Student Use of Self-Data for Out-of-Class Graphing Activities Increases Student Engagement and Learning Outcomes.

Two out-of-class graphing activities related to hormonal regulation of the reproductive cycle and stress responses are used to determine whether student use of self-data vs. provided data increases engagement, learning outcomes, and attitude changes. Comparisons of quizzes and surveys for students using self- vs. provided data suggest that while both activities increase learning outcomes, use of self-data compared with provided data has a greater impact on increasing learning outcomes, promotes recognition that hormones are relevant, and enhances confidence in graphing skills and graphing efficacy.

Pharmacological approaches to intervention in hypomyelinating and demyelinating white matter pathology.

White matter disease afflicts both developing and mature central nervous systems. Both cell intrinsic and extrinsic dysregulation result in profound changes in cell survival, axonal metabolism and functional performance. Experimental models of developmental white matter (WM) injury and demyelination have not only delineated mechanisms of signaling and inflammation, but have also paved the way for the discovery of pharmacological approaches to intervention. These reagents have been shown to enhance protection of the mature oligodendrocyte cell, accelerate progenitor cell recruitment and/or differentiation, or attenuate pathological stimuli arising from the inflammatory response to injury. Here we highlight reports of studies in the CNS in which compounds, namely peptides, hormones, and small molecule agonists/antagonists, have been used in experimental animal models of demyelination and neonatal brain injury that affect aspects of excitotoxicity, oligodendrocyte development and survival, and progenitor cell function, and which have been demonstrated to attenuate damage and improve WM protection in experimental models of injury. The molecular targets of these agents include growth factor and neurotransmitter receptors, morphogens and their signaling components, nuclear receptors, as well as the processes of iron transport and actin binding. By surveying the current evidence in non-immune targets of both the immature and mature WM, we aim to better understand pharmacological approaches modulating endogenous oligodendroglia that show potential for success in the contexts of developmental and adult WM pathology. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'.

Finding degrees of separation: experimental approaches for astroglial and oligodendroglial cell isolation and genetic targeting.

The study of CNS glial cell function requires experimental methods to detect, purify, and manipulate each cell population with fidelity and specificity. With the identification and cloning of cell- and stage-specific markers, glial cell analysis techniques have grown beyond physical methods of tissue dissociation and cell culture, and become highly specific with immunoselection of cell cultures in vitro and genetic targeting in vivo. The unique plasticity of glial cells offers the potential for cell replacement therapies in neurological disease that utilize neural cells derived from transplanted neural stem and progenitor cells. In this mini-review, we outline general physical and genetic approaches for macroglial cell generation. We summarize cell culture methods to obtain astrocytes and oligodendrocytes and their precursors, from developing and adult tissue, as well as approaches to obtain human neural progenitor cells through the establishment of stem cells. We discuss popular targeting rodent strains designed for cell-specific detection, selection and manipulation of neuroglial cell progenitors and their committed progeny. Based on shared markers between astrocytes and stem cells, we discuss genetically modified mouse strains with overlapping expression, and highlight SOX-expressing strains available for targeting of stem and progenitor cell populations. We also include recently established mouse strains for detection, and tag-assisted RNA and miRNA analysis. This discussion aims to provide a brief overview of the rapidly expanding collection of experimental approaches and genetic resources for the isolation and targeting of macroglial cells, their sources, progeny and gene products to facilitate our understanding of their properties and potential application in pathology.

FLT-3 expression and function on microglia in multiple sclerosis.

Inflammatory cell infiltration and resident microglial activation within the central nervous system (CNS) are pathological events in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). While MS therapies target the peripheral immune system, no treatment is currently known to also modulate microglia. FMS-like tyrosine-3 (FLT-3) is expressed on hematopoietic and dendritic cells. We reported that FLT-3 inhibition ameliorates early actively induced EAE by predominantly modulating dendritic cell function as compared to microglia. We demonstrate in this report that FLT-3 is expressed in perivascular cuffs, brain parenchyma and in non-lesioned gray and white matter within MS brain but not in these regions within control brain. Furthermore, we demonstrate that FLT-3 is expressed on two populations of cells within MS brain; one which expresses the dendritic cell marker CD209, and the other which does not, suggesting that FLT-3 within MS brain is expressed on infiltrating dendritic cells and a non-dendritic cell such as microglia. Additionally, we report that FLT-3 inhibition in murine microglia blocks, in a dose-dependent manner, IFN-γ-induced expression of MHC class II and CD86, and LPS-induced secretion of IL-6. These data suggest that FLT-3 is involved in microglial cells' capacity to respond to environmental cues to function as antigen presenting cells and mediate CNS inflammation. Furthermore these data suggest that FLT-3 may be a therapeutic target on microglia to mitigate CNS inflammation.

q-space and conventional diffusion imaging of axon and myelin damage in the rat spinal cord after axotomy.

