Premenopausal bilateral ovariectomy is considered to be one of the risk factors of Alzheimer's disease (AD). However, the underlying mechanisms remain unclear. Here, we aimed to investigate long-term neurological consequences of ovariectomy in a rodent AD model, TG2576 (TG), and wild-type mice (WT) that underwent an ovariectomy or sham-operation, using in vivo MRI biomarkers. An increase in osmoregulation and energy metabolism biomarkers in the hypothalamus, a decrease in white matter integrity, and a decrease in the resting-state functional connectivity was observed in ovariectomized TG mice compared to sham-operated TG mice. In addition, we observed an increase in functional connectivity in ovariectomized WT mice compared to sham-operated WT mice. Furthermore, genotype (TG vs. WT) effects on imaging markers and GFAP immunoreactivity levels were observed, but there was no effect of interaction (Genotype xSurgery) on amyloid-beta-and GFAP immunoreactivity levels. Taken together, our results indicated that both genotype and ovariectomy alters imaging biomarkers associated with AD. (C) 2021 Elsevier Inc. All rights reserved.

The abrupt loss of ovarian hormones before menopause, induced by oophorectomy, has been associated with accelerated aging, multimorbidity, and Alzheimer’s disease (AD). We investigated the impact of prolonged loss of ovarian hormones on AD progression in a transgenic mouse model of amyloid pathology using in vivo diffusion tensor MRI, resting‐state functional MRI (rsfMRI) and spectroscopy (MRS).

Alzheimer’s disease (AD) is the most common form of dementia in the elderly. According to the amyloid hypothesis, the accumulation and deposition of amyloid-beta (Aβ) peptides play a key role in AD. Soluble Aβ (sAβ) oligomers were shown to be involved in pathological hypersynchronisation of brain resting-state networks in different transgenic developmental-onset mouse models of amyloidosis. However, the impact of protein overexpression during brain postnatal development may cause additional phenotypes unrelated to AD. To address this concern, we investigated sAβ effects on functional resting-state networks in transgenic mature-onset amyloidosis Tet-Off APP (TG) mice. TG mice and control littermates were raised on doxycycline (DOX) diet from 3d up to 3 m of age to suppress transgenic Aβ production. Thereafter, longitudinal resting-state functional MRI was performed on a 9.4 T MR-system starting from week 0 (3 m old mice) up to 28w post DOX treatment. Ex-vivo immunohistochemistry and ELISA analysis was performed to assess the development of amyloid pathology. Functional Connectivity (FC) analysis demonstrated early abnormal hypersynchronisation in the TG mice compared to the controls at 8w post DOX treatment, particularly across regions of the default mode-like network, known to be affected in AD. Ex-vivo analyses performed at this time point confirmed a 20-fold increase in total sAβ levels preceding the apparition of Aβ plaques and inflammatory responses in the TG mice compared to the controls. On the contrary at week 28, TG mice showed an overall hypoconnectivity, coinciding with a widespread deposition of Aβ plaques in the brain. By preventing developmental influence of APP and/or sAβ during brain postnatal development, we demonstrated FC abnormalities potentially driven by sAβ neurotoxicity on resting-state neuronal networks in mature-induced TG mice. Thus, the Tet-Off APP mouse model could be a powerful tool while used as a mature-onset model to shed light into amyloidosis mechanisms in AD.

