EMIM 2018 ControlCenter

Online Program Overview Session: PW-12

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Brain: Structure, Function, and Networks

Session chair: Mathias Hoehn - Cologne, Germany; Elisabeth Jonckers - Antwerp, Belgium
 
Shortcut: PW-12
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 134

Defining the relationship between the brain vasculature and microglia cell development with fluorescence microscopy in neonate rodents. (#70)

C. Sussman1, J. Banting1, A. Rodriguez-Contreras1

1 CUNY, CIty College, Biology and CDI, New York, New York, United States of America

Introduction

Microglial cells are the resident macrophages of the brain, they originate in the yolk sac and migrate into the brain during embryonic development. Little is known about their early postnatal development when the population of microglia cells in the brain undergoes expansion. It is hypothesized that during early postnatal development microglia cells interact with the brain vasculature possibly to support cell proliferation and migration throughout the brain.

Methods

To address the hypothesis, changes in vascular network were examined with the plant lectin isolectin-B4, the thymidine analog EdU and confocal microscopy in 80 micrometers thick horizontal brainstem sections of rats at different postnatal ages (n=19 pups, P0 = birth). Additional histology experiments were performed in 80 micrometers thick coronal brainstem and forebrain sections of neonate mTmG; CX3CR1-GFP mice, which express red and green fluorescent proteins in endothelial and micro glial cells, respectively.

Results/Discussion

In neonate rats, vascular volume (in micrometers3) was constant between P0 and P5 (15,879 ± 1,147 and 16,654 ± 906, respectively), and a marked increase was detected at P10 (30,915 ± 2,502; students t test, p<0.00001). EdU labeling confirmed cell proliferation of vascular and perivascular cells at P6 and P7. Preliminary analysis of fixed tissue from mTmG; CX3CR1-GFP mice demonstrate that microglial cells interact with blood vessels as early as P0. Image segmentation analysis of confocal z-stacks suggests that there is an increase in the volume of both cell types between P0 and P10. Future experiments using transcranial two-photon microscopy in neonate mice that express fluorescent reporters will examine the dynamic behavior of microglia cells associated with blood vessels in vivo.

Conclusions

The data demonstrates that perivascular cells proliferate during a postnatal stage of angiogenesis in rats, and that microglia cells have a perivascular distribution in neonate mice.

Acknowledgement

Supported by NIH grants SC1HD068129 and G12-RR003060.

Keywords: Vascular development, angiogenesis, Fluorescent proteins, fluorescent histochemistry
# 135

[18F]DPA-714 PET-MRI imaging reveals pronounced innate immunity in (auto-)immune limbic encephalitis (#518)

W. Roll1, C. Strippel2, B. Zinnhardt1, 3, K. Golombeck2, P. Backhaus1, 3, R. Seifert1, 3, A. Dik2, C. Mönig2, A. Jacobs3, 4, S. Meuth2, N. Melzer2, M. Schäfers1, 3

1 University Hospital Münster, Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
2 University Hospital Münster, Neurology, Münster, North Rhine-Westphalia, Germany
3 University of Münster, European Institute of Molecular Imaging (EIMI), Münster, North Rhine-Westphalia, Germany
4 Johanniter Hospital Bonn, Geriatrics, Bonn, North Rhine-Westphalia, Germany

Introduction

(Auto-)immune limbic encephalitis (LE) usually presents with mesial temporal lobe epilepsy and cognitive changes and is thought to be mediated by adaptive neural immunity. Targeting the translocator protein (TSPO) with a second generation PET-Tracer (F-18-DPA-714) permits visualisation of TSPO overexpression in activated microglia and other innate immune cells. The aim of this study is to investigate the feasibility of a combined F-18-DPA-714 PET-MRI protocol for visualisation of innate inflammation in seropositive and seronegative LE patients.

Methods

Five LE patients underwent combined F-18-DPA-714-PET-MRI between 9/2017 and 2/2018. Dynamic images were acquired 0-60 min p.i. on a Siemens PET-MRI system (mMR). T1w, T2w and Flair MR-images were effected for correlation purposes. Inclusion criteria were recent diagnosis of limbic encephalitis (2 x seronegative; 1 x anti-GAD65 antibodies, 2 x anti-LGI1 antibodies) and naivety to immunotherapies. Inflammation to background ratios (SUVmax tumor/SUVmean reference region) measured in 30-60 min p.i. summation images were standardized to a cortical reference region including grey and white matter on the less-affected brain hemisphere. Time activity curves were implemented for the hippocampus in both hemispheres and the cortical reference region.

Results/Discussion

This study provides first insights into microglia activation in LE as assessed by DPA-714-PET-MRI. All LE patients showed increased uptake in the mesial temporal lobe (amygdala, hippocampus) on the affected side compared to the contralateral hemisphere and the reference region. TSPO expression correlated with signal alterations in T2 and FLAIR images. Time activity curves showed high activity in LE over time compared to the contralateral reference region.

