoxyuridine, injected one hour prior to
euthanasia, we found that 25% of all lesion-reactive microglia were
proliferating 3 days post-lesion, at which time the microglial cell
population had increased 3-fold in lesioned mice compared to controls.
Interestingly, at this time 50% of all the lesion-reactive microglia
expressed the co-stimulatory molecule the ICAM-1. Fluorescence-double
labeling immunohistochemistry for Mac-1 and ICAM-1 demonstrated
co-localisation.
Treatment with human (h)IFN-beta (Rebif) had no effect on the microglial
morphology and no statistically significant effect on the total number of
reactive microglia, the number of actively proliferating microglia, or the
ones coexpressing ICAM-1 (IFN-treated, n = 7; vehicle-treated, n = 7, P 0,05
for all comparisons). As the absence of effect might be explained by lack of
penetration of IFN- across the blood brain barrier or lack of effect of hIFN-
on murine cells, a conclusion about the effect of IFN- on axonal
lesion-induced microglial activation can therefore not yet be drawn.
TOP
STAT1 and STAT2 signaling pathway in activated microglia and
astrocytes
following
entorhinal cortex lesion
Khorooshi R, Owens T.
Medical Biotechnology Centre, University of Southern Denmark, Odense,
Denmark
Microglia and astrocytes in the outer molecular layer (OML)
of the hippocampal dentate gyrus are activated in response to entorhinal
cortex lesion (ECL). Signals for glial response are not well defined.
Several cytokines that may trigger these responses signal through receptor
associated signal transducer and activator of transcription (STAT). A role
for type 1 interferon has been proposed. Type 1 interferon act via a
receptor that is associated with STAT1 and STAT2 signaling pathway. The
present study was therefore designed to investigate whether ECL induces
activation of STAT1 and STAT2 in OML. The preliminary results showed
induction of STAT1 in reactive microglia and STAT2 in astrocytes within the
denervated hippocampus 3 days after ECL. These findings indicate the
involvement of type 1 interferon specific signaling pathways (STAT1 and
STAT2) in lesion-induced glial activities. In future studies we will ask
whether STAT signaling pathways play a role in the initiation of immune
responses to axonal injury at earlier time, and in different mouse models.
TOP
Peripheral benzodiazepine receptor expression and PK11195 binding
in lesion-induced reactive microgliosis in murine CNS
Mads D. Pedersena, Luciano Minuzzib, Martin Wirenfeldt 1,
Michael Meldgaard 1, Paul Cumming 2 and Bente Finsen 1
1 Medical Biotechnology Center, University of Southern
Denmark, Odense C, Denmark, and
2 Aarhus University PET Centre, and Center for Functionally Integrative
Neuroscience, Aarhus University Hospital, Aarhus, Denmark
The peripheral benzodiazepine receptor (PBR) binds the ligand
PK11195 with high affinity.
The receptor, which is located primarily to the
outer mitochondrial membrane, is composed of three subunits: the
isoquinoline-binding protein (18 kDa), the voltage dependent anion channel
(32 kDa), and the adenine nucleotide transporter (30 kDa). The receptor has
roles in steroid production, energy metabolism, cell survival and growth. A
large number of studies have reported an increase in PK11195 binding following damage to the CNS. This increase in binding has been attributed to
an increase in expression of the PBR by activated microglia and infiltrating
macrophages.
Adult C57BL/6 mice were subjected to a stereotactic transection of the
entorhinal-hippocampal perforant path (PP) projection resulting in a highly
characteristic microglial reaction in the PP-terminal zones in the dentate
gyrus and the hippocampus proper. PBR binding was analysed in brain sections
and PBR mRNA expression in hippocampal homogenates obtained from mice with
2, 5 and 10 days survival postlesion and sham-operated and unoperated controls.
For the PBR ligand PK11195, Bmax and Kd were established in the ipsi- and
contralateral dentate gyrus,
the ipsilateral CA3 and CA1, and in the cortex,
caudate nucleus, dorsolateral thalamus, superior colliculus, inferior
colliculus, fimbria, and cerebellum. Bmax displayed a 3-fold increase 5 days
post lesion in the dentate gyrus, which correlates with the time of maximal
activation of Mac-1 immunoreactive microglia. Quantitative rtPCR revealed
peak expression of PBR mRNA 5 days post lesioning, corresponding to maximal
expression of Mac-1 mRNA. In addition, rtPCR analysis using exon-specific
primers, indicated the presence of two splice forms, a long form consisting
of all 4 exons, and 3-fold more abundant short form consisting of exon 2 and
3. Comparison of Bmax in different brain regions revealed an up to 3-fold
difference between regions, the fimbria showing the strongest and the
inferior colliculi the smallest binding affinity.
