Microglia are perhaps the most underestimated cell type of our immune system. Not only were immunologists unaware of their capabilities until recently, but also, some neuroscientists denied their actual existence until the late 20th century. Nowadays, their presence is confirmed extensively, as demonstrated by numerous reports describing their involvement in virtually all neuropathologies. However, despite distinct approaches, their origin remains a point of controversy. Although many agree about their myeloid-monocytic ancestry, the precise progenitor cells and the differentiation mechanisms, which give rise to microglia in the different developmental stages of the CNS, are not unraveled yet. Mostly, this can be attributed to their versatile phenotype. Indeed, microglia show a high morphological plasticity, which is related to their functional state. This review about microglia aims to introduce the reader extensively into their ontogeny, cell biology, and involvement in different neuropathologies.

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H2DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.

BackgroundCell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.ResultsIn this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.ConclusionWe here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.

While neural stem cells (NSCs) are widely expected to become a therapeutic agent for treatment of severe injuries to the central nervous system (CNS), currently there are only few detailed preclinical studies linking cell fate with experimental outcome. In this study, we aimed to validate whether IV administration of allogeneic NSC can improve experimental autoimmune encephalomyelitis (EAE), a well-established animal model for human multiple sclerosis (MS). For this, we cultured adherently growing luciferase-expressing NSCs (NSC-Luc), which displayed a uniform morphology and expression profile of membrane and intracellular markers, and which displayed an in vitro differentiation potential into neurons and astrocytes. Following labeling with green fluorescent micron-sized iron oxide particles (f-MPIO-labeled NSC-Luc) or lentiviral transduction with the enhanced green fluorescent protein (eGFP) reporter gene (NSC-Luc/eGFP), cell implantation experiments demonstrated the intrinsic survival capacity of adherently cultured NSC in the CNS of syngeneic mice, as analyzed by real-time bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and histological analysis. Next, EAE was induced in C57BL/6 mice followed by IV administration of NSC-Luc/eGFP at day 7 postinduction with or without daily immunosuppressive therapy (cyclosporine A, CsA). During a follow-up period of 20 days, the observed clinical benefit could be attributed solely to CsA treatment. In addition, histological analysis demonstrated the absence of NSC-Luc/eGFP at sites of neuroinflammation. In order to investigate the absence of therapeutic potential, BLI biodistribution analysis of IV-administered NSC-Luc/eGFP revealed cell retention in lung capillaries as soon as 1-min postinjection, resulting in massive inflammation and apoptosis in lung tissue. In summary, we conclude that IV administration of NSCs currently has limited or no therapeutic potential for neuroinflammatory disease in mice, and presumably also for human MS. However, given the fact that grafted NSCs have an intrinsic survival capacity in the CNS, their therapeutic exploitation should be further investigated, and-in contrast to several other reports-will most likely be highly complex.

Cell transplantation has been suggested to display several neuroprotective and/or neuroregenerative effects in animal models of central nervous system (CNS) trauma. However, while most studies report on clinical observations, currently little is known regarding the actual fate of the cell populations grafted and whether or how the brain's innate immune system, mainly directed by activated microglia and astrocytes, interacts with autologous cellular implants. In this study, we grafted well-characterized neural stem cell, mouse embryonic fibroblast, dendritic cell, bone marrow mononuclear cell, and splenocyte populations, all isolated or cultured from C57BL/6-eGFP transgenic mice, below the capsula externa (CE) of healthy C57BL/6 mice and below the inflamed/demyelinated CE of cuprizone-treated C57BL/6 mice. Two weeks postgrafting, an extensive quantitative multicolor histological analysis was performed in order (i) to quantify cell graft localization, migration, survival, and toxicity and (ii) to characterize endogenous CNS immune responses against the different cell grafts. Obtained results indicate dependence on the cell type grafted: (i) a different degree of cell graft migration, survival, and toxicity and (ii) a different organization of the endogenous immune response. Based on these observations, we warrant that further research should be undertaken to understand-and eventually controlcell graft-induced tissue damage and activation of the brain's innate immune system. The latter will be inevitable before cell grafting in the CNS can be performed safely and successfully in clinical settings.