Conclusions

We demonstrate the feasibility of [18F]DPA -714-PET-MRI to visualize the innate immunity in LE. [18F]DPA -714-PET-MRI may be a promising future tool for the assessment of individual inflammation patterns in LE independent of the absence or presence and targets of an underlying adaptive immune response.

Keywords: DPA-714, PET-MRI, limbic encephalitis
# 136

Effect of illumination level on FDG-PET brain uptake: pilot study in mice (#137)

A. de Francisco1, 2, 4, Y. Sierra-Palomares1, 2, M. Felipe1, 4, L. Cussó1, 2, 3, D. Calle1, 2, M. Desco Menéndez1, 2, 3

1 Instituto de Investigación Sanitaria Gregorio Marañón, Experimental Medicine and Surgery Unit, Madrid, Spain
2 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Advanced Imaging Unit, Madrid, Spain
3 Universidad Carlos III de Madrid, Bioengineering and Aerospace Engineering Department, Madrid, Spain
4 Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Madrid, Spain

Introduction

FDG-PET neuroimaging is a widely spread preclinical research technique. Before image, uptake period is necessary to get a complete FDG distribution. During this uptake, the animals remains awake and exposed to standard environmental conditions (temperature, noise and light). In contrast, in clinical practice during uptake patients are kept in a dark and isolated room, without external stimuli. Previous studies have shown that rodents circadian cycle affects FDG brain uptake [1]. The aim of this work is to explore the effects of darkness during FDG brain uptake on mice.

Methods

Ten C57BL/6 mice of 11 weeks, half males and half females, were subjected to 8 h of fasting before FDG-PET imaging. Two scans were acquired from each animal, 48 ​​hours apart, at two illumination levels (lightness, 780 lx, and darkness, 0 lx) during the uptake (30 min) varying in each FDG-PET image. Animals remained under anesthesia (sevoflurano 3% - 300 cc / min O2) during PET acquisition (microPET-CT SuperArgus, SEDECAL). The images were analyzed using ROIs and voxelwise techniques (SPM).

Results/Discussion

SPM analysis of studies corresponding to FDG uptake in darkness (Figure 1) showed a relative FDG uptake increase in caudate, olfactory area, cortex, hippocampus and reticular nucleus, when FDG uptake was performed in darkness (p <0.05, t-test , k=25 neighboring voxels). Using manually selected ROIs, in darkness condition we observed an absolute increase in FDG uptake in whole brain and in manually selected regions based on SPM results (Figure 2), although the differences did not reach statistical significance (Wilcoxon test).

Conclusions

Our data shows that illumination level during FDG uptake affects the cerebral metabolism of glucose, being higher in darkness conditions. This may be in relationship with the active phase of the mouse (nocturnal) and points to the importance of controlling light conditions during uptake period for brain PET studies in mice.

References

[1] van der Veen D. R. et al. (2012) PloS one, 7(2), e31792.

Acknowledgement

The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015- 0505).

Figure 1. SPM and manual selected ROIs.

A: SPM analysis (p<0.05, t-test, k=25 neighboring pixels). Red represents the areas with higher FDG uptake in darkness condition. B: Cerebral areas manually selected for SUV analysis.

Figure 2. FDG uptake.

Mean SUV of the selected cerebral areas after lightness (yellow) and darkness (grey) conditions.

Keywords: brain glucose metabolism, FDG-PET
# 137

Connexin Isoforms Cx26, CX36 and Cx45 in the Developing Human Cortex (#327)

D. M. Kočović1, J. Tadić2, S. Vrzić-Petronijevic2, P. R. Andjus1, S. D. Antic3

1 Center for laser microscopy, Faculty of Biology, University of Belgrade, Department of Physiology and Biochemistry, Belgrade, Serbia
2 Clinical Center of Serbia, School of Medicine, University of Belgrade, Department of fetal and perinatal pathology, Belgrade, Serbia
3 University of Connecticut Health, Department of Neuroscience, Farmington, Connecticut, United States of America

Introduction

Connexin proteins are the membrane subunits of both single membrane channels (known as „hemichannels“) and gap junction channels, which allow passage of ionic currents and molecules between extracellular and intracellular space. Connexins play important roles in the generation of electrical activity in the human fetal cortex [1], generation and development of new neurons, cell proliferation and migration during the development of brain cortex [2]. The main goal of our study was to determine the distribution of the connexin isoforms throughout cortical zones of the developing human cortex.

Methods

Postmortem human fetal tissue was fixed in 4% paraformaldehyde for 24 hours, and then placed in 30% sucrose for 48 hours. Frozen tissue samples were cut into 30 µm-thick tissue sections and treated with polyclonal antibodies against three connexin (Cx) protein isoforms (Cx26, Cx36 and Cx45) and one neuronal marker (NeuN) to determine the location of postmitotic neurons expressing Cx isoforms. The nuclei of all cell types were stained by TO-PRO nuclear stain in order to assess the total number and density of cells per cortical zone. In this study, we used a laser scanning confocal microscope (objective lenses Plan-Apochromat 63X oil and Plan-Neofluar 20X) to follow the expression of Cx protein isoforms on new born human neurons.

