EMIM 2019
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Disease Models, Translational Approaches I | Neuroimaging

Session chair: Andreas Hess (Erlangen, Germany); Cornelius Faber (Münster, Germany)
 
Shortcut: PW08
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

909

Serial BLI of Hydrogel-Scaffolded Glial Progenitors Transplanted into Mouse Brain (#196)

Shreyas Kuddannaya1, Wei Zhu1, Chengyan Chu1, Anirudha Singh2, Piotr Walczak1, Jeff Bulte1

1 Johns Hopkins University School of Medicine, Dept. of Radiology, Baltimore, Maryland, United States of America
2 Johns Hopkins University School of Medicine, Dept. of Urology, BALTIMORE, Maryland, United States of America

Introduction

Neurorestorative cell therapies are prone to substantial cell loss (up to 90 %) owing to mechanical stress, hypoxia and immunorejection [1]. We assessed whether the fate and function of glial-restricted progenitor cells (GRPs), myelinating cells that support neuronal survival, can be improved following their scaffolding in hyaluronic acid (HA)-based composite hydrogels. Here, we determined the optimal composition of hydrogel formulations to facilitate injection, enhance survival, and aid with proper tissue integration of scaffolded GRPs.

Methods

Mouse GRPs (mGRPs) expressing GFP and transduced with firefly luciferase (pLenti4-CMV-Luc) were encapsulated within composite hydrogels containing thiolated hyaluronic acid (HA) and gelatin (Gelin-S) with polyethylene (glycol) diacrylate (PEGDA) as crosslinker. Using a 2:2:1 ratio of HA, GelinS and PEGDA, various composite formulations ranging from 5 to 20 mg/ml (all components) were synthesized. Rheological properties were determined using an ARES-G2 rheometer and the in vitro proliferation was assessed with a CCK-8 assay. The in vivo survival of (scaffolded) mGRPs (2x106 cells/100 µl hydrogel or saline) transplanted in immunocompetent BALB/c mice was assessed with bioluminescent imaging (BLI) using an IVIS-Spectrum CT imaging system and validated with histological fluorescence staining.

Results/Discussion

Small variations in the individual components of hydrogel formulations critically affect hydrogel stiffness (Fig. 1a). Scaffolded mGRPs showed enhanced proliferation compared to naked cells (Fig. 1b). In vitro BLI indicated differential cell survival in different hydrogel compositions (Fig. 1c) and with syringe injection a slight cell death was noted (Fig. 1d). On day 10, GFP-positive mGRPs showed distinct cell migration patterns away from the hydrogel scaffolding boundary (Fig. 1c). In vivo BLI readouts from sub-cutaneous and intrastriatal transplantation demonstrated a higher survival of scaffolded mGRPs (Fig. 2a,b) compared to the unscaffolded (naked) mGRPs. T2 weighted MRI and histology revealed a restricted localization of the mGRP-hydrogel graft at the injection/target site (Fig. 2c,d).

Conclusions

Composite hydrogel formulations can be customized to facilitate cell injection and enhance post-transplantation survival of scaffolded GRPs for use in neurorestorative therapies.

References

[1] Marquardt L.M., et al, Design of Injectable Materials to Improve Stem Cell Transplantation. Curr Stem Cell Rep., 2016. 2(3): p. 207–220.

Figure 1
(a) Storage modulus. (b) In vitro mGRP proliferation counts. (c) In vitro BLI of mGRPs scaffolded in different hydrogel formulations including PBS as control. (d) In vitro BLI for injection and normal pipetting conditions. (e) Cell migration from hydrogel boundary (red dotted line) to the surrounding regions. Images were obtained 10 days after plating. Scale bar=100 µm.
Figure 2

(a) Serial in vivo BLI of transplanted mGRPs scaffolded in various hydrogels or PBS. (b) Serial in vivo BLI of 3x105 mGRPs transplanted in mouse brain following scaffolding with optimized (10 mg/ml) hydrogel formulation or PBS as control. (c, d) T2-weighted MRI at day 1 and histological staining at day 16 post-intrastriatal transplantation of hydrogel-scaffolded GFP+ mGRPs.

Keywords: BLI, Cell therapy, Hydrogel
910

Erythropoietin receptors can be targeted in human and rat stroke tissue using [89Zr] ‑Deferoxamine-EPO (#496)

Kristin J. Patzwaldt1, Ramona Stumm1, Francesca Russo2, Laura Kuebler1, Dominik Seyfried1, Andreas Maurer1, Manuela Neumann4, Sven Poli2, Bernd Pichler1, Salvador Castaneda Vega1, 3

1 Eberhard Karls University of Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tübingen, Baden-Württemberg, Germany
2 Eberhard Karls University of Tuebingen, Department of Neurology & Stroke, and Hertie Institute for Clinical Brain Research, Tübingen, Baden-Württemberg, Germany
3 Eberhard Karls University of Tuebingen, Department of Nuclear Medicine and Clinical Molecular Imaging, Tübingen, Baden-Württemberg, Germany
4 Eberhard Karls University of Tuebingen, Division of Neuropathology, Tübingen, Baden-Württemberg, Germany

Introduction

Erythropoietin (EPO) and EPO receptor (EPOr) expression are directly linked to hypoxia in tissues including brain, heart, liver and kidney. EPO is a cytoprotective agent showing neuroprotective effects by inducing angiogenesis, neurogenesis and inhibiting apoptosis [1]. In this study, we prepared [89Zr]-Deferoxamine (Dfo)-EPO and evaluated EPOr expression in the stroke region of human and rat brains. The measurement of EPOr presence through [89Zr]-Dfo-EPO might be a potential tool to measure a tissue’s response to hypoxia.

Methods

Rats underwent surgery using the middle cerebral artery occlusion stroke model (n=9) or sham surgery (n=7). Animals were injected i.v. with [89Zr]-Dfo-EPO 3 h after stroke induction and scanned using PET and MRI at 24, 48 and 72 h after injection. Images were co-registered to MRI and evaluated using Pmod software.

Autoradiography (AR) was performed on human and rat ischemic brain tissues in the acute stroke phase. To determine total binding, tissue sections were incubated with 4.28 nM of [89Zr]-Dfo-EPO for 60 min. Blocking was performed on separate sections using 1320 nM of the nonradioactive EPO. After washing, the dried sections were exposed to AR plates for 24 h and read out with a phosphorimager at 50µm resolution. Image analysis was performed using ImageJ software.