Event Abstract Back to Event Increased Soluble Aβ in adult mice causes pathological brain network hypersynchronisation early after induction Inès Ben-Nejma1*, Aneta J. Keliris1, Jasmijn Daans2, Peter Ponsaerts2, Marleen Verhoye1, Annemie Van Der Linden1 and Georgios A. Keliris1 1 Bio-Imaging Lab, University of Antwerp, Belgium 2 University of Antwerp, Belgium Introduction: Soluble amyloid β (sAβ) before plaque deposition has been shown to be synaptotoxic and involved in pathological hypersynchronisation of brain networks by using resting state functional MRI (rsfMRI) in different mouse models overexpressing APP from birth (1). However, the impact of protein overexpression during brain development may cause additional phenotypes unrelated to AD. To address this, we investigated using rsfMRI whether sAβ causes synaptic deficits and impairment of networks before plaque formation in Tet-off APP mice, in which the APP overexpression was switched on in adulthood (2). Methods: Tet-off APP (TG) mice and control littermates have been treated with doxycycline (DOX) to suppress transgenic Aβ production from 3 days up to 3 months of age. The animals were longitudinally scanned on a 9.4T MR system at week 0, 8, 16 and 28 post DOX treatment. The data acquisition was performed using a mixture of medetomidine (0.05mg/kg s.c. bolus; 0.1mg/kg/h s.c. infusion) and a low dose of isoflurane (0.4%). Each imaging session consisted of a rsfMRI scan (2D GE-EPI; TR/TE 2000/17ms; 300 repetitions; 12 coronal slices; 0.208x0.313x0.4mm³) and a T2-weighted 3D anatomical scan (RARE, TR/TE 1800/42ms; RARE factor 16; 0.078x0.078x0.078mm³). Samples for ex vivo (histology, ELISA and electrospray ionization) analyses were collected at 8 and 28 weeks post DOX treatment. Results: Region-of-interest (ROI)-based Functional Connectivity (FC) analysis showed a pathological hypersynchronisation in the TG mice compared to the controls at 8 weeks post DOX treatment (Figure 1B). Ex vivo analyses performed at this time point confirmed the absence of amyloid plaques and showed a 20-fold increase of total sAβ levels in the TG mice compared to the controls. Furthermore, a pathological hyposynchronisation was observed in the TG mice compared to controls at 28 weeks post DOX treatment (Figure 1D) and TG mice presented amyloid plaques in the cortex and the hippocampus. The hyper- and hyposynchronisation were mainly observed in regions within the default mode-like network, which is known to be affected in AD (3). Further analyses (quantitative histology and 3D volume changes) are ongoing. Conclusion: Our results indicate that abnormalities in neuronal network FC, driven by the sAβ synaptotoxicity, also occur when APP expression is switched-on in adulthood, precluding that these effects were caused during development. Thus, the combination of this controllable APP expression model with early in-vivo (rsfMRI) readout for soluble Aβ toxicity sets the stage for future Aβ targeting preventative treatment studies. Figure 1. ROI-based functional connectivity (FC) analysis. A-D. Average Fisher z-transformed FC matrices for Ctrl (top) and Tet-Off APP (bottom) mice at different time point post DOX treatment. The color of each square indicates the correlation coefficient between each pair of ROIs. * p <.05; *** p <.001. Figure 1 Acknowledgements This research was supported by the Fund for Scientific Research Flanders (FWO) (grant agreement G067515N). References 1. Shah D, et al., (2016) Early pathologic amyloid induces hypersynchrony of BOLD resting-state networks in transgenic mice and provides an early therapeutic window before amyloid plaque deposition. Alzheimers Dement. 12, 964–976. doi: 10.1016/j.jalz.2016.03.010 2. Jankowsky JL, et al. (2005) Persistent amyloidosis following suppression of Ab production in a transgenic model of Alzheimer disease. PLoS Med 2(12):e355. 3. Sheline YI, Raichle ME (2013) Resting state functional connectivity in preclinical Alzheimer’s disease. Biol Psychiatry 74:340–347 Keywords: soluble amyloid beta, rsfMRI = resting-state fMRI, Alzheimer ' s disease, Functional Connectivity, Hypersynchronisation, tet-off APP mouse model Conference: 13th National Congress of the Belgian Society for Neuroscience , Brussels, Belgium, 24 May - 24 May, 2019. Presentation Type: Poster presentation Topic: Behavioral/Systems Neuroscience Citation: Ben-Nejma I, Keliris AJ, Daans J, Ponsaerts P, Verhoye M, Van Der Linden A and Keliris GA (2019). Increased Soluble Aβ in adult mice causes pathological brain network hypersynchronisation early after induction. Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience . doi: 10.3389/conf.fnins.2019.96.00038 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Mx. Inès Ben-Nejma, Bio-Imaging Lab, University of Antwerp, Wilrijk, Antwerp, Belgium, Ines.Ben-Nejma@uantwerpen.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Inès Ben-Nejma Aneta J Keliris Jasmijn Daans Peter Ponsaerts Marleen Verhoye Annemie Van Der Linden Georgios A Keliris Google Inès Ben-Nejma Aneta J Keliris Jasmijn Daans Peter Ponsaerts Marleen Verhoye Annemie Van Der Linden Georgios A Keliris Google Scholar Inès Ben-Nejma Aneta J Keliris Jasmijn Daans Peter Ponsaerts Marleen Verhoye Annemie Van Der Linden Georgios A Keliris PubMed Inès Ben-Nejma Aneta J Keliris Jasmijn Daans Peter Ponsaerts Marleen Verhoye Annemie Van Der Linden Georgios A Keliris Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX(3)CR1(+) iPSC-microglia (cultured within a neural environment) and round-shaped CX(3)CR1(-) iPSC-macrophages can easily be differentiated from newly established murine CX(3)CR1(eGFP/+)CCR2(RFP/+) iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX(3)CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharide + interferon gamma or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX(3)CR1(eGFP/+)CCR2(RFP/+) mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment.

Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35–55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. Treatment of MOG35–55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35–55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35–55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.

BACKGROUND:Subtle adjustment of the activation status of CNS resident microglia and peripheral macrophages, to promote their neuroprotective and neuroregenerative functions, may facilitate research towards curing neurodegenerative disorders. In the present study, we investigated whether targeted intracerebral delivery of the anti-inflammatory cytokine interleukin (IL)13, by means of transplanting IL13-expressing mesenchymal stem cells (IL13-MSCs), can promote a phenotypic switch in both microglia and macrophages during the pro-inflammatory phase in a mouse model of ischemic stroke.METHODS:We used the CX3CR1eGFP/+ CCR2RFP/+ transgenic mouse model to separately recognize brain-resident microglia from infiltrated macrophages. Quantitative immunohistochemical analyses were applied to characterize polarization phenotypes of both cell types.RESULTS:Distinct behaviors of both cell populations were noted dependent on the anatomical site of the lesion. Immunohistochemistry revealed that mice grafted with IL13-MSCs, in contrast to non-grafted and MSC-grafted control mice, were able to drive recruited microglia and macrophages into an alternative activation state, as visualized by a significant increase of Arg-1 and a noticeable decrease of MHC-II expression at day 14 after ischemic stroke. Interestingly, both Arg-1 and MHC-II were expressed more abundantly in macrophages than in microglia, further confirming the distinct behavior of both cell populations.CONCLUSIONS:The current data highlight the importance of controlled and localized delivery of the anti-inflammatory cytokine IL13 for modulation of both microglia and macrophage responses after ischemic stroke, thereby providing pre-clinical rationale for the application of L13-MSCs in future investigations of neurodegenerative disorders.

Over the last 30 years, numerous allogeneic and xenogeneic cell grafts have been transplanted into the central nervous system (CNS) of mice and men in an attempt to cure neurological diseases. In the early studies, human or porcine embryonic neural cells were grafted in the striatum of animals or patients in an attempt to replace lost neurons. Although the immune-privileged status of the brain as a recipient organ was widely accepted, it rapidly became evident that CNS-grafted allogeneic and xenogeneic cells could be recognized and rejected by the immune system, resulting in poor neural graft survival and limited functional recovery. Since then, the CNS transplantation field has witnessed a sharp rise in the number of studies in which allogeneic and xenogeneic neural or mesenchymal stem cells (NSCs or MSCs, respectively) are transplanted, predominantly aiming at providing trophic stimulation and promoting endogenous repair of the brain. Interestingly, in many recent NSC and MSC-based publications functional improvement was used as the principal measure to evaluate the success of cell transplantation, while the fate of transplanted cells remained largely unreported. In this review, we first attempt to understand why primary neural cell isolates were largely substituted for NSCs and MSCs in cell grafting studies. Next, we review the current knowledge on the immune mechanisms involved in the recognition and rejection of allogeneic and xenogeneic cellular grafts in the CNS. Finally, we propose strategies to reduce graft immunogenicity and to improve graft survival in order to design improved cell-based CNS therapies. Stem Cells Translational Medicine 2017;6:1434-1441.

Over the past two decades, several cell types with fibroblast-like morphology, including mesenchymal stem/stromal cells, but also other adult, embryonic and extra-embryonic fibroblast-like cells, have been brought forward in the search for cellular therapies to treat severe brain injuries and/or diseases. Although current views in regenerative medicine are highly focused on the immune modulating and regenerative properties of stromal cell transplantation in vivo, many open questions remain regarding their true mode of action. In this perspective, this study integrates insights gathered over the past 10 years to formulate a unifying model of the cellular events that accompany fibroblast-like cell grafting in the rodent brain. Cellular interactions are discussed step-by-step, starting from the day of implantation up to 10 days after transplantation. During the short period that precedes stable settlement of autologous/syngeneic stromal cell grafts, there is a complex interplay between hypoxia-mediated cell death of grafted cells, neutrophil invasion, microglia and macrophage recruitment, astrocyte activation and neo-angiogenesis within the stromal cell graft site. Consequently, it is speculated that regenerative processes following cell therapeutic intervention in the CNS are not only modulated by soluble factors secreted by grafted stromal cells (bystander hypothesis), but also by in vivo inflammatory processes following stromal cell grafting. Copyright © 2016 John Wiley & Sons, Ltd.