Results/Discussion

We quantified the expression of the neuronal marker NeuN in cortical plate (CP), subplate (SP) and subventricular zone (SVZ) of the human fetal brain during gestational weeks 16 – 26. Our results revealed a decreasing trend in the number of nuclei (i.e.cells) and putative neurons (NeuN+ cells) with gestational weeks (GW), probably due to uncorrected growth of cell somata. At the fetal age of 20 GW, ~100% cells in CP were neurons, while ~90% of cells in SP and ~40% of cells in SVZ were putative neurons. We quantified the expression of the Cx isoforms Cx26, Cx36 and Cx45 among the NeuN-positive cells. From 16th to 22nd GW, the number of Cx-positive neurons decreased in SVZ (proliferation zone) and increased in CP (cortical precursor zone), consistent with the notion that neurons are born in SVZ, migrate through the SP zone and assume their final positions in CP zone.

 

Conclusions

During the second trimester of gestation (16 – 26 GW) all three major neuronal Cx isoforms, Cx26, Cx36 and Cx45 were present in the postmitotic neurons of the human fetal cortex. The greatest number of Cx-positive neurons was in the period 18-22 GW, followed by a decreasing trend in their expression in later gestational weeks from 24th to 26th, in all cortical zones.

 

 

References

[1] A.R. Moore, W.L. Zhou, C.L. Sirois, G.S. Belinsky, N. Zecevic and S.D. Antic (2014) PNAS 16;111(37): E3919 – E3928.

[2] D. Niculescu and C. Lohmann (2014) Cereb Cortex 24:3097-106.

Keywords: Connexins, Human fetal brain, Neurons, Development
# 138

FMRI as a Valuable Tool to Evaluate the Central Anti-Nociceptive Effects of Acupuncture Analgesia in an Animal Model (#134)

I. Wank1, 2, J. Zhang3, 4, S. Chen4, V. Nagy3, 5, L. Zhang6, J. Penninger3, A. Hess1

1 University of Erlangen-Nuremberg, Institute of Pharmacology and Toxicology, Erlangen, Bavaria, Germany
2 University Hospital Erlangen, Department of Medicine 3 Rheumatology and Immunology, Erlangen, Bavaria, Germany
3 Austrian Academy of Sciences, Institute of Molecular Biotechnology, Vienna, Austria
4 China Academy of Chinese Medical Sciences, Institute of Acupuncture and Moxibustion, Beijing, China
5 Austrian Academy of Sciences, Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria
6 Vienna General Hospital, Clinical Institute of Laboratory Medicine, Vienna, Austria

Introduction

In Traditional Chinese Medicine, it is thought that blockade of a body’s energy flows may evoke many different pathologies including chronic pain. Disturbed flows can be restored by inserting acupuncture needles (N) into specified acupoints (AP). Using fMRI, the analgesic effects of two different acupuncture (ACU) treatments (insertion of needles and electro-acupuncture (EA)) at Zusanli acupoint (ST36) were compared in a mouse model to evade the placebo-effect.

Methods

The brain’s response to 50°C heat stimuli (right hindpaw; dur. 20 sec, in-between 3min40sec) was assessed via BOLD fMRI (4.7T Bruker Biospec, GE single-shot EPI (TR=200ms; TEef=25.3ms); matrix 64×64, voxels 0.234x0.234mm, 22 axial slices of 0.5mm) in healthy ♂ C57Bl/6J WT mice.

The FMRI experiment was split into 3 time windows to check for time-dependent effects:
pre-phase 0-18min
middle-phase 19-34min (application of EA (1mA 180μs pulses; 2Hz frequency) in H/N/EA & N/EA group)
post-phase 35-50min.
Mice were randomly assigned to 4 groups:
H: only heat stimuli
H/N: 1 N in left ST36 AP
H/N/EA: 2 N, inserted as above & 2nd ~2mm lateral connected to electric stimulator for EA
N/EA: pseudo-resting state (p-RS) measurement, no heat stimuli.

GLM analyses and graph-theoretical analyses [1] were performed.

Results/Discussion

Stimulus-evoked response amplitudes and activated volumes, compared between groups 1-3, revealed a time-dependent analgesia for both ACU modalities even for the whole brain: compared to H, both parameters were reduced about 50% in middle-phase, EA being more effective. Analgesia remained for H/N/EA during post-phase.

Graph-theory revealed ACU-induced changes in prefrontal / limbic areas: amygdala, hypothalamus, n. accumbens in H to more cognitive regions in both ACU groups (sensory / cingulate cortex, hippocampus). EA furthermore engaged thalamus and c. putamen. This shift lead to significant functional connectivity (FC) decreases in a similar subset for both ACU groups in pre- and middle-phase: thalamus, sensory / association cortex, hippocampus and basalganglia. Only in H/N/EA decreased thalamic FC persisted into post-phase.