Results/Discussion

Our in vivo results show significant accumulation of [89Zr]-Dfo-EPO starting at 24 h after stroke induction in the stroke area of rats in comparison to healthy controls (p = 0.05) and contralateral hemispheres (p = 0.02). A significant increment continued at 48 and 72 h in comparison to sham (p < 0.01, at 48 & 72 h) and contralateral hemispheres (p < 0.01, at 48 & 72 h).

A specific binding to the stroke area in human and rat samples was observed. Human tissue sections showed binding in stroke tissue of 64.47 ± 18.9 fmol of [89Zr]-Dfo-EPO per mg of brain tissue. Blocked tissue sections showed a significantly lower binding (14.1 ± 4.0 fmol per mg of tissue, p < 0.01).  The stroke area presented significantly increased binding of [89Zr]-Dfo-EPO in contrast to the unspecific background and healthy non-stroke brain tissue (p < 0.01).

Conclusions

In this study we show for the first time that human stroke tissues can be targeted using [89Zr]-Dfo-EPO. The amount of [89Zr]-Dfo-EPO binding in human and rat stroke tissues was consistent between both species. These results demonstrate the potential of [89Zr]-Dfo-EPO to evaluate tissue response to hypoxia [2].

References

[1] M. Buemi et al., “The pleiotropic effects of erythropoietin in the central nervous system,” J. Neuropathol. Exp. Neurol., vol. 62, no. 3, pp. 228–36, Mar. 2003.

[2] M. Brines and A. Cerami, “The receptor that tames the innate immune response,” Mol. Med., vol. 18, no. 3, p. 1, Jan. 2012.

Keywords: Erythropoietin, Stroke, [89Zr]-Desferoxamine-EPO, Hypoxia
911

Lasting effects of adolescence cannabinoid exposure on the brain glucose metabolism and endogenous cannabinoid system after adult morphine consumption (#377)

Nicolás Lamanna1, Marta Casquero-Veiga1, 2, Alejandro Higuera-Matas4, Karina MacDowell5, 6, Emilio Ambrosio4, Manuel Desco1, 2, 3, Maria Luisa Soto-Montenegro1, 2

1 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
2 CIBER de Salud Mental (CIBERSAM), Madrid, Spain
3 Universidad Carlos III de Madrid, Departamento de Bioingeniería e Ingeniería Aeroespacial, Leganés, Spain
4 Universidad Nacional de Educación a Distancia (UNED), Departament of Psychobiology, School of Psychology, Madrid, Spain
5 Complutense University, Department of Pharmacology, Faculty of Medicine, Madrid, Spain
6 Instituto de Investigación Sanitaria Hospital 12 de Octubre (I+12), Madrid, Spain

Introduction

The endocannabinoid system (ECS) is the target of the psychoactive component of cannabis and modulates the rewarding effects of addictive drugs. This study examines the influence of a chronic peripuberal treatment with the synthetic cannabinoid CP-55,940 (CP) on morphine (M) self-administration at adulthood in rats.  Our work benefits from the detection of the brain glucose metabolism through positron emission tomography (PET) studies, as well as from the evaluation of EC receptors and enzymes by means of western blot (WB) analyses.

Methods

37 male and 35 female Wistar rats were daily injected with CP or its vehicle (VH) from postnatal day (PND) 28 to 38. At PND100, rats were trained in an operant task and then in a self-administration  (SA) procedure,  for 15 d. After MSA they underwent extinction sessions for 15 d with saline (sal) instead of M. 4 study groups were evaluated according to 2 factors: pretreatment (CP, VH) and treatment (M, sal). PET (Argus PET/CT, SEDECAL) and MR (Bruker, 7T) images were acquired after 16 weeks of withdrawal. [18F]FDG was injected and, after 45 min of uptake, animals were scanned for 45 min. PET data were analyzed by SPM12 analyses. ROI analyses were performed in amygdala and Caudate-Putamen (CPu). CB1R, CB2R, DAGL, MAGL and FAAH protein expression was measured with WB assays in brain tissue.

Results/Discussion

PET data: Global analysis of brain metabolism revealed an interaction between the pre-treatment and M in both males and females, with a reduction of brain metabolism after MSA in CP-treated animals. M-treatment reduced [18F]FDG uptake in nucleus accumbens (nacc), amygdala, entorhinal cortex (Ent) and CPu in males; and reduced metabolism in the cerebellum and Ent in females as compared to sal-treated rats.  On the other hand, M-treatment in CP pre-exposed animals induced an opposite metabolic pattern. WB data: We found an effect of the sex factor in CB1 and CB2 receptors and the enzymes studied. An effect of the phenotype was found in CB2-CPu and MAGL-nacc in males, CB2-st and CB1-nacc in females. An interaction between pre-treatment and treatment was found in CB1-nacc and DAGL-nacc in males and CB2-st and CB2-nacc in females

Conclusions

M altered the metabolism in limbic and reward areas in a sex- dependent manner. This pattern turned opposite when CP was present; hence suggesting that CP may cause permanent metabolic changes. Also, CP modulated the ECS mainly through receptors and enzymes in the nacc. Together, our results suggest that CP pre-exposure contributes to modulate reward and EC systems, influencing the different vulnerability to abuse drugs such as the opiates.

 

Acknowledgement

This work was partially supported by the Ministry of Economy and Competitiveness ISCIII-FIS grants (PI14/00860, CPII/00005, PI17/01766), co-financed by ERDF (FEDER) Funds from the European Commission, “A way of making Europe”, Fundación Alicia Koplowitz, Fundación Tatiana Pérez de Guzmán el Bueno and Delegación del Gobierno para el Plan Nacional sobre Drogas.

