minimally-invasive-technique-for-injection-into-rat-optic-nerve

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Johns Hopkins University

Agar-gelatin for embedding tissues prior to paraffin processing.

CSF IL-6 is elevated in transverse myelitis (TM) and predicts disability. Since IL-17 regulates cytokines (TNFalpha, IL-1beta and IL-6) known to stimulate IL-6 production by astrocytes, we sought to determine whether IL-17 was increased in TM and MS compared to healthy controls (HC) and other neurologic diseases (OND). IL-17 and IL-6 levels were measured in stimulated peripheral blood mononuclear cell (PBMC) supernatants from HC, MS, TM and OND. IL-17 was increased in TM compared to HC, MS, and OND (mean pg/ml+/-standard error; HC: 36.1+/-11.7, MS: 89.4+/-23.3, TM: 302.6+/-152.5, OND: 41.2+/-13.0, p=0.01). IL-6 was increased in TM relative to MS and HC (HC: 2624 pg/ml+/-641, MS: 6129+/-982, TM: 12,536+/-2657, OND: 6920+/-1801, p<0.002). MS patients with early disease (<2 years) also had increased levels of IL-17 (p<0.04) and IL-6 (p<0.05). Cytokine neutralization experiments demonstrated that IL-6 was the main inducer of astrocyte IL-6 production. We conclude that IL-17 and IL-6 production from PBMC in TM and early MS are increased and induce astrocyte IL-6 production through IL-6.

Interleukin-17 in transverse myelitis and multiple sclerosis.

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.

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

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.

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

High resolution diffusion tensor images of the mouse brain were acquired using the pulsed gradient spin echo sequence and the oscillating gradient spin echo sequence. The oscillating gradient spin echo tensor images demonstrated frequency-dependent changes in diffusion measurements, including apparent diffusion coefficient and fractional anisotropy, in major brain structures. Maps of the rate of change in apparent diffusion coefficient with oscillating gradient frequency revealed novel tissue contrast in the mouse hippocampus, cerebellum, and cerebral cortex. The observed frequency-dependent contrasts resembled neuronal soma-specific Nissl staining and nuclei-specific 4',6-diamidino-2-phenylindole (DAPI) staining in the mouse brain, which suggests that the contrasts might be related to key features of cytoarchitecture in the brain. In the mouse cuprizone model, oscillating gradient spin echo-based diffusion MRI revealed significantly higher frequency-dependence of perpendicular diffusivity (λ(⊥) ) in the demyelinated caudal corpus callosum at 4 weeks after cuprizone treatment when compared with control mice and mice at 6 weeks after cuprizone treatment. The elevated frequency-dependence of λ(⊥) coincided with the infiltration of activated microglia/macrophages and disruption of axons during acute demyelination in the caudal corpus callosum. The results demonstrate the potential of oscillating gradient spin echo-based diffusion MRI for providing tissue contrasts complimentary to conventional pulsed gradient spin echo-based diffusion MRI.

Probing mouse brain microstructure using oscillating gradient diffusion MRI.

Diffusion tensor imaging has been widely used in studying rodent models of white matter diseases. In this study, we examined the differences between in vivo and ex vivo fractional anisotropy and diffusivity measurements in the mouse cuprizone model. In the control mouse corpus callosum, ex vivo diffusivities were significantly lower than in vivo measurements, but ex vivo fractional anisotropy values were not significantly different from in vivo fractional anisotropy values. With cuprizone induced demyelination and accompanying pathology in the corpus callosum, changes in in vivo and ex vivo fractional anisotropy and diffusivity measurements were not always in agreement. Our results suggest that ex vivo λ(⟂) was a more reliable indicator of white matter demyelination than in vivo λ(⟂) and in vivo λ(‖) was a more reliable indicator of axonal injury than ex vivo λ(‖) in this model. When comparing in vivo and ex vivo diffusion tensor imaging results of axon and myelin pathology in the rodent models, potential changes in tissue microstructures associated with perfusion fixation should be considered.

In vivo and ex vivo diffusion tensor imaging of cuprizone-induced demyelination in the mouse corpus callosum.