Detrimental inflammatory responses in the central nervous system are a hallmark of various brain injuries and diseases. With this study we provide evidence that lentiviral vector-mediated expression of the immune-modulating cytokine interleukin 13 (IL-13) induces an alternative activation program in both microglia and macrophages conferring protection against severe oligodendrocyte loss and demyelination in the cuprizone mouse model for multiple sclerosis (MS). First, IL-13 mediated modulation of cuprizone induced lesions was monitored using T2 -weighted magnetic resonance imaging and magnetization transfer imaging, and further correlated with quantitative histological analyses for inflammatory cell influx, oligodendrocyte death, and demyelination. Second, following IL-13 immune gene therapy in cuprizone-treated eGFP+ bone marrow chimeric mice, we provide evidence that IL-13 directs the polarization of both brain-resident microglia and infiltrating macrophages towards an alternatively activated phenotype, thereby promoting the conversion of a pro-inflammatory environment toward an anti-inflammatory environment, as further evidenced by gene expression analyses. Finally, we show that IL-13 immune gene therapy is also able to limit lesion severity in a pre-existing inflammatory environment. In conclusion, these results highlight the potential of IL-13 to modulate microglia/macrophage responses and to improve disease outcome in a mouse model for MS. GLIA 2016;64:2181-2200.

Background Promoting the neuroprotective and repair-inducing effector functions of microglia and macrophages, by means of M2 polarisation or alternative activation, is expected to become a new therapeutic approach for central nervous system (CNS) disorders in which detrimental pro-inflammatory microglia and/or macrophages display a major contribution to the neuropathology. In this study, we present a novel in vivo approach using intracerebral grafting of mesenchymal stem cells (MSC) genetically engineered to secrete interleukin 13 (IL13-MSC). Methods In the first experimental setup, control MSC and IL13-MSC were grafted in the CNS of eGFP + bone marrow chimaeric C57BL/6 mice to histologically evaluate IL13-mediated expression of several markers associated with alternative activation, including arginase1 and Ym1, on MSC graft-recognising microglia and MSC graft-infiltrating macrophages. In the second experimental setup, IL13-MSC were grafted on the right side (or on both the right and left sides) of the splenium of the corpus callosum in wild-type C57BL/6 mice and in C57BL/6 CX 3 CR1 eGFP/+ CCR2 RFP/+ transgenic mice. Next, CNS inflammation and demyelination was induced by means of a cuprizone-supplemented diet. The influence of IL13-MSC grafting on neuropathological alterations was monitored by non-invasive T 2 -weighted magnetic resonance imaging (MRI) and quantitative histological analyses, as compared to cuprizone-treated mice with control MSC grafts and/or cuprizone-treated mice without MSC injection. Results In the first part of this study, we demonstrate that MSC graft-associated microglia and MSC graft-infiltrating macrophages are forced into alternative activation upon grafting of IL13-MSC, but not upon grafting of control MSC. In the second part of this study, we demonstrate that grafting of IL13-MSC, in addition to the recruitment of M2 polarised macrophages, limits cuprizone-induced microgliosis, oligodendrocyte death and demyelination. Furthermore, we here demonstrate that injection of IL13-MSC at both sides of the splenium leads to a superior protective effect as compared to a single injection at one side of the splenium. Conclusions Controlled and localised production of IL13 by means of intracerebral MSC grafting has the potential to modulate cell graft- and pathology-associated microglial/macrophage responses, and to interfere with oligodendrocyte death and demyelinating events in the cuprizone mouse model.

Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissue-for the in situ delivery of a wide variety of MSC-secreted therapeutic proteins-is an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point-of-view, allogeneic MSCs have numerous advantages over patient-specific autologous MSCs since "off-the-shelf" cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen-presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the host's immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin-13 (IL13)-expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13-expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen-reactive IFNγ- and/or IL2-producing CD8(+) T cells compared to nonmodified allografts. Similarly, intracerebral IL13-expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. Stem Cells 2016;34:1971-1984.