Brain metabolic changes
[A] SPM results in T-maps overlaid on a T2 MR image, showing the brain metabolic changes in morphine and CP-morphine-treated animals in male (left side) and females (right side). Color bars show the T values (lower and higher FDG uptake). Right (R) and Left (L). [B] ROI analysis. Values expressed as mean ± EEM.  ***p<0.001, **p<0.01, *p<0.05 vs saline; &&p<0.01, &p<0.05 vs VH-saline, 2-way ANOVA .
Changes in the endocannabinoid system
Effects of morphine on the expression of receptors [A] and enzymes [B] in males and females pre-exposed to CP55,940 during adolescence. [A] CB1 and CB2 receptor expression in the caudate-putamen (CPu) and nucleus accumbens (nacc). [B] Synthesis enzyme (DAGL) and degradation enzymes (MAGL, FAAH) in the nacc. Data are expressed as the mean ± SEM. ***p<0.001, **p<0.01, *p<0.05 2-way ANOVA.
Keywords: drug addiction, opiates, THC, endocannabionid system, FDG-PET
912

Detection of accumulated iron and microglia in the striatum of Huntington’s disease patients: evidence from post-mortem MRI and histology (#155)

Marjolein Bulk1, Ernst Suidgeest1, Ingrid Hegeman-Kleinn3, Sjoerd van Duinen3, Jan Lewerenz2, Bernhard Landwehrmeyer2, Itamar Ronen1, Louise van der Weerd1

1 Leiden University Medical Center, Radiology, Leiden, Netherlands
2 Ulm University Hospital, Neurology, Ulm, Germany
3 Leiden University Medical Center, Pathology, Leiden, Netherlands

Introduction

T2*-weighted MRI and quantitative susceptibility mapping (QSM) in Huntington’s Disease (HD) patients showed iron accumulation in the striatum correlated with disease state1,2. Although previous studies showed the usefulness of iron-sensitive MRI scans in detecting iron accumulation in HD patients and its potential as a biomarker for disease progression, the underlying pathological substrates have never been investigated. Here we investigated the correlation between 7T MRI and histopathology in the striatum of HD patients and their association with disease severity.

Methods

Formalin-fixed brain material, one whole HD brain and coronal brain slabs including the striatum of HD patients (N=6) and controls (N=3), were obtained respectively from Ulm University Hospital, the local neuropathology tissue collection LUMC, and the Normal Ageing Brain Collection Amsterdam. The whole HD brain and individual brain slabs were scanned on a whole body human 7T MR system. Smaller tissue blocks including the caudate nucleus and putamen were resected and scanned at ultra-high resolution (100μm isotropic) on a 7T Bruker system (Bruker Biospin, Germany). R2*-maps were calculated using an in-house written Matlab pipeline. The same tissue blocks as used for MRI were used for iron histochemistry (Meguro staining).

Results/Discussion

On MRI, lowest signal amplitude was found in the myelin-rich areas of the white matter, followed by the striatum. Additionally, large focal hypointensities were observed in the striatum in HD patients but not in controls (Fig. 1). Striatal R2*-values were significantly increased in HD patients compared to controls and were correlated with neuropathological disease severity as defined by the Vonsattel grade. Currently, we are quantifying the susceptibility changes using QSM. Striatal hypointense regions on MRI colocalized macroscopically with increased iron staining intensity (Fig.1). Microscopic analysis showed an increase of iron in regions which most likely represented the matrix component of the striatum and in cells morphologically resembling glial cells (Fig. 2). Specifically in controls, the microglial-like positive stainings had the appearance of densely packed cells with long, thin processes. In contrast, in HD patients these cells had fewer, thicker, more twisted processes.

Conclusions

Using high-field MRI, we clearly distinguished HD patients from controls, which is mirrored in the histology, mostly in differences in iron. Clinically, the role of iron accumulation is increasingly recognized and shown to be a potential imaging biomarker for disease progression, possibly reflecting neuroinflammation. Susceptibility-based MRI methods could therefore play an important role for further clinical and mechanistic studies.

References

1.            van Bergen JM, Hua J, Unschuld PG, Lim IA, Jones CK, Margolis RL, et al. Quantitative susceptibility mapping suggests altered brain iron in premanifest huntington disease. AJNR Am J Neuroradiol. 2016;37:789-796

2.            Dominguez JF, Ng AC, Poudel G, Stout JC, Churchyard A, Chua P, et al. Iron accumulation in the basal ganglia in huntington's disease: Cross-sectional data from the image-hd study. J Neurol Neurosurg Psychiatry. 2016;87:545-549

Figure 1. Post-mortem 7T MRI of brain slabs of an HD patient and a control.
Lowest signal amplitude was found in the white matter followed by the striatum. In HD patients large focal hypo intensities were found in the striatum. High-resolution 7T MRI ((a,d) magnitude and (b,e) R2*-maps) of smaller tissue blocks of the striatum showed spatial colocalization with histological iron (c,f) in both HD patients and controls. Color bars in R2*-maps range from 0-0.2. CN = caudate nucleus; P = putamen.
Figure 2. Microscopic images of striatal regions with high staining intensity.
Microscopic images of striatal regions with high staining intensity showed a general increase of iron in the matrix and in cells which morphologically closely resemble glial cells (arrows). In HD patients (a) these cells have fewer, thicker, more twisted processes than controls (b). Scale bars: 50 μm.
Keywords: Huntington's disease, iron, microglia, MRI, neuroinflammation
913

N-acetylcysteine and physical exercise as preventive therapies in the schizophrenia onset: A behavioral and imaging study in an animal model (#384)

Diego Romero-Miguel1, Marta Casquero-Veiga1, 2, Vanessa Gómez-Rangel1, Alejandro Higuera-Matas4, Emilio Ambrosio4, Manuel Desco3, 2, 1, María Luisa Soto-Montenegro1, 2

1 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
2 CIBER de Salud Mental (CIBERSAM), Madrid, Spain
3 Universidad Carlos III de Madrid, Departamento de Bioingeniería e Ingeniería Aeroespacial, Madrid, Spain
4 Universidad Nacional de Educación a Distancia, School of Psychology, Department of Psychobiology, Madrid, Spain

Introduction

Oxidative stress and inflammation play an important role in the biochemical mechanisms underlying schizophrenia and other mental disorders. In this sense, N-acetylcysteine (NAC) and physical exercise (Ex) have been reported to induce anti-inflammatory and antioxidant effects. Therefore, the aim of this study is to evaluate whether the combined treatment with NAC and exercise during adolescence in the maternal immune stimulation (MIS) rodent model of schizophrenia could prevent 1) the behavioral deficits and 2) the metabolic and volumetric brain changes described at adulthood.