P-RS networks showed that EA alone modulates bottom-up interaction of thalamus, amygdala, hypothalamus and basalganglia, indicating control over higher regions.

Conclusions

Both acupuncture methods showed a significant analgesic effect in WT mice verified by BOLD-fMRI. Electro-acupuncture was found to be more effective and longer-lasting than acupuncture needles alone. Analgesia is mediated by modulation of a specific subset of subcortical brain regions that regulates the activity of higher-order brain structures.

References

[1] Sporns O, Chialvo DR, Kaiser M, Hilgetag CC. Organization, development and function of complex brain networks. Trends in cognitive sciences 2004;8(9):418-425.

Acknowledgement

BMBF (NEUROIMPA, TP4 01EC1403C)

Influence of acupuncture on BOLD parameters (whole brain).
Analysis of the classical BOLD parameters response amplitude and activated brain volume showed that both ACU modalities yield a significant analgesic effect (~50% reduction of both parameters). The effect was found to be time-dependent and was greatest in the middle-phase (19-34min). EA was more effective with a greater reduction in middle-phase and prolonged analgesic effect in post-phase.
Influence of electro-acupuncture on pseudo-resting state brain networks
Pseudo-resting state measurements were conducted to assess which brain regions are directly influenced by electro-acupuncture (EA).
EA was found to modulate a specific subset of limbic brain regions: enhanced functional connectivity (FC) was found between amygdala, hypothalamus and the basalganglia. This in turn reduced thalamic FC and enhanced cortical FC, mediating the lasting analgesic effect.
Keywords: fmri, functional connectivity, acupuncture, acupuncture analgesia, graph theory, brain networks, resting state
# 139

A general protocol of ultra-high resolution MR angiography to image the cerebro-vasculature in 6 different rats strains. (#304)

G. Pastor1, M. Beraza1, S. Plaza-García1, A. Egimendia1, D. Padro1, P. Ramos-Cabrer1, 2

1 CIC biomaGUNE, Magnetic Resonance Imaging, Donostia-San Sebastián, Spain
2 Ikerbasque, Basque Foundation for Science, Bilbao, Spain

Introduction

3D time of flight (TOF) magnetic resonanance angiography (MRA) is a highly interesting tool for the evaluation of the brain vasculature on animal models of vascular diseases such as stroke. A common animal model of this disease, the occlusion of the middle cerebral artery (MCA) yields different infarct sizes in different rat strains1. Thus, we decided to use the advantages of high magnetic fields (such as 11.7 T) to perform MR angiographies at high spatial resolution of six of the most commonly used rat strains, to evaluate the utility of this imaging modality in animal models of stroke.

Methods

Animals: Sprague Dawley (SD), Wistar Kyoto, Wistar SHR, Long Evans, Fisher and Lister Hooded male rats were used (240-320g). MRI: experiments performed on 11.7T Bruker Biospec. Angiography: a 3D-FLASH-flow-compensated (FC) method was used. Ultra-high resolution angiograms were acquired with the following imaging parameters: TE=4.45ms; TR=60ms, 30º flip angle, 512*512*256 matrix, a 32*32*32mm field of view leading to a  resolution of 0.062mm/voxel in 100 minutes. The vessels were visualized using maximal intensity projection (MIP) algorithm following a manual segmentation of intra-cranial blood vessels. Infarct sizes were detected with a T2 weighted RARE image. Infarct volumes were calculated with ImageJ.

Results/Discussion

High resolution MR angiograms were successfully obtained in six different rat strains (n=2 each) before and 24-48 h after MCAO surgery . In three of those animals the MCA presented a double branching to the circle of Willis and resulted in smaller infarct sizes. The post-processing of the 3D data substantially improved the clarity of the angiogram (figure 1). Angiograms obtained 24-48 h after MCA surgery were valid, despite the presence of brain oedema, using same imaging parameters valid before MCA surgery. No significant differences in brain vessel architecture was observed for the rat strains analyzed.

Conclusions

We have optimized a FC-3D TOF-MRA to produce high-resolution angiograms in six different rat strains without using a contrast agent. Visualization of the brain vasculature was further improved by removing extra-cranial vessels. The method has been successful in detecting one source of variability in the cerebro-vasculature in SD rats subjected to transient MCAO.

References

1. A. Sauter and M. Rudin, JPET, 1995, 274, No. 2, 1008-1013.

2. Y. Sato et al., Med Image Anal., 1998, 2, No. 2, 143-168.

Acknowledgement

We want to acknowledge the Spanish ministry of economy and competence (SAF2014-53412-R) and the Basque Government (PC2015-1-05 (53-80)) for financial support. We also thank IKERBASQUE (the Basque foundation for Science) for funding PRC.

