Multiple sclerosis is a chronic inflammatory disease of the CNS leading to focal destruction of myelin, still the earliest changes that lead to lesion formation are not known. We have studied the geneexpression pattern of 12 samples of normal appearing white matter from 10 post‐mortem MS brains. Microarray analysis revealed upregulation of genes involved in maintenance of cellular homeostasis, and in neural protective mechanisms known to be induced upon ischemic preconditioning. This is best illustrated by the upregulation of the transcription factors such as HIF‐1α and associated PI3K/Akt signalling pathways, as well as the upregulation of their target genes such as VEGF receptor 1. In addition, a general neuroprotective reaction against oxidative stress is suggested. These molecular changes might reflect an adaptation of cells to the chronic progressive pathophysiology of MS. Alternatively, they might also indicate the activation of neural protective mechanisms allowing preservation of cellular and functional properties of the CNS. Our data introduce novel concepts of the molecular pathogenesis of MS with ischemic preconditioning as a major mechanism for neuroprotection. An increased understanding of the underlying mechanisms may lead to the development of new more specific treatment to protect resident cells and thus minimize progressive oligondendrocyte and axonal loss.

Recent evidence suggests that nerve growth factor (NGF), in addition to its neurotrophic functions, acts as an immunomodulator mediating "cross-talk" between neuronal and immune cells, including T lymphocytes. We have analyzed murine CD4+ T-cell clones for their ability to express transcripts encoding NGF, low-affinity NGF receptor, and trk protooncogene, the signal-transducing receptor subunit for NGF. We show that two CD4+ T-helper (Th) Nerve growth factor (NGF), the best characterized member of a family of neurotrophic factors, is required for the differentiation and survival of sympathetic and some sensory and cholinergic neuronal populations (1). In addition to its neurotrophic effects, there is accumulating evidence that NGF is involved in inflammatory responses. For example, NGF increases the number of mast cells in neonatal rats (2), leads to a massive degranulation of rat peritoneal mast cells (3-5), promotes differentiation of specific granulocytes (6), and stimulates wound healing (7). Treatment of young rats with NGF before and after immunization with sheep erythrocytes results in an enhancement of T-lymphocytedependent antibody synthesis (8). In addition, NGF has been shown to enhance lymphocyte proliferation in both B-and T-cell populations (9-11) and to stimulate production of IgG4 (12). These data implicate NGF in the modulation of humoral and cell-mediated immune responses.Studies on the regulatory mechanisms involved in NGF production reveal that inflammatory stimuli contribute significantly to an increased NGF production (13). Interleukin 1 (IL-1), a mediator of inflammation and tissue degradation, is a potent inducer of NGF synthesis in the rat sciatic nerve (14) and in the central nervous system (15). IL-1 (16) and IL-6 (17) both induce increased production and secretion ofbiologically active NGF by astrocytes. The findings that activated rat peritoneal macrophages (18)

Multiple sclerosis is a chronic inflammatory disease of the CNS. Although progressive axonal injury and diffuse inflammatory damage has been shown in the chronic phase of the disease, little is known about the molecular mechanisms underlying these pathological processes. In order to identify these mechanisms, we have studied the gene expression profile in non-lesion containing tissue, the so-called normal-appearing white matter (NAWM). We performed differential gene expression analysis and quantitative RT-PCR on subcortical white matter from 11 multiple sclerosis and 8 control cases. Differentially expressed genes were further analysed in detail by in situ hybridization and immunofluorescence studies. We show that genes known to be involved in anti-inflammatory and protective mechanisms such as STAT6, JAK1, IL-4R, IL-10, Chromogranin C and Hif-1alpha are consistently upregulated in the multiple sclerosis NAWM. On the other hand, genes involved in pro-inflammatory mechanisms, such as STAT4, IL-1beta and MCSF, were also upregulated but less regularly. Immunofluorescence colocalization analysis revealed expression of STAT6, JAK1, IL-4R and IL-13R mainly in oligodendrocytes, whereas STAT4 expression was detected predominantly in microglia. In line with these data, in situ hybridization analysis showed an increased expression in multiple sclerosis NAWM of HIF-1alpha in oligodendrocytes and HLA-DRalpha in microglia cells. The consistency of the expression levels of STAT6, JAK1, JAK3 and IL-4R between the multiple sclerosis cases suggests an overall activation of the STAT6-signalling pathway in oligodendrocytes, whereas the expression of STAT4 and HLA-DRalpha indicates the activation of pro-inflammatory pathways in microglia. The upregulation of genes involved in anti-inflammatory mechanisms driven by oligodendrocytes may protect the CNS environment and thus limit lesion formation, whereas the activation of pro-inflammatory mechanisms in microglia may favour disease progression. Altogether, our data suggests an endogenous inflammatory reaction throughout the whole white matter of multiple sclerosis brain, in which oligodendrocytes actively participate. This reaction might further influence and to some extent facilitate lesion formation.

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

Traumatic spinal cord injury (SCI) disrupts the blood-spinal cord barrier and reduces the blood supply caused by microvascular changes. Vessel regression and neovascularization have been observed in the course of secondary injury contributing to microvascular remodeling after trauma. Spatio-temporal distribution of blood vessels and modulation of gene expression of several angiogenic factors have been investigated in rats after spinal cord compression injury. Rarefaction of vessels was detectable at the injury site 2 days after SCI before they disappeared in the developing cavity after 2 and 4 weeks, whereas no changes were observed in the penumbra. Investigation of the temporal expression of angiogenic genes using quantitative RT-PCR disclosed a constant down-regulation of the vascular endothelial growth factor (VEGF), and transient decreases of angiopoietin-1 (Ang-1), platelet-derived growth factor-BB (PDGF-BB), as well as placental growth factor (PlGF), with the lowest values obtained 3 days after injury, when compared to the expression levels obtained in sham-operated rats. Hepatocyte growth factor (HGF) was the only angiogenic factor with a constant increased gene expression when compared with controls, starting at day 3 post-SCI. mRNA levels of transforming growth factor-beta 1 (TGF-β1) were elevated at every time point following SCI, whereas those encoding for the cysteine-rich protein CCN1/CYR61 were upregulated after 2 h, 6 h, and 1 week only. Our data provide an overview of the temporal modulated expression of the major angiogenic factors, hampering revascularization in the lesion during the phase of secondary injury. These findings should be considered in order to improve therapeutic interventions.