Methods

At gestational day 15, Poly I:C  or saline were injected to pregnant Wistar rats. 6 study groups were evaluated attending to the factors: phenotype (VH, MIS) & treatment (saline, NAC, NAC+Ex). Treatments started at postnatal day (PND) 35. NAC was orally administered (500mg/kg) for 15 days, and then in the drinking water until PND60. Exercise lasted 12 weeks1. At PND60, sensorimotor gating was tested by prepulse inhibition (PPI) test2. Animals were imaged at PND120. After 45 min of 18FDG uptake, rats were scanned for 45 min (Argus PET/CT, SEDECAL). 2D-OSEM reconstruction was applied and PET images were analyzed by SPM12. T2W-MRI images were acquired with a 7T MRI scanner (Bruker), and ROIs were studied. MRI and PPI data were analyzed with 2 way-ANOVAs and Bonferroni post-hoc test.

Results/Discussion

PET: In VH-offspring, NAC reduced the metabolism in the brain stem (BS), entorhinal cortex and piriform cortex (Pir), while increased it in caudate-putamen and motor and primary somatosensory cortex. In MIS-offspring, NAC reduced the metabolism in the Pir and septum and increase it in the BS. Comparing to VH-saline, VH-NAC+Ex group revealed lower FDG uptake in substantia nigra-hypothalamus-Pir area and BS-cerebellum, and cortical FDG increase. Also, higher cortical FDG uptake was shown in MIS-NAC+Ex animals compared to MIS-saline rats.

MRI: MIS-offspring showed significant volumetric reductions in whole brain, cortex and hippocampus, and volumetric enlargement in ventricles (V), compared to VH. Both hippocampus and V differences were reverted after NAC or NAC+Ex in MIS animals.

PPI: MIS-offspring showed significant PPI deficit (120 ms PP) comparing with VH-offspring. NAC and NAC+Ex treatments induced PPI improvements in both phenotypes, although they were no significant.

Conclusions

NAC and NAC+Ex were able to slightly improve some of the attentional, metabolic and volumetric alterations present in the MIS model3. Thus, both approaches have shown a moderate capability as preventive strategies of the functional and structural schizophrenia deficits, making necessary further research to uncover their real potential in this field.

References

  1. Sastre E, Caracuel L, Balfagón G, & Blanco-Rivero J (2015). Aerobic exercise training increases nitrergic innervation function and decreases sympathetic innervation function in mesenteric artery from rats fed a high-fat diet. Journal of Hypertension, 33(9): 1819–1830.
  2. Santos-Toscano R, Borcel É, Ucha M, et al (2016). Unaltered cocaine self-administration in the prenatal LPS rat model of schizophrenia. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 69: 38-48.
  3. Hadar R, Soto-Montenegro ML, Götz T, et al. (2015). Using a maternal immune stimulation model of schizophrenia to study behavioral and neurobiological alterations over the developmental course. Schizophrenia Research, 166(1-3), 238–247.

Acknowledgement

This work was partially supported by the Ministry of Economy and Competitiveness ISCIII-FIS grants (PI14/00860, CPII/00005, PI17/01766), co-financed by ERDF (FEDER) Funds from the European Commission, “A way of making Europe”, Fundación Alicia Koplowitz, Fundación Tatiana Pérez de Guzmán el Bueno and Delegación del Gobierno para el Plan Nacional sobre Drogas.

Prepulse inhibition protocol
Figure 1 - Behavioural results. Prepulse inhibition (PPI) percentage in two sets of prepulses (12 db 30 ms; 12 db 120 ms) for the 6 groups: VH-saline (n=15), VH-NAC (n=11), VH-NAC+Ex (n=10), MIS-saline (n=13), MIS-NAC (n=13), MIS-NAC+Ex (n=12). Data are expressed as mean ± SEM, p<0.05.
Metabolic and structural imaging results
A) Voxel-based SPM results in T-maps overlaid on a T2 MR image. Color bars in the right represent T values corresponding to lower and higher FDG uptake. Statistical threshold: k>50; p<0.01 (unc.). B) Region of interest (ROI) analysis in the whole brain (WB), cortex (Cx), hippocampus (Hi) and ventricles (V). Values are expressed as mean ± SEM. *p<0.05; ***p<0.001 (Bonferroni post-hoc test).
Keywords: positron emission tomography (PET), magnetic resonance (MRI), N-acetylcysteine, physical exercise, MIS model
914

Multimodal combination of in vivo bioluminescence and magnetic resonance imaging reveales enhanced elements of repair after ischemic brain lesion in Tlr2-deficient mouse (#480)

Srecko Gajovic1, Dunja Gorup1, Siniša Škokić1, Jasna Križ2

1 University of Zagreb School of Medicine, Croatian Institute for Brain Research, Zagreb, Croatia
2 Laval University Faculty of Medicine, Department of Psychiatry and Neuroscience, Quebec, Québec, Canada

Introduction

Previous studies of brain ischemia using Tlr2-deficient mice, including our own, have shown that altering neuroinflammatory response did not result in either beneficial or harmful consequences with regards to the lesion size, but in a combination of both, depending on the time or phase following ischemia. In the acute phase, Tlr2-deficiency reduces the volume of the ischemic lesion, however in the later phase, the resulting modified inflammation leads to delayed apoptosis and a larger sized ischemic lesion at later time points compared to the wild type (WT) animals (1,2).

Methods

To clarify the consequences of the modulated neuroinflammation due to Tlr2-deficiency, this study monitored evolution of ischemic brain lesion through time by multimodal in vivo imaging combining magnetic resonance imaging (MRI), bioluminescence imaging (BLI) and caged-luciferin-BLI (cagedBLI). Ischemic lesion was obtained by temporary middle cerebral artery occlusion. MRI was done using T2 modality, BLI used Gap43-luc/GFP transgenic animals crossbred to Tlr2-deficient mice, and cagedBLI on the same animals but using sensitive to caspases 3 and 7 DEVD-luciferin (VivoGlo, Promega) as a substrate (3). The animals were functionally assessed by neruological scoring, accelerating rotarod, Y-maze, Schallert’s cylinder, and bilateral tactile stimulation.

Results/Discussion

When used as a single modality Gap43 BLI and cagedBLI in 4 different time points (3, 7, 14 and 28 days after ischemia) were comparable between Tlr2-deficient and wild type animals. As the same animals were imaged in the same time points by MRI, we could show the positive correlation of the bioluminescence signal with the MRI-obtained volume of the ischemic lesion. Subsequently, this allowed to normalise the BLI to the lesion size, which revealed statistically significantly higher Gap43 and Casp3 activity in the brains of Tlr-2 deficient animals. The imaging data were confirmed by Western blot, which as well showed the higher levels of synaptic markers DLG and synaptophysin.