Conventional MRI is frequently used during the diagnosis of multiple sclerosis but provides only little additional pathological information. Proton MRS (1H‐MRS), however, provides biochemical information on the lesion pathology by visualization of a spectrum of metabolites. In this study we aimed to better understand the changes in metabolite concentrations following demyelination of the white matter. Therefore, we used the cuprizone model, a well‐established mouse model to mimic type III human multiple sclerosis demyelinating lesions. First, we identified CX3CL1/CX3CR1 signaling as a major regulator of microglial activity in the cuprizone mouse model. Compared with control groups (heterozygous CX3CR1+/− C57BL/6 mice and wild type CX3CR1+/+ C57BL/6 mice), microgliosis, astrogliosis, oligodendrocyte cell death and demyelination were shown to be highly reduced or absent in CX3CR1−/− C57BL/6 mice. Second, we show that 1H‐MRS metabolite spectra are different when comparing cuprizone‐treated CX3CR1−/− mice showing mild demyelination with cuprizone‐treated CX3CR1+/+ mice showing severe demyelination and demyelination‐associated inflammation. Following cuprizone treatment, CX3CR1+/+ mice show a decrease in the Glu, tCho and tNAA concentrations as well as an increased Tau concentration. In contrast, following cuprizone treatment CX3CR1−/− mice only showed a decrease in tCho and tNAA concentrations. Therefore, 1H‐MRS might possibly allow us to discriminate demyelination from demyelination‐associated inflammation via changes in Tau and Glu concentration. In addition, the observed decrease in tCho concentration in cuprizone‐induced demyelinating lesions should be further explored as a possible diagnostic tool for the early identification of human MS type III lesions. Copyright © 2015 John Wiley & Sons, Ltd.

Transplantation of neural stem cells (NSC) in diseased or injured brain tissue is widely studied as a potential treatment for various neurological pathologies. However, effective cell replacement therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers after transplantation. In this context, it is hypothesized that structural support for grafted NSC will be of utmost importance. With this study, we present a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular three-dimensional (3D) brain sections. Next, the obtained 3D brain sections were seeded with murine NSC expressing both the eGFP and luciferase reporter proteins (NSC-eGFP/Luc). Using real-time bioluminescence imaging, the survival and growth of seeded NSC-eGFP/Luc cells was longitudinally monitored for 1-7 weeks in culture, indicating the ability of the acellular brain sections to support sustained ex vivo growth of NSC. Next, the organization of a 3D maze-like cellular structure was examined using confocal microscopy. Moreover, under mitogenic stimuli (EGF and hFGF-2), most cells in this 3D culture retained their NSC phenotype. Concluding, we here present a novel protocol for decellularization of mouse brain sections, which subsequently support long-term 3D culture of undifferentiated NSC.

Although implantation of cellular material in the central nervous system (CNS) is a key direction in CNS regenerative medicine, this approach is currently limited by the occurrence of strong endogenous immune cell responses. In a model of mesenchymal stem cell (MSC) grafting in the CNS of immune-competent mice, we previously described that MSC grafts become highly surrounded and invaded by Iba1(+) myeloid cells (microglia and/or macrophages). Here, following grafting of blue fluorescent protein (BFP)-expressing MSC in the CNS of CX(3)CR1(+/-) and CX(3)CR1(-/-) mice, our results indicate: (1) that the observed inflammatory response is independent of the fractalkine signalling axis, and (2) that a significant spatial distribution of Iba1(+) inflammatory cells occurs, in which Iba1(+) CX(3)CR1(+) myeloid cells mainly surround the MSC graft and Iba1(+) CX(3)CR1(+) myeloid cells mainly invade the graft at 10 days post transplantation. Although Iba1(+) CX(3)CR1(+) myeloid cells are considered to be of resident microglial origin, Iba1(+) CX(3)CR1(+) myeloid cells are most likely of peripheral monocyte/macrophage origin. In order to confirm the latter, we performed MSC-BFP grafting experiments in the CNS of eGFP(+) bone marrow chimeric C57BL/6 mice. Analysis of MSC-BFP grafts in the CNS of these mice confirmed our observation that peripheral monocytes/macrophages invade the MSC graft and that resident microglia surround the MSC graft site. Furthermore, analysis of major histocompatibility complex class II (MHCII) expression revealed that mainly macrophages, but not microglia, express this M1 pro-inflammatory marker in the context of MSC grafting in the CNS. These results again highlight the complexity of cell implantation immunology in the CNS.