Parallel and perpendicular diffusion properties of water in the rat spinal cord were investigated 3 and 30 days after dorsal root axotomy, a specific insult resulting in early axonal degeneration followed by later myelin damage in the dorsal column white matter. Results from q-space analysis (i.e., the diffusion probability density function) obtained with strong diffusion weighting were compared to conventional anisotropy and diffusivity measurements at low b-values, as well as to histology for axon and myelin damage. q-Space contrasts included the height (return to zero displacement probability), full width at half maximum, root mean square displacement, and kurtosis excess of the probability density function, which quantifies the deviation from gaussian diffusion. Following axotomy, a significant increase in perpendicular diffusion (with decreased kurtosis excess) and decrease in parallel diffusion (with increased kurtosis excess) were found in lesions relative to uninjured white matter. Notably, a significant change in abnormal parallel diffusion was detected from 3 to 30 days with full width at half maximum, but not with conventional diffusivity. Also, directional full width at half maximum and root mean square displacement measurements exhibited different sensitivities to white matter damage. When compared to histology, the increase in perpendicular diffusion was not specific to demyelination, whereas combined reduced parallel diffusion and increased perpendicular diffusion was associated with axon damage.

Diffusion tensor magnetic resonance imaging of Wallerian degeneration in rat spinal cord after dorsal root axotomy.

Diffusion tensor imaging (DTI) and immunohistochemistry were used to examine axon injury in the rat spinal cord after unilateral L(2)-L(4) dorsal root axotomy at multiple time points (from 16 h to 30 d after surgery). Three days after axotomy, DTI revealed a lesion in the ipsilateral dorsal column extending from the lumbar to the cervical cord. The lesion showed significantly reduced parallel diffusivity and increased perpendicular diffusivity at day 3 compared with the contralateral unlesioned dorsal column. These findings coincided with loss of phosphorylated neurofilaments, accumulation of nonphosphorylated neurofilaments, swollen axons and formation of myelin ovoids, and no clear loss of myelin (stained by Luxol fast blue and 2'-3'-cyclic nucleotide 3'-phosphodiesterase). At day 30, DTI of the lesion continued to show significantly decreased parallel diffusivity. There was a slow but significant increase in perpendicular diffusivity between day 3 and day 30, which correlated with gradual clearance of myelin without further significant changes in neurofilament levels. These results show that parallel diffusivity can detect axon degeneration within 3 d after injury. The clearance of myelin at later stages may contribute to the late increase in perpendicular diffusivity, whereas the cause of its early increase at day 3 may be related to changes associated with primary axon injury. These data suggest that there is an early imaging signature associated with axon transections that could be used in a variety of neurological disease processes.

High resolution diffusion tensor imaging of axonal damage in focal inflammatory and demyelinating lesions in rat spinal cord.

Inflammation, demyelination, gliosis and axonal degeneration are pathological hallmarks of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis. Axonal damage is thought to contribute to irreversible damage and functional impairment, but is difficult to quantify. Conventional MRI has been used to assess the inflammatory and demyelinating aspects of MS lesions, but more sensitive and specific methods are needed to identify axonal damage to monitor disease progression and to determine efficacy of putative neuroprotective agents. We used high resolution diffusion tensor imaging (DTI) and fibre tracking to examine the spinal cord in rats with focal dorsal column inflammatory or demyelinating lesions to determine whether DTI measures can be used to detect pathology at the site of the focal lesion and to measure axonal damage in tracts distal to the focal lesion. Distant from the focal lesion, total axon counts, degenerating axon counts and SMI-31 staining, but not Luxol fast blue staining, were significantly correlated with fractional anisotropy, axial diffusivity and radial diffusivity, all of which are derived from the DTI data. These data suggest that high resolution DTI may be a more sensitive method than conventional imaging for detecting axonal damage at sites distant from inflammation.

Immune-mediated neuroprotection of axotomized mouse facial motoneurons is dependent on the IL-4/STAT6 signaling pathway in CD4(+) T cells.