Figure 1.
MR angiogram of a Fisher 344 rat, before (A) and after (B) removal of extracranial signal. Angiogram acquired in less than 100 min with a resolution up of 62.5 μm. Post-processing allows the direct identifications of the smaller vessels branching from the circle of Willis.
Keywords: MRA., Angiography, Rat strains, stroke, MCAo
# 141

Food as a Modulator of Resting State Networks in Rodents and Humans. (#225)

A. Mendez-Torrijos1, S. Kreitz1, C. Ivan1, L. Konerth1, J. Rösch2, M. Pischetsrieder3, G. Moll4, O. Kratz4, A. Dörfler2, S. Horndasch4, A. Hess1

1 Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Experimental and Clinical Pharmacology and Toxicology, Erlangen, Bavaria, Germany
2 Universitätsklinikum Erlangen, Department of Neuroradiology, Erlangen, Bavaria, Germany
3 Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Chemistry and Pharmacy, Food Chemistry Division, Erlangen, Bavaria, Germany
4 Universitätsklinikum Erlangen, Department of Child and Adolescent Mental Health, Erlangen, Bavaria, Germany

Introduction

Non-homeostatic hyperphagia contributes to obesity. Food intake is associated with diet’s composition e.g. energy content and snack food may induce satiety independent from intake. First, we tested in rats the tendency to consume snack food (chips vs. standard chow, additionally controlling the fat/carbohydrates ratio). Following, human Resting State fMRI (RS-fMRI) was investigated after visually presenting different foods (high-caloric: chips and low-caloric: zucchini) followed by the ingestion of on of the foods presented to explore changes in RS networks of healthy individuals [1]. 

Methods

Manganese-enhanced magnetic resonance imaging (MEMRI) was used for mapping the whole rat brain activity related standard chow and chips intake over one week.

Following, 17 healthy human subjects with BMI 19 to 27 underwent two different fMRI sessions where an initial resting state scan was acquired, followed by visual presentation of different potato chips and zucchini images. There was then a 5 minutes pause to ingest food (day 1=chips, day 3=zucchini), followed by a second resting state scan. Human FMRI data was further analyzed using graph theory analysis [2, 3] and support vector machine techniques.

Results/Discussion

Results in rats showed that the intake of potato chips manganese accumulation increased in certain areas related to the reward system as well as to locomotor activity indicating higher neural activity in these areas [4]. Furthermore a specific mixture of 35% fat and 50% carbohydrate in food but not the pure energy content leads to this hedonic hyperphagia [5].

In humans, chips vs. zucchini stimulation lead to significant connectivity changes. The support vector machine was able to accurately categorize the two types of food stimuli with 100% accuracy. Visual, auditory and somatosensory structures, as well as, thalamus, insula and basal ganglia were found to be important for food classification. After chips consumption, the BMI was positively correlated with the path length and degree in nucleus accumbens, middle temporal gyrus and thalamus (Figure 1).

Conclusions

Our human study showed that high vs. low caloric food in healthy individuals can induce significant changes in RS which are detectable by graph theory and support vector machine. Nucleus accumbens connectivity changes in humans directly relate to changes in the rats. However, some human connectivity differences (e.g. visual cortex) cannot be found in rats, as no visual stimulation occurred. Furthermore, our results in humans involve higher order brain functions such as semantic reasoning not present in rats. However, our human RS-fMRI study nicely translated results from rats MEMRI studies.

References

1.            Mendez-Torrijos, A., et al., Snack food as a modulator of human resting-state functional connectivity. [In press]. CNS spectrums, 2017.

2.            Watts, D.J. and S.H. Strogatz, Collective dynamics of 'small-world' networks. Nature, 1998. 393(6684): p. 440-2.

3.            Zalesky, A., A. Fornito, and E.T. Bullmore, Network-based statistic: identifying differences in brain networks. Neuroimage, 2010. 53(4): p. 1197-207.

4.            Hoch, T., et al., Manganese-enhanced magnetic resonance imaging for mapping of whole brain activity patterns associated with the intake of snack food in ad libitum fed rats. PLoS One, 2013. 8(2): p. e55354.

5.            Hoch, T., M. Pischetsrieder, and A. Hess, Snack food intake in ad libitum fed rats is triggered by the combination of fat and carbohydrates. Frontiers in psychology, 2014. 5: p. 250.

Acknowledgement

Neurotrition Project by FAU Emerging Fields Initiative.

Figure 1
Brain structures that significantly correlate between BMI with (a) path length and (b) degree, after chips consumption (p<0.05). Regions with positive correlations are highlighted in red, while negatively correlated regions in blue.
Keywords: Manganese-enhanced magnetic resonance imaging, Resting State fMRI, Resting State Networks, Support Vector Machine, graph theory
# 142

Increased hippocampal volume in exercising mice: comparison of control conditions with in vivo voxel based morphometry (#442)

G. Becker1, L. - F. Lespine2, M. A. A. Bahri1, A. Luxen1, E. Tirelli2, A. Plenevaux1

1 University of Liege, GIGA - Cyclotron Research Center, LIEGE, Belgium
2 University of Liege, Psychology Faculty, LIEGE, Belgium

Introduction

Both human and animal studies have shown that physical exercise (primarily aerobic exercise) may have facilitating effects on brain plasticity and cognition (1). In rodents, improvements of various forms of learning and memory induced by wheel-running have been associated with numerous neuroplastic changes such as increased hippocampal neurogenesis (2). A few studies, using magnetic resonance imaging (MRI), consistently reported hippocampal volumetric increase relative to non-exercising mice (3, 4, 5, 6). However, the control group is commonly limited either to a locked wheel or no wheel.