These preliminary data are presently being supported by inclusion of larger
numbers of mice in the experiment and inclusion of information on the
cell-specific expression of PBR.
TOP
Reactive microgliosis engages distinct responses by microglial
subpopulations
after minor CNS injury
Martin Wirenfeldt1, Alicia Anne Babcock2, Rune Ladeby1,
Kate Lykke Lambertsen1, Frederik Dagnaes-Hansen3, Robert Graham Quinton
Leslie4, Trevor Owens1,2 and Bente Finsen1
1 Medical Biotechnology Center, University of Southern Denmark, Winsløwparken
25, DK-5000 Odense C, Denmark
2 Neuroimmunology Unit, Montreal Neurological Institute, Montreal, Quebec,
Canada H3A 2B4
3 Department of Medical Microbiology and Immunology, University of Aarhus, Wilhelm Meyers Allé 240, DK-8000 Aarhus C, Denmark
4 Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark,
Winsløwparken 21, DK-5000 Odense C, Denmark
Microglia are bone marrow-derived cells that constitute a
facultative macrophage population when activated by trauma or pathology in
the CNS. Endogenous CNS-resident microglia as well as exogenous (immigrant)
bone marrow-derived cells contribute to reactive microgliosis. This
phenomenon raises fundamental questions about the cellular composition,
kinetics and functional characteristics of the reactive microglial cell
population in the CNS. Bone marrow chimeric mice reconstituted with green
fluorescent protein expressing (GFP+) donor bone marrow cells were subjected
to a selective axonal degeneration resulting in a localized microglial
reaction in the hippocampus. Flow cytometric evaluation of individual
microdissected hippocampi demonstrated small, but consistent, increases in
the proportion of immigrant GFP+ microglia (CD11b+CD45dim) by 3 and 5 days
post-lesion. Seven days post-lesion, however, approximately 13% of microglia
in lesion-reactive hippocampi were bone marrow-derived. Immigrant microglia
expressed lower levels of CD11b than resident microglia in bone marrow
chimeric mice, forming a distinct subpopulation on CD11b/CD45 profiles. In
addition a subpopulation of resting and activated CD11b+ microglia,
primarily of resident origin, expressed the haematopoietic stem cell marker
CD34. Our results show that distinct subpopulations of microglia respond to
minor CNS injury, and suggest that this heterogeneity in microglial response
encloses functional consequences for repair and possibly therapy.
TOP
Neuroinflammation after transient cerebral ischemia
Jesper Kelsen, MD1, 2
1The Water and Salt Research Centre, University of Aarhus, DK-8000 Aarhus,
Denmark
2 Clinical Institute, University Hospital of Aarhus, DK-8200 Aarhus N,
Denmark
There has been a static view upon the inability of human
adult brain tissue to renew itself. However, the discovery of neurogenesis
in the adult human hippocampus has led to considerable research in the
potential to regulate human neurogenesis. Both the hippocampal dentate gyrus
Granule Cell Layer (GCL) and the Subventricular Zone (SVZ) house neural stem
cells with the potential of differentiating into glia or neuronal cells.
Incorporation of the thymidine analog, 5-bromo-2´-deoxy-uridine (BrdU) is
the classical method used to prove the proliferation of neural stem cells in
adult brain tissue. Recently, infiltration with endogenous precursor cells
in an infarcted area, has been documented in an adult rat model of transient
Middle Cerebral Artery occlusion (tMCAo). The proces of neurogenesis is
stimulated by brain damage like degeneration, inflammation and ischemia.
The innate neuroinflammatory response to brain injury delivered by activated
microglia is considered as a doubleedged sword. A complex interplay between
astrocyte glutamate release and activated microglia is proposed to cause
neurotoxicity. Recently, the deleterious effects of microglial secretion of
the proinflammatory mediator, IL-6 have been adressed in regard to
hippocampal neurogenesis. Blockage of neuronal inflammation with
indomethacin or microglial activation with minocycline has
effectively restored hippocampal neurogenesis in rats subjected to
lipopolysaccharide (LPS) induced systemic inflammatory respons syndrome
(SIRS) or cranial irradiation.