The CD4(+) T lymphocyte has recently been found to promote facial motoneuron (FMN) survival after nerve injury. Signal Transducer and Activator of Transcription (STAT)4 and STAT6 are key proteins involved in the CD4(+) T cell differentiation pathways leading to T helper type (Th)1 and Th2 cell development, respectively. To determine which CD4(+) T cell subset mediates FMN survival, the facial nerve axotomy paradigm was applied to STAT4-deficient (-/-) and STAT6-/- mice. A significant decrease in FMN survival 4 weeks after axotomy was observed in STAT6-/- mice compared to wild-type (WT) or STAT4-/- mice. Reconstituting STAT6-/- mice with CD4(+) T cells obtained from WT mice promoted WT levels of FMN survival after injury. Furthermore, rescue of FMN from axotomy-induced cell death in recombination activating gene (RAG)-2-/- mice (lacking T and B cells) could be achieved only by reconstitution with CD4(+) T cells expressing functional STAT6 protein. To determine if either the Th1 cytokine, interferon-gamma (IFN-gamma) or the Th2 cytokine IL-4 is involved in mediating FMN survival, facial nerve axotomy was applied to IFN-gamma-/- and IL-4-/- mice. A significant decrease in FMN survival after axotomy occurred in IL-4-/- but not in IFN-gamma-/- mice compared to WT mice, indicating that IL-4 but not IFN-gamma is important for FMN survival after nerve injury. In WT mice, intracellular IFN-gamma vs. IL-4 expression was examined in CD4(+) T cells from draining cervical lymph nodes 14 days after axotomy, and substantial increase in the production of both CD4(+) effector T cell subsets was found. Collectively, these data suggest that STAT6-mediated CD4(+) T cell differentiation into the Th2 subset is necessary for FMN survival. A hypothesis relevant to motoneuron disease progression is presented.

CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy.

CD4+ T cells rescue facial motoneurons (FMN) from axotomy-induced cell death. The objective of this study is to determine if the CD4+ T regulatory subsets, CD4+CD25+ T or CD1d-restricted NKT cells are critical for FMN survival after facial nerve axotomy. Surviving FMN within facial motor nuclei from axotomized and control sides 4 weeks after axotomy were counted to determine percent FMN survival. Data generated by applying this paradigm to recombination activating gene-2-deficient mice reconstituted with CD4+ T cells depleted of CD4+CD25+ T cells and to CD1-/- mice, deficient in CD1d-restricted NKT cells, suggest that neither regulatory CD4+ T subset is critical for FMN survival.

Role of the immune system in the maintenance of mouse facial motoneuron viability after nerve injury.

In the field of neuroimmunology, an emerging area of research involves the role that the immune system plays in neural injury and repair. Such immune:neural interactions may involve both neuroprotective and neurodestruction actions. To begin to address the compelling, and clinically relevant, issue of how the immune system impacts neural reparative processes, we combined the well described facial nerve injury paradigm, a simple neural injury model, with various immunodeficient mouse models, in order to delineate the contributing immune cells/factors involved in neural injury and repair. We have discovered a role for the CD4+ T cell in mediating facial motoneuron survival after facial nerve injury in the mouse. In this review, we present an overview of our work to date in this field and discuss future directions relevant to understanding key elements in the crosstalk between the immune:neural systems that develops subsequent to injury and/or trauma.

CD4-positive T cell-mediated neuroprotection requires dual compartment antigen presentation.

Our laboratory discovered that CD4-positive (CD4+) T cells of the immune system convey transitory neuroprotection to injured mouse facial motoneurons (FMNs) (Serpe et al., 1999, 2000, 2003). A fundamental question in the mechanisms responsible for neuroprotection concerns the identity of the cell(s) that serves as the antigen-presenting cell (APC) to activate the CD4+ T cells. Here, we first establish that CD4+ T cells reactive to non-CNS antigen fail to support FMN survival and, second, demonstrate a two-compartment model of CD4+ T cell activation. Mouse bone marrow (BM) chimeras were developed that discriminate between resident antigen-presenting host cell and BM-derived antigen-presenting donor cell expression of major histocompatibility complex II within central and peripheral compartments, respectively. After facial nerve transection, neither compartment alone is sufficient to result in activated CD4+ T cell-mediated FMN survival. Rather, CD4+ T cell-mediated neuroprotection appears to depend on both resident microglial cells in the central compartment and a BM-derived APC in the peripheral compartment. This is the first in vivo report demonstrating a neuroprotective mechanism requiring APC functions by resident (i.e., parenchymal) microglial cells.