High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)–Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations

This note is to report how histological preparation techniques influence the extravasation pattern of the different molecular sizes of fluorescein isothiocyanate (FITC)–dextrans, typically used as markers for blood-brain barrier leakage. By using appropriate preparation methods, false negative results can be minimized. Wistar rats underwent a 2-h middle cerebral artery occlusion and magnetic resonance imaging. After the last imaging scan, Evans blue and FITC–dextrans of 4, 40, and 70 kDa molecular weight were injected. Different histological preparation methods were used. Sites of blood-brain barrier leakage were analyzed by fluorescence microscopy. Extravasation of Evans blue and high molecular FITC–dextrans (40 and 70 kDa) in the infarcted region could be detected with all preparation methods used. If exposed directly to saline, the signal intensity of these FITC–dextrans decreased. Extravasation of the 4-kDa low molecular weight FITC–dextran could only be detected using freshly frozen tissue sections. Preparations involving paraformaldehyde and sucrose resulted in the 4-kDa FITC–dextran dissolving in these reactants and being washed out, giving the false negative result of no extravasation. FITC–dextrans represent a valuable tool to characterize altered blood-brain barrier permeability in animal models. Diffusion and washout of low molecular weight FITC–dextran can be avoided by direct immobilization through immediate freezing of the tissue. This pitfall needs to be known to avoid the false impression that there was no extravasation of low molecular weight FITC–dextrans

Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b+ and CD90+ within the CD45+ cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5 days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM

Characterization of the murine myeloid precursor cell line MuMac-E8

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies

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Relative mRNA expression of several pluripotency and differentiation markers.

<p>(A) Relative expression of the marker of pluripotency Nanog and Nucleostemin (NST) determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. Nanog was hardly regulated. There were only small increases or decreases in expression. Nucleostemin (NST) showed on day 10 and 25 a 4-fold increase in expression. On all other days of culture variations were found around the control value. (B) Relative expression of the hematopoietic markers EPCR, Sca-1 and the mesenchymal marker CD105 determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. EPCR showed in the first days after synchronization a significant decrease in expression and approached back to the expression level of control cells. In Sca-1 a slightly increased expression was observed, while it approached to the control value 30 days after treatment. CD105 expression rate decreased slightly within 30 days. (C) Relative expression of the hematopoietic markers CD11b and CD45 determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. CD11b expression showed a clear increase up to day 2. Until day 30 the expression levels of CD11b approached to the control value. For CD45, only minor fluctuations were determined around the control value. (D) Relative expression of the neuronal markers Ezrin and Pax-6 determined by quantitative real-time RT-PCR (n = 6 per gene). Shown is the resultant of two independent experiments, mean ± SD. The expressions are shown in relation to control cells (prior to synchronization). Negative values stand for a reduced expression, positive values for an increased expression of each gene compared to control cells. Ezrin showed very moderate expression increases and reductions, which followed no clear pattern. Pax-6 expression was found to be decreased up to 5-fold of the control level between days 3 and 30.</p

CFC assay after 12 days of culture.

<p>(A) MuMac-E8 cells were cultured for 12 days in methylcellulose-based semi-solid medium. The initial cell number was 2×10⁵ cells (magnification 100-fold). Cells revealed a distinct colony formation. The colonies consist of numerous cells of predominantly round shape. By comparison with example images from the manufacturer's instructions MuMac-E8 colonies were identified as CFU-M. (B) May-Grünwald-Giemsa stained MuMac-E8 cells harvested from bulk culture and allowed to adhere in chamber slides.</p

Collagen and methylene blue staining of MuMac-E8 cells.

<p>Used cell number was 1×10⁴. (A) The cultivation was carried out in osteogenic differentiation medium and in (B) normal medium. Isolated yellow-colored cells are visible in both cases. (C) The cultivation was carried out in osteogenic differentiation medium and in (D) normal medium. Isolated blue-colored cells are visible in both cases. There was no colony formation.</p

Optimum cell culture conditions and morphology of cultured MuMac-E8 cells.

<p>By means of real-time cell analysis using the xCELLigence system the optimum cell density (n = 12 per value of cell density, mean ± SD; A) and the optimum serum supplementation for cell synchronization through serum deprivation (B) were determined. Typical morphology of MuMac-E8 cells in culture was shown by inverse phase contrast microscopy (Axiovert, Zeiss). The majority of the cells were found to grow adherent until confluence (C). Visualisation of cell spreading after fluorescence staining of F-actin with Phalloidin-Alexa was performed by confocal laser-scanning microscopy (LSM 510 Meta, Zeiss) (D). Figure was reprint from Biochemica, 4, 14–16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113743#pone.0113743-Pfefferkorn1" target="_blank">[12]</a> with permission from the journal.</p