Neuropathologic and biochemical changes during disease progression in liver X receptor beta-/- mice, a model of adult neuron disease.

In amyotrophic lateral sclerosis (ALS), there is selective degeneration of motor neurons that leads to paralysis and death. Although the etiology of ALS is unclear, its heterogeneity suggests that a combination of factors (endogenous and/or environmental) may induce progressive motor neuron stress that results in the activation of different cell death pathways. Alterations of brain cholesterol homeostasis have recently been considered as possible cofactors in many neurodegenerative disorders, including ALS. The liver X receptor beta (LXRbeta) receptor is involved in lipogenesis and cholesterol metabolism, and we previously found that adult-onset motor neuron pathology occurs in LXRbeta mice. Here, we investigated neuromuscular alterations of LXRbeta mice from ages 3 to 24 months. Increased cholesterol levels, gliosis, and inflammation preceded motor neuron loss and clinical disease onset; the mice showed progressivemotor neuron deficits starting from age 7 months. The numbers ofmotor neurons and neuromuscular junctions were decreased in 24-month-old mice, but neither paralysis nor reduced life span was observed. Moreover, other spinal neurons were also lost in these mice. These results suggest that LXRbeta may inhibit neuroinflammation and maintain cholesterol homeostasis, and that LXRbeta mice represent a potential model for investigating the role of cholesterol in ALS and other neurodegenerative disorders

Endogenous erythropoietin as part of the cytokine network in the pathogenesis of experimental autoimmune encephalomyelitis

Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS

MRI of labeled hAFCs. Representative pictures of MRI of healthy (A) and wobbler (B) mice one day before hAFC transplantation.

<p>Axial MRI analysis of the same healthy (<b>C</b>) and wobbler mice (<b>D</b>) at 1, 14 and 56 days after graft. For each panel, the coronal slices are indicative of different regions of the ventricular system including the site close to cell administration (L.V.), the region corresponding to the ventral hippocampus (HP), the region between the brainstem and the cerebellum (CB) and the cervical spinal cord region (S.C.).</p

<i>Ex vivo</i> analyses.

<p>(<b>A</b>) MRI pictures showing axial slices corresponding to the lateral ventricle close to the site of administration. (<b>B</b>) Histological section observed by UV shows the presence of Hoechst 33258 positive nuclei in the ventricular area. (<b>C</b>) MRI axial slice close to the ventral hippocampus. (<b>D</b>) Fluorescent microscopy of histological section revealed a co-localization between Hoechst 33258 and MRI signal. (<b>E</b>) Iron accumulation in the brain section adjacent to that shown in figure D. (<b>F</b>) High magnification picture showing the merge between iron accumulation and Hoechst 33258 positive nuclei. (<b>G</b>) Merge between the carboxydextran immunoreactivity (green) and Hoechst 33258 positive nuclei (blue). (<b>H</b>) Merge between the HLA-I (red) and Hoechst 33258 positive nuclei (blue). Scale bars: B–D–E = 600 µm; F–G = 45 µm; H = 30 µm.</p

<i>In vitro</i> measurements of SPIOn.

<p>(<b>A</b>) Size distribution and (<b>B</b>) zeta-potential measured in distilled water, saline solution and Amniomax II by DLLS. (<b>C–D</b>) Representative images of SPIOn spotted on the mica and visualized by AFM. (<b>C</b>) SPIOn are mainly monodispersed although a small number of chain-like clusters (red arrow) were found. (<b>D</b>) AFM picture shows the spheroid shape of SPIOn. (<b>E</b>) EFTEM images of SPIOn. Single particles were detectable, despite aggregation caused by magnetic forces, confirming data obtained by AFM. Red spots indicate the ESI analysis for iron. Scale bars: C = 1 µm; D–E = 40 µm.</p

Relative quantification of MRI signal.

<p>(<b>A</b>) Acquisition scheme of serial slices selected for the quantification of volumes. (<b>B</b>) Representative slice showing the ROI manually defined for the volume quantification. Light blue areas show the hypo-intense signal, while yellow area show the brain parenchyma. (<b>C</b>) Quantification of the percentage of hypo-intense volume in the brain. No significant difference was found between wobbler mice and healthy controls 1, 14 and 56 days after hAFC transplantation. Data are expressed as mean ± SD (n = 4) for each group. Statistical analysis: Two-Way ANOVA and Bonferroni post test for multiple comparisons.</p

Detection of SPIOn internalization in hAFCs.

<p>(<b>A</b>) Representative picture of Prussian blue staining. Internalization process of SPIOn in hAFCs involved the whole cell population regardless the different morphologies. (<b>B</b>) Higher magnification figure of Prussian blue staining. (<b>C</b>) Representative figure of Hoechst 33258 (blue) and anti-carboxydextran staining (green). (<b>D</b>) Higher magnification picture of carboxydextran and Hoechst 33258 internalization. (<b>E</b>) Aggregates of SPIOn not enclosed by a membrane in the cytoplasm of hAFCs (red arrows). (<b>F</b>) TEM analysis in iron-free hAFCs (F). Scale bars: A–C = 100 µm; B–D = 20 µm. E = 1 µm.</p

FACS analysis of hAFCs.

<p>Evaluation of cell cycle revealed that there were no differences between unlabeled (<b>A</b>) and SPIOn/Hoechst 33258 positive hAFCs (<b>B</b>). In all, 50,000 events per histogram were analyzed (horizontal axis: linear fluorescence intensity; vertical axis: relative cell number). Additional data are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032326#pone-0032326-t002" target="_blank">Table 2</a>.</p

Cytofluorimetric results comparing marker expression, apoptotic/dead cells and number of hAFCs in the different cell cycle phases between controls (unlabeled) and SPIOn/Hoechst 33258 labeled cells.

<p>Data are expressed as the percentage of three independent experiments ± SD. Statistical analysis: Welch's t-test.</p

Experimental groups for <i>in vivo</i> analyses.

<p>Days are referred as days after transplantation (corresponding to Day 0).</p><p>H: healthy mice; W: wobbler mice, M: MRI; L hAFCs: SPIOn/Hoechst 33258 labeled cells; U hAFCs: unlabeled cells; S: sacrifice.</p