Methods

In the present study, we intended to test whether 6 weeks of voluntary wheel-running exercise during adulthood induced a detectable volumetric change in mice brain in comparison to non-exercised control mice housed either with a locked wheel or without such wheel.

54 C57Bl6 males were randomly assigned to one of the three groups and individually housed for 6 weeks before imaging session. MRI (Agilent 9.4T) acquisition consisted in 3D T2 volume sequence (voxel size: 0.21 mm isotropic) using a dedicated surface coil receiver. We used Dartel software for the preprocessing of the data, and the Voxel Based Morphometry was done with SPM mouse toolbox (F test, threshold p < .001 uncor). A small volume correction was applied to limit the analysis to the hippocampus.

Results/Discussion

VBM analysis shows significant clusters with increased grey matter volume in the hippocampus (cluster sizes 1531 and 3460, puncor < .001) when we compare the wheel vs locked wheel groups. Regarding the wheel vs no wheel comparison, significant clusters were observed in the hippocampus (cluster sizes 955 and 238, puncor < .001). Interestingly, no differences were found when we compare the two control groups (locked wheel vs no wheel).

Conclusions

In this study, we replicate previous studies depicting an increased hippocampal volume under physical exercise in mice using VBM. Moreover, we certified here that attempting to study the impact of physical exercise on brain volume, control groups with a locked wheel or no wheel are equivalent.

References

  1. Hötting and Röder, 2013
  2. Vivar et al. 2012
  3. Bindermann et al. 2012
  4. Pereira et al. 2007
  5. Fuss et al. 2014
  6. Cahill et al. 2015

Acknowledgement

We thank F. Moes for excellent technical assistance in data acquisition.

Keywords: MRI, mice, VBM, exercise
# 143

Cerebral Reflections of Conditioned Pain Modulation in the Rat: An fMRI study (#282)

S. Kreitz1, 2, T. Klasfauseweh1, S. Strobelt1, J. Kaesser1, I. Wank1, M. Uder2, A. Hess1

1 Friedrich-Alexander-University Erlangen-Nuremberg, Institute for Pharmacology and Toxicology, Erlangen, Germany
2 University Hospital of the Friedrich-Alexander University Erlangen-Nuremberg, Department of Radiology, Erlangen, Germany

Introduction

Conditioned pain modulation (CPM) describes the phenomenon of a conditioning stimulus altering the pain perception of a noxious test stimulus1. Most CPM studies are psychophysically based human studies and do have clinical relevance in diagnostics and treatment2. However, the underlying neuronal mechanisms of CPM are not well understood. In animal models a comparable mechanism of endogenous pain inhibition was observed on the level of spinal neurons3. Using fMRI we present an innovative animal model to investigate the involvement of specific brain areas of the central nervous system in CPM.

Methods

Wistar ♂ rats anesthetized with isoflurane were scanned in a 4.7T BRUKER BioSpec magnet having their right hind paw placed in a custom-made cooling tube lined with a latex glove. Via barometric pressure warm or cold water was pumped into the tube. Each scan session consisted of 3x20min phases with warm (30°C), cold (10°C) and warm (30°C) water, respectively. During each phase 6 thermal stimuli were applied at the left hind paw with alternating innocuous 42°C and noxious 52°C. Two scans were performed (EPI, TR=2s, TE=25.3ms, matrix 64x64, FOV 0.391x0.391 mm, 22 axial slices of 1mm): first a resting state scan without, and second a CPM scan with thermal stimulation. A control group was scanned identical but with no cold phase. Data were analyzed using GLM and graph-theoretical approaches4.

Results/Discussion

As expected, non-nociceptive heat stimulation (42°C) revealed no conclusive cold stimulation effects. However, the conditioning cold stimulus influences the neural processing of the nociceptive heat stimulus in various pain related brain structures. These modulations included response amplitude, activated volume and connectivity (Fig. 1). Especially affected were brainstem, tegmentum and hypothalamus (indicating modulation of the descending pathways in pain processing), sensory cortex and lateral thalamus (responsible for the sensory-discriminative dimension of pain), frontal association cortex (the centre of the cognitive dimension of pain) and the limbic system (involved in emotional pain processing and learning).

Resting state connectivity was modulated after the cold stimulus, with increasing extensiveness over time. Here, functional connectivity of the sensorimotor system, cingulum and brainstem were reduced whereas amygdala connections were enhanced (Fig. 2).