Conclusions

Altered inflammation in Tlr2-deficient mice was accompanied by enhanced elements of poststroke repair, in particular during the chronic phase of recovery, but also with delayed apoptosis and final consolidation of the brain lesion. Multimodal imaging allowed to compare the animal groups in a more informative way than single modalities only.

References

  1. Bohacek I, et al. J Neuroinflammation. 2012 Aug 8;9:191.
  2. Winters L,  et al. Neuroscience. 2013;238:87-96.
  3. Gorup D et al. Neurosci Lett. 2015;597:176-82.

Acknowledgement

This study was supported by EU European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant agreement No.KK.01.1.1.01.0007, CoRE – Neuro, and by the Croatian Science Foundation under the project IP-06-2016-1892 (RepairStroke). Multimodal imaging was done at Laboratory for Regenerative Neuroscience - GlowLab, University of Zagreb School of Medicine.

Keywords: MRI, BLI, stroke, ischemia
915

[11C]leucine PET imaging to measure cerebral protein synthesis rate in rats. (#478)

Daniela Bochicchio2, 3, Matthias Vandesquille1, 3, Karl Herholz1, 3, Christine Parker4, Rainer Hinz2, 3, Hervé Boutin1, 3

1 University of Manchester, Faculty of Biology, Medicine and Health, School of Biological Sciences, Division of Neuroscience and Experimental Psychology, Manchester, United Kingdom
2 University of Manchester, Faculty of Biology, Medicine and Health, School of Health Sciences, Division of Informatics, Imaging and Data Sciences, Manchester, United Kingdom
3 University of Manchester, Faculty of Biology, Medicine and Health, Wolfson Molecular Imaging Centre, Manchester, United Kingdom
4 GlaxoSmithKline Research and Development Limited, Experimental Medicine Imaging, Stevenage, United Kingdom

Introduction

Alzheimer’s disease (AD) is characterised by severe alterations of cognitive function and memory which depend on protein synthesis1. Protein synthesis is essential to neuronal plasticity and new connections. The various protein synthesis pathways have been shown to be altered in animal models of AD2 and patients3-5. Here we set up a measurement of protein synthesis rate (PSR) by [11C]leucine PET in rats to characterise the arterial input function (AIF) and determine an image-derived input function (IDIF). These methods will be applied to a longitudinal study in the TgF344-AD rat model of AD.

Methods

Wistar rats (n=6) were anaesthetised with isoflurane (2-2.5% in O2/NO2 30%/70%). A CT scan was first performed; [11C]leucine was injected in the tail vein at the start of the PET scan. Arterial blood activity was monitored continuously over the 60min scans using a Twilite Swisstrace. Discrete blood samples were collected at 2, 5, 10, 20, 30, 40, and 60min post-injection. Whole-blood and plasma samples were then counted in a γ-counter. Free leucine in plasma was measured by γ-counting after precipitation of plasma proteins using 5% perchloric acid. the images. ROIs for the heart left ventricle and vena-cava were segmented automatically using local means analysis (LMA) in the BrainVisa/Anatomist framework6. The [11C]leucine uptake in the brain was quantified with an MRI rat brain atlas7.

Results/Discussion

The PET [11C]leucine time-activity curve (TAC) in the heart ventricle peaked at 22s post-injection (SUV=7.7±1.61), followed by a rapid wash-out, a plateau between 7 and 20min (0.67±0.11) and then a slight increase from 20 to 60min (0.82±0.06) post-injection (Fig. 1a). Whole-blood and plasma γ-counting confirmed these observations. The ratio of free/protein-bound [11C]leucine decreased with time (Fig. 1b). These data are in agreement with previous studies in human1,8 and in rat9.
[11C]leucine uptake in various brain regions showed similar pharmacokinetics with a rapid uptake within 5 minutes post-injection, reaching a plateau from 10 minutes onwards (Fig. 2). PET analysis and γ-counting of different brain regions were in good agreement:  highest [11C]-leucine uptake in cerebellum and cortical regions (0.62±0.07), intermediate in the hippocampus region (0.59±0.06), thalamus (0.54±0.06) and lowest in the striatum (0.51±0.03). These data are also in agreement with previously results1,10,11.

Conclusions

The biodistribution and pharmacokinetics of [11C]-leucine in blood, plasma and brain observed here are similar to those previously reported. γ-Counting and heart-ventricular values as measured by PET are also in good agreement, highlighting the accuracy of PET measurements, and support the feasibility of modelling an IDIF for [11C]-leucine, which we are currently performing. A longitudinal study in the TgF344-AD rat model of AD is also ongoing.

References

1. Bishu S. et al. (2008). J Cereb Blood Flow Metab.28:1502-1513.

2. Pasini S. et al. (2015). Cell Rep.11:183-191.

3. Hoozemans J. J. et al. (2005). Acta Neuropathol.110:165-172.

4. Ma T. et al. (2013). Nat Neurosci.16:1299-1305.

5. Garcia-Esparcia P. et al. (2017). Am J Neurodegener Dis.6:15-25.

6. Maroy R. et al. (2008). IEEE Trans Med Imaging.27:342-354.

7. Schwarz A. J. et al. (2006). Neuroimage.32:538-550.

8. Sundaram S. K. et al. (2006). J Nucl Med.47:1787-1795.

9. Lauenstein L. et al. (1987). Neurosurg Rev.10:147-150.

10. Smith C. B. et al. (2005). J Cereb Blood Flow Metab.25:629-640.

11. Hawkins R. A. et al. (1989). J Cereb Blood Flow Metab.9:446-460.

Acknowledgement

CB and this study are funded by a GSK-University of Manchester PhD studentship.

MV was funded by the EPSRC project EP/M005909/1.

The authors wish to thank all the personnel of the WMIC, especially Ms. Lidan Christie, Ms. Carol Brough, Mr Michael Green and Mr Hamza Al-qasmi for facilitating the study.

Conflict of interest:

Christine A. Parker is an employee of GSK. GSK was not involved in the study design or data analysis.