Preclinical animal studies involving intracerebral (stem) cell grafting are gaining popularity in many laboratories due to the reported beneficial effects of cell grafting on various diseases or traumata of the central nervous system (CNS). In this chapter, we describe a histological workflow to characterize and quantify cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue. First, we provide standardized protocols to isolate and culture eGFP(+) neural and fibroblast(-like) stem cells from embryonic mouse tissue. Second, we describe flow cytometric procedures to determine cell viability, eGFP transgene expression, and the expression of different stem cell lineage markers. Third, we explain how to induce reproducible demyelination in the CNS of mice by means of cuprizone administration, a validated mouse model for human multiple sclerosis. Fourth, the technical procedures for cell grafting in the CNS are explained in detail. Finally, an optimized and validated workflow for the quantitative histological analysis of cell graft survival and endogenous astroglial and microglial responses is provided.

While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.

Although implantation of cellular material in the central nervous system (CNS) is a key direction in CNS regenerative medicine, this approach is currently limited by the occurrence of strong endogenous immune cell responses. In a model of mesenchymal stem cell (MSC) grafting in the CNS of immune-competent mice, we previously described that MSC grafts become highly surrounded and invaded by Iba1 + myeloid cells (microglia and/or macrophages). Here, following grafting of blue fluorescent protein (BFP)-expressing MSC in the CNS of CX 3 CR1 +/− and CX 3 CR1 −/− mice, our results indicate: (1) that the observed inflammatory response is independent of the fractalkine signalling axis, and (2) that a significant spatial distribution of Iba1 + inflammatory cells occurs, in which Iba1 + CX 3 CR1 + myeloid cells mainly surround the MSC graft and Iba1 + CX 3 CR1 − myeloid cells mainly invade the graft at 10 days post transplantation. Although Iba1 + CX 3 CR1 + myeloid cells are considered to be of resident microglial origin, Iba1 + CX 3 CR1 − myeloid cells are most likely of peripheral monocyte/macrophage origin. In order to confirm the latter, we performed MSC-BFP grafting experiments in the CNS of eGFP + bone marrow chimeric C57BL/6 mice. Analysis of MSC-BFP grafts in the CNS of these mice confirmed our observation that peripheral monocytes/macrophages invade the MSC graft and that resident microglia surround the MSC graft site. Furthermore, analysis of major histocompatibility complex class II (MHCII) expression revealed that mainly macrophages, but not microglia, express this M1 pro-inflammatory marker in the context of MSC grafting in the CNS. These results again highlight the complexity of cell implantation immunology in the CNS.

Stem cell transplantation in the central nervous system (CNS) is currently under intensive investigation as a novel therapeutic approach for a variety of brain disorders and/or injuries. However, one of the main hurdles at the moment is the lack of standardized procedures to evaluate cell graft survival and behavior following transplantation into CNS tissue, thereby leading to the publication of confusing and/or conflicting research results. In this chapter, we therefore provide validated in vivo bioluminescence and postmortem histological procedures to quantitatively determine: (a) the survival of grafted stem cells, and (b) the microglial and astroglial cell responses following cell grafting.

Although intracerebral transplantation of various fibroblast(-like) cell populations has been shown feasible, little is known about the actual in vivo remodeling of these cellular grafts and their environment. In this study, we aimed to compare the in vitro and in vivo behavior of two phenotypically similar—but developmentally distinct—fibroblast-like cell populations, namely, mouse embryonic fibroblasts (mEFs) and mouse fetal membrane-derived stromal cells (mFMSCs). While both mEFs and mFMSCs are readily able to reduce TNF-α secretion by LPS/IFN-γ-activated BV-2 microglia, mFMSCs and mEFs display strikingly opposite behavior with regard to VEGF production under normal and inflammatory conditions. Whereas mFMSCs downregulate VEGF production upon coculture with LPS/IFN-γ-activated BV-2 microglia, mEFs upregulate VEGF production in the presence of LPS/IFN-γ-activated BV-2 microglia. Subsequently, in vivo grafting of mFMSCs and mEFs revealed no difference in microglial and astroglial responses toward the cellular grafts. However, mFMSC grafts displayed a lower degree of neoangiogenesis compared to mEF grafts, thereby potentially explaining the lower cell number able to survive in mFMSC grafts. In summary, our results suggest that physiological differences between fibroblast-like cell populations might lie at the basis of variations in histopathological and/or clinical outcome following cell grafting in mouse brain.