In this PhD study neuroinflammation and neurogenesis after tMCAo is being
investigated in two different rat strains. Unbiased stereological methods
are used for the immunohistochemical quantification of hippocampal BrdU
incorporation and an activated microglia marker (ED-1). Further the possible
beneficial effects of selective COX-2 inhibition after tMCAo is evaluated
with immunohistochemistry, quantitative RT-PCR and MRI.
TOP
Neurogenesis in the
adult rat hipocampus
Torsten M. Madsen, M.D., Ph.D.
Center for Basic Psychiatric Research, Aarhus University Hospital Stereology
and Electron Microscopy Res. Lab.,
Aarhus University
Psychiatric treatment and neural plasticity.
Hippocampal neurogenesis is increased after administration of antidepressant
drugs as well as seizures, indicating a dynamic regulation of this process.
To further investigate this important phenomenon, studies are underway to
quantify changes in the number of synapses in discrete regions of the rat
brain after antidepressant treatment.
Also, the molecular underpinnings of the neural plasticity are investigated:
After electroconvulsive seizures, b-catenin, an important component of the
Wnt pathway, is increased in the hippocampal dentate gyrus, in places where
cell proliferation also occurs. Given that electroconvulsive seizures are
commonly used for the treatment of intractable depression, b-catenin and the
Wnt pathway may be potential therapeutic targets for psychiatric disorders.
TOP
Chronic antidepressant treatment increases hippocampal
neurogenesis
in rodents
Anders B. Marcussen1,2, Peter Flagstad2, Flemming F.
Johansen1, Paul E.G Kristjansen1 and Ulrica Englund2
1 Institute of Molecular Pathology, University of Copenhagen, Denmark
2 H. Lundbeck A/S, Pharmacology Target Research, Copenhagen, Denmark
Recently, it has been hypothesized that generation of new
nerve cells (neurogenesis) in the hippocampus is necessary for the effect of
antidepressant treatment. This hypothesis is based on findings from
pre-clinical studies, showing that several modalities of chronic
antidepressant treatment increase hippocampal neurogenesis (e.g. Malberg et
al., 2000).
In the current study, we treated mice and rats with fluoxetine (10 mg/kg) or
vehicle for 28 days prior to being tested in the NSF assay. Proliferating
cells were detected using the thymidine analogue BrdU, which becomes
incorporated into the DNA of dividing cells. The level of neurogenesis was
analysed at day 28 using immunohistochemical techniques and microscopical
analysis.
The behavioural effect of chronic as well as acute antidepressant treatment
was examined using the NSF test. This anxiety-related behavioural test is
able to detect an effect of chronic, but not acute, antidepressant treatment
in rats and mice (Bodnoff et al. 1989; Santarelli et al. 2003).
The results showed that fluoxetine-treatment had a significant neurogenic
effect in mice. In rats, a tendency towards an increase in neurogenesis was
observed after the fluoxetine treatment, although this did not reach
statistical significance. The chronic drug-treatment resulted in a
significant behavioral effect using the NSF test in rats, but not in mice.
No behavioral effect after acute (nor sub-chronic) fluoxetine treatment was
observed.
Our findings strengthen previous results demonstrating that chronic
antidepressant treatment increases hippocampal neurogenesis in rodents.
However, we could not correlate this to a behavioural outcome in the NSF
test. Thus, the current data indicate the requirement for validated and
stable methods in order to elucidate the functional significance of
increased hippocampal neurogenesis.
TOP
Unbiased estimation of the total number of NeuN positive neurons in
the developing neocortex
Thomas Krøigård, Lise Lyck, Nina Drøjdahl, and Bente
Finsen
Medical Biotechnology Centre, University of Southern Denmark, Odense,
Denmark
Stereology provides an unbiased and precise estimate of the
total number of cells within a specific region.
In the investigation of neocortical development this technique offers an
excellent means of quantifying the temporal changes in total cell number and
changes in the number of neurons using cell specific markers. Neurons were
visualized immunohistochemically using an antibody against NeuN, which is
found in the nuclei of all neocortical neurons. NeuN positive neurons were
counted in the neocortex of postnatal day 8 C57Bl6/J mice using thick (80
mm) vibratome sections cut in the saggital plane. Following mounting the
sections had a mean thickness of 22.3 ± 2.6 mm (mean ± SD) which allowed us
to use a 10 mm disector starting at 5 mm from the bottom of the section.
Neocortex was defined as the neocortex proper, proisocortex, and
periallocortex whereas allocortex and piriform cortex were excluded.