Figure 1

[11C]leucine in whole-blood (WB) and plasma (P). (A) Whole blood (PET in blue and γ-counting in purple) and plasma (γ-counting in red) time-activity curves. Data shown as standard uptake values (mean±SD, n=6). (B) Percentage of free and protein-bound leucine in plasma at different time point (n=1).

Figure 2

Time-activity curves of [11C]leucine in different brain regions: somatosensory cortex, posterior hippocampus and thalamus (A), frontal cortex and anterior hippocampus (B) and caudate-putamen and cerebellum (C) in Wistar rats. Data shown as SUV (mean±SD, n=6). (D) Coronal, axial and sagittal view of a representative PET scan (20-60min sum image in SUV).

Keywords: brain metabolism, protein synthesis, PET, Input function, Alzheimer's disease
916

Identifying the cerebrovascular changes associated with lipopolysaccharide treatment in a mouse model of Alzheimer’s disease (#461)

Sebastien Serres1, Nerissa Culi1, Faiza Bukenya3, Malcom Prior2, Alessandra Agostini1, Bai Li3, Marie-Christine Pardon1

1 University of Nottingham, School of Life Sciences, Nottingham, United Kingdom
2 University of Nottingham, School of Medicine, Nottingham, United Kingdom
3 University of Nottingham, School of Computer Sciences, Nottingham, United Kingdom

Introduction

Systemic inflammation is regarded as a key contributor to Alzheimer’s disease (AD) with proinflammatory markers associated with cognitive decline and amyloid plaque load. This is in part mediated by vascular inflammation, and can be mimicked in mouse models of AD using lipopolysaccharide (LPS). We therefore tested the hypothesis that LPS, administered systemically to model peripheral inflammation, or intranasally to overcome the blood-brain barrier (BBB) will damage cerebral blood vessels in a mouse model of AD using in vivo magnetic resonance imaging and ex vivo histological analysis. 

Methods

APP/PS1 mice and their wildtype littermates were challenged with LPS (100μg/kg, i.v.) or its vehicle PBS as follows: (i) one injection was given (LPS or PBS) to 16 APP/PS1 and 16 WT littermates and two injections were given. (LPS/LPS, LPS/PBS, PBS/PBS, 7 days apart) to 24 APP/PS1 and 24 WT littermates, all aged 4.5-months-old at the start of the experiment. Histological analysis of cerebral vessel diameter in the cortex and hippocampus was performed using anti-collagen IV. (ii) To assess blood-brain barrier permeability, one injection was given i.n. (LPS, on one side, PBS on the other side) to 4 APP/PS1 and 4 WT females, aged 4 months and 5-month-old APP/PS1 females. Mice underwent T2 map and contrast enhanced T1-weighted MRI to assess oedema and BBB permeability, respectively.

Results/Discussion

Transverse assessment of blood vessels (Fig1A) showed in the single challenge, that females exhibiting significantly larger vessels than males (p<0.05) regardless of genotype and treatment. Longitudinal measurement of blood vessels (Fig1B) showed that a single systemic challenge with LPS significant increased vessel diameters (p<0.001), but this was only significant in WT males. This was also the case in the double challenge experiment (p<0.01). MRI analysis after intranasal administration of PBS and LPS, showed a significant decrease in intensity between hippocampus and cortex on T1 scans in 4-month-old APP/PS1 vs WT mice (p<0.001, Fig2A) and 5-month-old APP/PS1 mice (p<0.05, Fig2C), but a significant increase on T2 map in these mice, regardless of age, treatment and genotype (p<0.05 in all cases, Fig2B-C).

Conclusions

Our findings suggest that vessel diameter respond to systemic inflammation in a sex-dependent manner, with males showing smaller inner lumen diameters than females whilst responding to systemic infection by increasing vessel diameters. MRI scans suggest that BBB is more permeable in the cortex than in the hippocampus, whilst oedema is more likely to occur in the hippocampus rather than the cortex but not affected by intranasal LPS administration.

Acknowledgement

This work was funded by a British Association of Psychopharmacology in vivo training award NRS and MCP and an University of Nottingham Vice-Chancellor scholarship to AA.

Fig1: The effect of systemic injection of LPS on cerebral blood vessels.
(A) Representative image of transverse measurement using collagen IV staining and graphs showing vessel diameter for one or two challenges in APP and WT mice. (B) Representative image of longitudinal measurement using collagen IV staining and graphs showing vessel diameter for one or two challenges in APP and WT mice. N=16-24, mean ± SEM, ANOVA and post-hoc t test; *p<0.05, **p<0.01 and ***p<0.001
Fig2: The effect of systemic injection of LPS on blood-brain barrier and tissue oedema

(A) Representative contrast enhanced T1-weighted MR images of WT (top), APP (4 months; middle), and APP (5 months, bottom) mice. Graphs showing signal intensity for T1-weighted Gadolinium (Gd) enhanced MRI and T2 map in WT and APP (4 months) mice (B) and in APP (4 months) and APP (5 months) mice (C) after LPS or PBS challenge (male or female; N=3-4; mean ± SEM, ANOVA and post-hoc t test;*p <0.05).

Keywords: Alzheimer's disease, MRI, cerebral blood vessels, systemic inflammation, amyloid-beta
917

Plasma pharmacokinetic and metabolism of (S)-[18F]THK5117 and [18F]THK5351 are dependent on sex (#120)

Severin Mairinger1, Thomas Filip1, Michael Sauberer1, Stefanie Flunkert2, Thomas Wanek1, Johann Stanek1, Nobuyuki Okamura3, Claudia Kuntner1

1 AIT Austrian Institute of Technology, Center for Health & Bioresources, Seibersdorf, Austria
2 QPS Austria, Neuropharmacology, Grambach, Austria
3 Tohoku Medical and Pharmaceutical University, Division of Pharmacology, Sendai, Japan

Introduction

Tau deposition is one of the hallmarks of Alzheimer’s disease (AD). The 2-arylquinoline derivatives (S)-[18F]THK5117 and [18F]THK5351 show high affinity for neurofibrillary tangles [1, 2]. Kinetic modelling has been proposed to determine tau binding. However, kinetic modelling is precluded in mouse AD models by their small blood volume, thus rat models with tau pathology may offer the possibility to perform arterial blood sampling and kinetic modelling. To determine the feasibility of this approach, we measured blood pharmacokinetics and radiotracer metabolism in female and male rats.