Conclusions

In conclusion, the introduced model is suitable to demonstrate the central effects of CPM in rats. The presented results contribute to the understanding of the underlying neural mechanisms of CPM.

References

  1. Yarnitsky D. Conditioned pain modulation (the diffuse noxious inhibitory control-like effect): its relevance for acute and chronic pain states. Curr Opin Anaesthesiol. 2010;23(5):611-5.
  2. Nir RR, Yarnitsky D. Conditioned pain modulation. Curr Opin Support Palliat Care. 2015;9(2):131-7.
  3. Le Bars D, Dickenson AH, Besson JM. Diffuse noxious inhibitory controls (DNIC). I. Effects on dorsal horn convergent neurones in the rat. Pain. 1979;6(3):283-304.
  4. Kreitz S, de Celis Alonso B, Uder M, Hess A. A new analysis of resting state connectivity and graph theory reveals distinctive short-term modulations due to whisker stimulation in rats. bioRxiv 223057; doi: https://doi.org/10.1101/223057

Acknowledgement

This work was supported by BMBF Neurorad (02NUK034D)

Brain regions showing significant CPM during cold phase

(A) Significant modulation of activated volume and response amplitude during noxious heat stimulation in the cold phase relative to the control group. (B) Networks of significant connectivity differences in heat activated regions between first warm and cold phase for control and CPM group. Sizes of nodes (i.e. brain regions) are proportional to the amount of modulated connections. t-test, p<0.05.

Resting state connectivity modulation due to the tonic cold stimulation
(A) Resting state subdivision windows in the experimental design. (B) Number of modulated nodes and connections for all resting state windows compared to the pre early window. (C) Significant modulated components compared to the control group( paired t-test). Sizes of nodes (i.e. brain regions) are proportional to the amount of modulated connections.
Keywords: conditioned pain modulation, animal model, BOLD fMRI, resting state
# 144

Functional connectivity changes during epileptogenesis: a longitudinal resting-state fMRI study (#87)

E. Christiaen1, M. - G. Goossens2, B. Descamps1, P. Boon2, R. Raedt2, C. Vanhove1

1 Ghent University-IMEC, MEDISIP, Department of Electronics and Information Systems, Ghent, Belgium
2 Ghent University, Laboratory for Clinical and Experimental Neurophysiology, Neurobiology and Neuropsychology (LCEN3), Department of Neurology, Ghent, Belgium

Introduction

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults. Research has shown that abnormal functional brain networks could be involved in the development of epilepsy and its comorbidities1. Gaining more insight into these networks can be useful for the development of new therapies. Resting-state functional MRI can visualize changes in functional networks on a whole-brain level2. In this study, we aim to map changes in functional networks during epileptogenesis in the intraperitoneal kainic acid (IPKA) rat model for TLE using longitudinal resting-state fMRI and graph theory.

Methods

Twenty-four adult male Sprague-Dawley rats (276±15g) were used in this study. Seventeen animals were intraperitoneally injected with kainic acid (KA) according to the protocol of Hellier et al.3 resulting in status epilepticus (SE). The other 7 animals were injected with saline and used as a control group. Rs-fMRI images were acquired before and at 5 time points during the development of epilepsy on a 7T system, while the animals were anesthetized with medetomidine. The images were preprocessed using SPM12. The Pearson correlation coefficient was calculated between the fMRI time series of 38 regions of interest (ROIs) and stored in a correlation matrix. Several network measures were calculated using a graph theoretical network analysis toolbox (GRETNA)4, and plotted as a function of time.

Results/Discussion

In Fig. 1 the distribution of the correlation coefficients is shown at different time points during the development of epilepsy in the IPKA rat model and in control animals. The correlation coefficients shift to smaller values during epileptogenesis and their distribution becomes wider. This indicates that network connections progressively become weaker during the development of epilepsy. Four network measures can be seen in Fig. 2: clustering coefficient, local efficiency, characteristic path length and global efficiency. In Fig. 2A and 2B clustering coefficient and local efficiency are shown. Both decrease during epileptogenesis, indicating a decrease in segregation or local interconnectivity in the functional brain network. Fig. 2C and 2D show that characteristic path length increases and global efficiency decreases during epileptogenesis. This indicates that the integration in the brain network decreases, so there is a decrease in overall communication efficiency.

Conclusions

The results of this study show that functional brain network connections progressively become weaker and that segregation and integration of the network are decreased during epileptogenesis. In the next phase of this study, EEG monitoring will be used to characterize the severity of epilepsy in these rats to investigate how changes in functional brain networks during epileptogenesis correlate with epilepsy severity.