Knowledge of the three-dimensional (3D) architecture of blood vessels in the brain is crucial because the progression of various neuropathologies ranging from Alzheimer's disease to brain tumors involves anomalous blood vessels. The challenges in obtaining such data from patients, in conjunction with development of mouse models of neuropathology, have made the murine brain indispensable for investigating disease induced neurovascular changes. Here we describe a novel method for "whole brain" 3D mapping of murine neurovasculature using magnetic resonance microscopy (μMRI). This approach preserves the vascular and white matter tract architecture, and can be combined with complementary MRI contrast mechanisms such as diffusion tensor imaging (DTI) to examine the interplay between the vasculature and white matter reorganization that often characterizes neuropathologies. Following validation with micro computed tomography (μCT) and optical microscopy, we demonstrate the utility of this method by: (i) combined 3D imaging of angiogenesis and white matter reorganization in both, invasive and non-invasive brain tumor models; (ii) characterizing the morphological heterogeneity of the vascular phenotype in the murine brain; and (iii) conducting "multi-scale" imaging of brain tumor angiogenesis, wherein we directly compared in vivo MRI blood volume measurements with ex vivo vasculature data.

Three-dimensional imaging of the mouse neurovasculature with magnetic resonance microscopy.

Myelin-associated glycoprotein (MAG) expressed by oligodendrocytes promotes the stability of axons but also impedes neural repair by inhibiting axon extension through lesioned white matter. We previously reported exacerbated axon losses in MAGKO as compared to wild type mice, 30days into experimental autoimmune encephalitis (EAE). Here, we report the time course of axon losses in EAE and show this occurs as early as 7days post-immunization, confirming MAG is protective against immune-mediated axon transection events. MAGKO mice also exhibit increased microglial activation prior to EAE, which is not seen in B4galnt1KO mice that also have axon loss, suggesting that the microglial activation may be a consequence of the loss of MAG inhibitory influence, and not a simple result of axonal degeneration.

Accelerated axon loss in MOG35-55 experimental autoimmune encephalomyelitis (EAE) in myelin-associated glycoprotein-deficient (MAGKO) mice.

Neuromyelitis optica (NMO) is a devastating inflammatory disorder of the optic nerves and spinal cord characterized by frequently recurring exacerbations of humoral inflammation. NMO is associated with the highly specific NMO-IgG biomarker, an antibody that binds the aquaporin-4 water channel. Aquaporin-4 is present on glial endfeet in the central nervous system (CNS). In humans, the NMO-IgG portends more frequent exacerbations and a worse long-term clinical outcome.

Passively transferred human NMO-IgG exacerbates demyelination in mouse experimental autoimmune encephalomyelitis.

Neuromyelitis optica (NMO) is a recurrent neuroinflammatory disease of the optic nerves and spinal cord associated with the anti-aquaporin-4 (AQP4) antibody biomarker, NMO-IgG. As clinical and scientific research interest in NMO grows, the need for an animal model becomes more urgent. Over the past few years, several groups have developed rodent models that partially represent human NMO disease. Passive transfer of the NMO-IgG is not pathogenic alone, but in certain contexts can recruit granulocytes and lead to increased inflammation. Studies of the cellular immune response against AQP4 have also shed light on the roles of B and T cells in NMO, especially focusing on the role of Th17 T helper cells. This review discusses the contribution of the available NMO animal models to the understanding of NMO disease pathogenesis.

Article	Year
Agar-gelatin for embedding tissues prior to paraffin processing. BioTechniques\| PubMed ID: 17515193	2007
Interleukin-17 in transverse myelitis and multiple sclerosis. Journal of neuroimmunology\| PubMed ID: 18417225	2008
q-space and conventional diffusion imaging of axon and myelin damage in the rat spinal cord after axotomy. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine\| PubMed ID: 20432303	2010
FLT-3 expression and function on microglia in multiple sclerosis. Experimental and molecular pathology\| PubMed ID: 20566414	2010
Probing mouse brain microstructure using oscillating gradient diffusion MRI. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine\| PubMed ID: 21590726	2012
In vivo and ex vivo diffusion tensor imaging of cuprizone-induced demyelination in the mouse corpus callosum. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine\| PubMed ID: 21656567	2012
Three-dimensional imaging of the mouse neurovasculature with magnetic resonance microscopy. PloS one\| PubMed ID: 21818357	2011
Accelerated axon loss in MOG35-55 experimental autoimmune encephalomyelitis (EAE) in myelin-associated glycoprotein-deficient (MAGKO) mice. Journal of neuroimmunology\| PubMed ID: 23899666	2013
Passively transferred human NMO-IgG exacerbates demyelination in mouse experimental autoimmune encephalomyelitis. BMC neurology\| PubMed ID: 23927715	2013
Review of Animal Models of Neuromyelitis Optica. Multiple sclerosis and related disorders\| PubMed ID: 24555175	2012
Evidence for classic complement activity in neuromyelitis optica. Clinical neuropathology\| PubMed ID: 24569172	2014

Melina V. Jones

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