Methods

Female and male rats (n=10-12) were cannulated via the femoral artery for continuous blood sampling. Blood sampling was performed every 5-6 sec for the first 3 min and then at 5, 10, 20, 30, 40, 50 and 60 min after intravenous injection of (S)-[18F]THK5117 or [18F]THK5351. After collection of the 60 min blood sample, animals were sacrificed and organs were excised. Blood from minute 5, 20 and 60 was centrifuged to obtain plasma. Radiolabelled metabolites in plasma, brain, liver and urine were analyzed by radio-thin-layer chromatography (radio-TLC).

Results/Discussion

Plasma pharmacokinetics and metabolism were significantly different between female and male rats for both radiotracers. (S)-[18F]THK5117 plasma clearance was faster in female (0.66±0.08 mL/h/kg BW) than in male (0.52±0.11 mL/h/kg BW) rats (p=0.005). For (S)-[18F]THK5117, the percentage of unmetabolized parent in plasma was different between both sexes (5 min: 63% (f) vs 69% (m), ns; 20 min: 49% (f) vs 28% (m), p<0.001; 60 min: 32% (f) vs 13% (m), p<0.001) see Fig 1. [18F]THK5351 plasma clearance was faster in female compared to male rats (1.07±0.23 mL/h/kg BW vs 0.67±0.04 mL/h/kg BW) and percentage of unmetabolized parent was also different (5 min: 83% (f) vs 76% (m), ns; 20 min: 50% (f) vs 19% (m), p=0.01; 60 min: 37% (f) vs 4% (m), p<0.001). In the liver, 7% (f) vs 6% (m) unchanged parent was measured for (S)-[18F]THK5117 and 16% (f) vs 3% (m) for [18F]THK5351 (Fig 2). In the brain, 90-98% and 84-90% of total radioactivity consisted of (S)-[18F]THK5117 and [18F]THK5351, respectively.

Conclusions

Our results show pronounced sex differences in blood pharmacokinetics and metabolism of (S)-[18F]THK5117 and [18F]THK5351 in rats. Female animals showed a faster plasma clearance of both radiotracers. These results underline the importance of investigating both sexes and also support the notion that individual input functions are needed for kinetic modelling analyses.

References

[1] Okamura N, Furumoto S, Harada R, Tago T, Yoshikawa T, Fodero-Tavoletti M, et al. Novel 18F-labeled arylquinoline derivatives for noninvasive imaging of tau pathology in Alzheimer disease. J Nucl Med 2013;54:1420-7.

[2] Harada R, Okamura N, Furumoto S, Furukawa K, Ishiki A, Tomita N, et al. 18F-THK5351: A Novel PET Radiotracer for Imaging Neurofibrillary Pathology in Alzheimer Disease. J Nucl Med 2016;57:208-14.

Acknowledgement

The research leading to these results has been funded by the Austrian Research Promotion Agency (FFG) through project 853256.

Figure 1.
Unchanged parent fraction determined in plasma at 5, 20 and 60 min after injection.
Figure 2
Unchanged parent fraction determined in brain, liver and urine at 60 min after injection.
Keywords: tau tracer, plasma PK, metabolism
918

MRI investigation of neuro-anatomical and neuro-functional differences in models of Rett Syndrome (#575)

Sara Carli1, Clarissa Butti2, Angelisa Frasca2, Nicoletta Landsberger2, 1, Linda Chaabane1, 3

1 IRCCS San Raffaele Scientific Institute, Dept. of Neuroscience, Milano, Italy
2 University of Milan, Dept. of Medical Biotechnology and Translational Medicine, Milano, Italy
3 San Raffaele Hospital, INSPE-CIS, Milano, Italy

Introduction

Rett syndrome (RTT) is a devastating neurological disorder caused by mutations in the Methyl-CpG-binding protein-2 (MECP2) gene. Interestingly, metabolic dysregulations have been found in RTT patients and mice models using 1H-MR-Spectroscopy (MRS)1,2. Thus, in order to identify the key brain areas to study by MRS, we initially used manganese contrast (Mn) to highlight the neuronal dysfunctions in two models of RTT, Mecp2-null and knock-in Mecp2-Y120D mice that have similar behavioural phenotypes, but different molecular expressions3.

Methods

Ex-vivo MRI experiments were conducted on a 7-Tesla MRI scanner (Biospec, Bruker-biospin). Brains were taken and fixed 24h after the administration of manganese in all transgenic mice (P150 females and P39 males) and their respective littermate controls. Brains from mice untreated with manganese were also used as negative controls. To quantify Mn uptake, T1 maps were acquired by varying the repetition time (294-4000 ms). To evaluate the anatomical differences, serial transversal sections covering the entire brain (except olfactive bulbs) were acquired with T2-weighted fast spin-echo sequence with a relative high resolution (0.083x0.083x0.7 mm).

Results/Discussion

For both genders, the volume of brains were clearly smaller for both Mecp2-null and Mecp2-Y120D mice (Fig. 1). Interestingly, brain's atrophy is more caudal in the Mecp2-null mice while Mecp2-Y120D animals showed a diffused atrophy throughout the whole brain (Fig. 1). From T1 maps, significant manganese contrast uptake compared to negative controls were measured in several brain's area as the striatum and hippocampus, as expected. Different responses to Mn contrast were observed between genotypes and genders. A diffuse hypo-neuronal activity was found in Mecp2-Y120D males while significant hyperactivity was measured in the pons and regions of the medulla in Mecp2-null males. In females, hypo and hyperactive areas were measured in the mid-brain with a more significant activity of the somatosensorial cortex in Mecp2-Y120D females.

Conclusions

Differences in brain size and manganese uptakes were observed between mice genotypes and gender that are supporting the molecular differences previously found in these models of RTT. According to these results, we will further investigate the metabolic profile of these different brain areas with major impairments with 1H-MR spectroscopy to monitor the cerebral dysregulation at different age.

References

1. Orska A, et al. Quantitative 1H MR spectroscopic imaging in early Rett syndrome. Neurology 2000, 54, 715–722. doi:10.1212/WNL.54.3.715

2. De Filippis B, et al. Modulation of RhoGTPases improves the behavioral phenotype and reverses astrocytic deficits in a mouse model of Rett syndrome. Neuropsychopharmacology. 2012 Apr;37(5):1152-63. doi: 10.1038/npp.2011.301.