References

  1. Chiang, S. & Haneef, Z. Graph theory findings in the pathophysiology of temporal lobe epilepsy. Clin. Neurophysiol. 125, 1295–1305 (2014).
  2. Hutchison, R. M., Mirsattari, S. M., Jones, C. K., Gati, J. S. & Leung, L. S. Functional Networks in the Anesthetized Rat Brain Revealed by Independent Component Analysis of Resting-State fMRI. J. Neurophysiol. 103, (2010).
  3. Hellier, J. L., Patrylo, P. R., Buckmaster, P. S. & Dudek, F. E. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res. 31, 73–84 (1998).
  4. Wang, J. et al. GRETNA: a graph theoretical network analysis toolbox for imaging connectomics. Front. Hum. Neurosci. 9, 386 (2015).
Fig. 1.
Distribution of correlation coefficients during epileptogenesis in IPKA animals and control animals
Fig. 2.

Changes in network measures during epileptogenesis: A) Mean clustering coefficient, B) Mean local efficiency, C) Mean characteristic path length, D) Mean global efficiency

Keywords: resting-state fMRI, temporal lobe epilepsy, graph theory
# 145

Non-invasive bioluminescence imaging for optimization of hyaluronic acid-based hydrogel to support the survival of embedded glial-restricted progenitors (#221)

I. Malysz-Cymborska1, L. Kalkowski1, D. Golubczyk1, U. Kozlowska2, M. Janowski3, 4, 5, M. Kurpisz2, W. Maksymowicz1, P. Walczak1, 4, 5

1 University of Warmia and Mazury, School of Medicine, Neurology and Neurosurgery, Olsztyn, Poland
2 Institute of Human Genetics Polish Academy of Sciences, Reproductive Biology and Stem Cells, Poznan, Poland
3 Mossakowski Medical Research Centre, Polish Academy of Sciences, NeuroRepair Dept., Warsaw, Poland
4 Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Cellular Imaging Section, Baltimore, Maryland, United States of America
5 The Johns Hopkins University School of Medicine, Russell H. Morgan Dept. of Radiology and Radiological Science, Baltimore, Maryland, United States of America

Introduction

Due to the lack of effective treatment for a variety of neurological disorders,  cell-based therapies  are being actively pursued. Using glial restricted progenitors is one of the most spectacular examples where robust therapeutic effect has been observed (Lyczek et al. 2017). However, the challenge is poor survival of transplant in adult recipients. To address this, we propose to embed the cells in an injectable hydrogel with supplementation to provide optimal support for transplantation. For screening and to optimize the hydrogel composition we used bioluminescence imaging (BLI).

Methods

We used hyaluronic acid-based hydrogels with and without heparin (Glycosil vs. Heprasil; Esi Bio), in cross-linked phase. Luciferase-expressing mouse glial restricted progenitors (GRPs) were isolated as described (Phillips et al. 2012). In vitro toxicity was assessed using luciferase assay for monolayer culture with hydrogel components, 0,5% hydrochloric acid being a reference. Optimization was made with the same manner. Cells were embedded in hydrogels, supplemented with: Gelin-S, Laminin, Fibronectin and a combination of Laminin, Fibronectin and Collagen IV; for 5 days in culture. Monolayer without hydrogel was used as a control. For in vivo viability assay, 1x105 of hydrogel-embedded cells were injected subdermally into NOD mice. Relative luminescence was measured for up to 14 days.

Results/Discussion

Our study has shown that GRP cells tolerated well all components of commercially-available HA hydrogel with no evidence of toxicity (Fig. 1a). Our viability study has shown differences in viability of GRPs between Glycosil and Heprasil, when supplemented with laminin, fibronectin and laminin/fibronectin/collagen IV (Fig. 1b). Relative luminescence was higher in all supplemented-hydrogels groups, compared to monolayer cultures as well as cells embedded in non-supplemented hydrogels. In vivo study with subcutaneous injection of GRPs, revealed highest viability when hydrogels were supplemented with laminin/fibronectin and collagen IV (Fig 1c,d).

Conclusions

In conclusion, HA-based commercial hydrogels with proper ECM supplementation support GRPs survival and promote proliferation. These hydrogels, with potential for utilizing their injectable formulations are promising for improving cell viability for transplantation studies.

References

Lyczek A, Arnold A, Zhang J, Campanelli JT, Janowski M, Bulte JWM, Walczak P. 2017. Transplanted human glial-restricted progenitors can rescue the survival of dysmyelinated mice independent of the production of mature, compact myelin. Experimental Neurology 291:74-86.

Phillips AW, Falahati S, DeSilva R, Shats I, Marx J, Arauz E, Kerr DA, Rothstein JD, Johnston MV, Fatemi A. 2012. Derivation of Glial Restricted Precursors from E13 mice. Journal of Visualized Experiments : JoVE:3462.

Acknowledgement

The study was supported by Strategmed 1/233209/12/NCBIR/2015 and NanoTech4ALS 12/EuroNanoMed/2016.

Figure 1
Relative luminescence comparison for hydrogel formulations; in vitro toxicity (a) and viability (b)  assay after 5 days, in vivo viability at day 0 (c) and 14 (d).
Keywords: bioluminescence, hydrogel, luciferase, regenerative medicine