Counting was performed in the right hemisphere which had been cut into five
series yielding a section sampling fraction of 0.1. The area sampling
fraction was 0.0008 based on a step length of 500 mm which gave a step area
of 250,000 mm2 and the counting frame which had an area of 193 mm2. Counting
six animals resulted in an estimate of 4.8 ± 0.5 (mean ± SD) million
neurons. These data will be supplemented with estimates of the total number
of NeuN positive neurons at postnatal days 0, 16, and in the adult mouse and
estimates of total cell numbers in order to put this result into a
developmental perspective.
TOP
FRET FLIM microscopy
Jens R. Nyengaard1
Stereology and Electron Microscopy Research Laboratory, Institute of
Clinical Medicine, University of Aarhus, Denmark
We are interested in “bridging” the gap between cellular and
ultrastructural quantification to neurobiochemistry and
neurophysiology by introducing two-photon excitation FRET (fluorescence
resonance energy transfer) and FLIM (microscopy. Through advances in light
microscopical technology, the ability to image specific protein molecules
within neurobiological specimens will take basic neuroscience research to
the next level. Historically neurobiology had been conducted at an
impressive microscopic level. Through the utilization of clever methods for
labeling brain areas of interest with standard staining protocols including
immunohistochemistry, in-situ hybridization, fluorescent tagging, among
others, great discoveries have been made. However, these discoveries
inevitably lead to new questions. Interacting proteins assemble into
molecular machines that control cellular homeostasis in living neurons.
These protein assemblies were traditionally studied using biophysical or
biochemical methods such as affinity chromatography or
co-immunoprecipitation. While these in vitroscreening methods have the
advantage of providing direct access to the genetic information encoding
unknown protein partners, they do not however, allow direct access to
interactions of these protein partners in their natural environment inside
the living neuron. But using the approach of FRET/FLIM microscopy, this
information can be obtained from single living neurons with nanometer
resolution. However, many problems remain with 3- and 4-D quantification of
the signals and mathematical manipulation of the spectral bleedthrough
signal in two-photon FRET/FLIM microscopy.
With our interest and experience in microscopy and
computer-assisted-stereological-toolbox microscopical systems as well as GFP
mice we hope to foster an environment which would be ideal for the
introduction of FRET/FLIM microscopy at Aarhus University.
TOP
High sensation seeking men have increased dopamine release
in the right
putamen
during
gambling.
J. Linnet; A. Gjedde; Y. Kumakura; P. Cumming; A. Møller*
PET-centre & CFIN, Aarhus University, Aarhus, Denmark
Sensation seeking is a personality trait based on Eysenck’s
theory of optimal arousal, which has been associated with the dopaminergic
system. High sensation seekers have a hedonic response to a higher level of
stimulation, while low sensation seekers may react with aversion to the same
level of stimulation. It is a common claim that high
sensation seekers have a lower activity in the dopaminergic system which may
make them susceptible for dopamine enhancing stimulants such as amphetamine
and possibly gambling. This PET study focus on [11C]raclopride binding
potential (pB) in high sensation seekers compared to low and mid-range
sensation seekers in a control version and a gambling version of the Iowa
Gambling Task. From a normally distributed sample of 243 individuals we
defined cut-points of high and low sensation seekers as the top and bottom
20% of the distribution. This corresponded to a score of 25 and greater for
high sensation seekers, 14 and lower for low sensation seekers, and 15 24
for mid-range sensation seeking. In the current study we compared 8 high
sensation seeking men with 12 low to normal ranging sensation seeking men.
Dynamic emission recordings were obtained during 60 minutes after i.v. [ C]raclopride
(190 366 MBq) using the ECAT HR tomography. Maps of [ C]raclopride pB were
calculated by the Logan reference tissue method with the cerebellum serving
as reference and the mean pB calculated in templates for the putamen. High
sensation seekers had a significantly lower [ C]raclopride binding potential
in the right putamen (Mean SD: 3.0 0.1 vs. 2,9 0.1, p = 0.025), indicating
increased dopamine occupancy of dopamine D2 and D3 receptors during the
gambling session. No changes in the binding potential was observed in the
normal/low sensation group. The increase in pB (contrast gamling task) in
the right putamen was significantly larger in the high sensation seekers
compared to normal/low sensation seekers (F = 5,74; p = 0,028) These
preliminary results show increased dopamine release in the right putamen in
high sensation seeking men while gambling.
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