3. Gandaglia A, et al. Novel Mecp2(Y120D) Knock-in Model Displays Similar Behavioral Traits But Distinct Molecular Features Compared to the Mecp2-Null Mouse Implying Precision Medicine for the Treatment of Rett Syndrome. Mol Neurobiol. 2018 Nov 6. doi: 10.1007/s12035-018-1412-2.

Figure 1
Example of T2 weighted section of brains from a wild type, Mecp2 null and Y120D mice showing the reduced size of transgenic mice. The brain size measured along the different MRI sections (slice 1 to 10: rostral to caudal) differs between both genotypes (Left graph: Mecp2 null female; Right graph: Mecp2 Y120D female). Statistical analysis were done using Two-way ANOVA, Bonferroni post-hoc test.
Keywords: Rett syndrome, Mecp2 models, MRI anatomy, Manganese Enhanced MRI, Brain atrophy
919

In vivo longitudinal tracking of anti-inflammatory, cerebrovascular and functional effects of a hydroxytyrosol enriched diet after stroke (#559)

Cristina Barca1, 2, Bastian Zinnhardt1, 2, 3, Maximilian Wiesmann4, Claudia Foray1, 2, Michael Schäfers1, 3, Amanda J. Kiliaan4, Andreas H. Jacobs1, 2, 5

1 Westfälische Wilhelms University , European Institute for Molecular Imaging (EIMI) , Münster , North Rhine-Westphalia, Germany
2 PET imaging in Drug Design and Development , PET3D, Münster, Germany
3 Universitätklinikum Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
4 Radboud University medical center , Department of Anatomy, Nijmegen, Netherlands
5 Johanniter Hospital , Department of Geriatrics, Bonn, Germany

Introduction

Novel dietary therapies aim to facilitate stroke recovery by e.g. counteracting immune cells activation/neuroinflammation1,2,3,4. Hydroxytyrosol (HT) shows broad anti-inflammatory and anti-oxidant properties in vitro5, 6.  The aim of this project is therefore to investigate the effects of a long-term HT diet in a murine ischemic stroke on neuroinflammation, cerebrovascular and functional parameters using non-invasive multimodal imaging. We hypothetized HT may positively alter neuroinflammation and so help tissue recovery and functional outcomes. 

Methods

A total of N=10 C57BL/6 mice were provided either a HT-enriched (n=6) or control diet (n=4) for 35 days after a 30 min tMCAo. They underwent combined [18F]DPA-714 (TSPO) PET-CT and MR imaging (T2w imaging, perfusion-/diffusion weighted imaging) to assess immune cell activity, cerebral blood flow and water diffusion respectively at 3, 7, 14, 21 and 30/34 days after tMCAo, as well as behavioural parameters. All imaging datasets were co-registered and analysed using an atlas-based approach. The percentage of injected tracer (%ID/mL) and mean lesion-to-contralateral ratios were calculated (mean±sd).

Results/Discussion

Peak [18F]DPA-714 uptake was observed between day 14 and 21 post ischemia with no significant difference between control and HT fed mice over time, indicating that HT has no effects on TSPO levels.

HT fed mice exhibited enhanced water diffusion (HT: 1.10 ± 0.08, control: 0.97 ± 0.098, ANOVA on ranks, p = 0.038), a sign of altered tissue integrity. However, PW imaging data indicated improved reperfusion of the lesioned compared to control mice at day 34 post-stroke (HT: 0.98 ± 0.13, control: 0.81 ± 0.080, ANOVA on ranks, p = 0.049).

Ex vivo validation and analysis of behavioural data are currently ongoing.

Conclusions

This study indicates that a HT diet may modulate tissue recovery: we didn't observe any change in inflammation in vivo but mice fed with HT showed improved perfusion of the lesioned tissue along with higher water diffusivity, indicative of altered tissue. Still, behavioural and ex vivo data must be added to better appreciate therapeutic effects of HT.

References

1 Lourbopoulos A, Ertürk A, Hellal F. Microglia in action: how aging and injury can change the brain's guardians. Front Cell Neurosci. 2015;9:54

2 Wiesmann M, Zinnhardt B, Reinhardt D, Eligehausen S, Wachsmuth L, Hermann S, Dederen PJ, Hellwich M, Kuhlmann MT, Broersen LM, Heerschap A, Jacobs AH, Kiliaan AJ. A specific dietary intervention to restore brain structure and function after ischemic stroke. Theranostics 2017; 7(2):493-512.

3 Aquilani R, Scocchi M, Boschi F, Viglio S, Iadarola P, Pastoris O. et al. Effect of calorie-protein supplementation on the cognitive recovery of patients with subacute stroke. Nutr Neurosci. 2008;11:235-40.

4 Gardener H, Wright CB, Gu Y, Demmer RT, Boden-Albala B, Elkind MS. et al. Mediterranean-style diet and risk of ischemic stroke, myocardial infarction, and vascular death: the Northern Manhattan Study. Am J Clin Nutr. 2011;94:1458-64

5 Fuccelli R, Fabiani R, Rosignoli P. Hydroxytyrosol Exerts Anti-Inflammatory and Anti-Oxidant Activities in a Mouse Model of Systemic Inflammation. Molecules. 2018 Dec 5;23(12).

6 Illesca P, Valenzuela R, Espinosa A, Echeverría F, Soto-Alarcon S, Ortiz M, Videla LA. Hydroxytyrosol supplementation ameliorates the metabolic disturbances in white adipose tissue from mice fed a high-fat diet through recovery of transcription factors Nrf2, SREBP-1c, PPAR-γ and NF-κB. Biomed Pharmacother. 2019 Jan;109:2472-2481

Acknowledgement

This research was funded by the Horizon2020 Programme under grant agreement n°675417 (PET3D).

Fig 1.
Similar temporal [18F]DPA-714 uptake (%ID/mL) in mice fed with the control diet (green) versus mice fed with the HT-enriched diet (red) after a 30 min tMCAo.
Fig. 2
Water diffusion and cerebral blood flow (CBF) determined by diffusion weighted (DWI)- and perfusion weighted (PWI)- MR imaging indicate hyperdiffusion and higher CBF in HT fed mice compared to control mice at day 34 post ischemia. (*p< 0.05, **p<0.01)
Keywords: Stroke, hydroxytyrosol, neuroinflammation, multimodal imaging