Effect of CXCR2 Inhibition on Behavioral Outcomes and Pathology in Rat Model of Neuromyelitis Optica.

OBJECTIVE: To reduce immune-mediated damage in a rat model of neuromyelitis optica (NMO) by blocking neutrophil migration using SCH527123, a drug that inhibits CXCR2. BACKGROUND: Neuromyelitis optica is a relapsing autoimmune disease that preferentially targets the optic nerves and spinal cord leading to blindness and paralysis. Part of the immunopathogenesis of this disease is thought to involve neutrophils, which are present within NMO lesions. We tested the effect of blocking neutrophil migration in an NMO rat model. METHODS: The Lewis rat model of NMO uses a myelin-reactive experimental autoimmune encephalomyelitis (EAE) background with passive transfer of pooled human antibody from patients with aquaporin-4 (AQP4) seropositive NMO at onset of EAE symptoms. We treated rats early in the course of EAE with CXCR2 inhibitor and assessed the extent of neutrophil infiltration into the spinal cord and the extent of AQP4 depletion. RESULTS: CXCR2 inhibitor decreased neutrophil migration into the spinal cord of AQP4 IgG-treated EAE rats. However, there was no difference in the acute behavioral signs of EAE or the extent and distribution of AQP4 lesions. This suggests that neutrophils are not centrally involved in the immunopathogenesis of the Lewis rat NMO disease model. CONCLUSIONS: CXCR2 inhibitor blocks neutrophil migration into the spinal cord during EAE but does not significantly reduce inflammation or AQP4 lesions in the Lewis rat model of NMO.

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 nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Pathogenic aquaporin-4 reactive T cells are sufficient to induce mouse model of neuromyelitis optica.

INTRODUCTION: Neuromyelitis Optica (NMO) is an autoimmune disease primarily targeting the spinal cord and optic nerve leading to paralysis and blindness. The discovery of an antibody against the astrocytic water channel, aquaporin-4 (AQP4), in the majority of patients, has led to the presumption that the antibody was necessary for disease pathogenesis. The potential role of T cells in the central nervous system, however, has not been thoroughly examined. RESULTS: We generated an anti-AQP4 antibody seronegative model of NMO using pathogenic AQP4-reactive T cells in mice by immunizing AQP4 null mice with peptides corresponding to the second extracellular loop of AQP4, loop C. When polarized to a Th17 phenotype and transferred to wild-type mice, these cells caused tail and limb weakness. Histology showed demyelination and T cell infiltration in the spinal cord, optic nerve and brain. Animals receiving cells re-stimulated in culture with non-specific proteins resulted in no behavioral disease, indicating that specific targeting of AQP4 is essential for this phenotype. CONCLUSIONS: In summary, we show that AQP4-reactive T cells are sufficient to trigger an NMO-like disease in mice, independent of antibodies, indicating that pathogenic AQP4-reactive T cells may play a similar role in humans.

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

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.

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

BACKGROUND: 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. METHODS: We tested the longer-term outcome of mice with EAE injected with NMO-IgG and followed them for 60 days. Clinical exams and pathology of the spinal cord and optic nerves were compared to mice that received control human IgG. RESULTS: Passively transferred human NMO-IgG leads to more severe neurology disability over two months after onset of disease. Clinical worsening is associated with an increased concentration of large demyelinating lesions primarily to subpial AQP4-rich regions of the spinal cord. CONCLUSIONS: NMO-IgG is pathogenic in the context of EAE in mice.

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

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.

Probing mouse brain microstructure using oscillating gradient diffusion MRI.

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.

Review of Animal Models of Neuromyelitis Optica.

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.

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

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.

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.

Axonal protective effects of the myelin-associated glycoprotein.

Progressive axonal degeneration follows demyelination in many neurological diseases, including multiple sclerosis and inherited demyelinating neuropathies, such as Charcot-Marie-Tooth disease. One glial molecule, the myelin-associated glycoprotein (MAG), located in the adaxonal plasmalemma of myelin-producing cells, is known to signal to the axon and to modulate axonal caliber through phosphorylation of axonal neurofilament proteins. This report establishes for the first time that MAG also promotes resistance to axonal injury and prevents axonal degeneration both in cell culture and in vivo. This effect on axonal stability depends on the RGD domain around arginine 118 in the extracellular portion of MAG, but it is independent of Nogo signaling in the axon. Exploiting this pathway may lead to therapeutic strategies for neurological diseases characterized by axonal loss.

Interleukin-17 in transverse myelitis and multiple sclerosis.

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.

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.

Granzyme B mediates neurotoxicity through a G-protein-coupled receptor.

Neuroinflammatory diseases such as multiple sclerosis (MS) are characterized by focal regions of demyelination and axonal loss associated with infiltrating T cells. However, the role of activated T cells in causing neuronal injury remains unclear. CD4 and CD8 T cells were isolated from normal donors and polyclonally activated using plate-bound anti-CD3 and soluble anti-CD28. The conditioned T cell supernatants caused toxicity to cultured human fetal neurons, which could be blocked by immunodepleting the supernatants of granzyme B (GrB). Recombinant GrB also caused toxicity in neurons by caspase-dependent pathways but no toxicity was seen in astrocytes. The neurotoxicity was independent of perforin and could not be blocked by mannose-6-phosphate. However, GrB-induced neurotoxicity was sensitive to pertussis toxin, implicating the stimulation of Gialpha protein-coupled receptors. GrB caused a decrease in cAMP levels but only modest increases in intracellular calcium. The effect on intracellular calcium could be markedly potentiated by stromal-derived factor 1alpha. GrB-induced neurotoxicity could also be blocked by vitamin E and a neuroimmunophilin ligand. In conclusion, GrB may be an important mediator of neuronal injury in T cell-mediated neuroinflammatory disorders.

Cleavage of cystatin C in the cerebrospinal fluid of patients with multiple sclerosis.

OBJECTIVE: The diagnosis of multiple sclerosis (MS) can be challenging because of the lack of a specific diagnostic test. Recent advances in proteomics, however, offer new opportunities for biomarker discovery and the study of disease pathogenesis. METHODS: We analyzed cerebrospinal fluid (CSF) samples from 29 patients with MS or clinically isolated syndromes (CIS), 27 patients with transverse myelitis (TM), 50 patients with human immunodeficiency virus (HIV) infection, and 27 patients with other neurological diseases (ONDs) by surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy. RESULTS: We found a unique protein of 12.5 kDa that was 100% specific for MS/CIS compared with TM or OND. Low levels of this protein were found in some patients with HIV infection. Tandem mass spectroscopy of a tryptic digest of this 12.5 kDa protein identified it as a cleavage product of full-length cystatin C (13.4 kDa), an important inhibitor of cysteine proteases including the cathepsins. Although total cystatin C levels in the MS patients was not different compared with controls, the patients with the highest 12.5/13.4 peak ratios also had the greatest cathepsin B inhibitory activity. INTERPRETATION: This suggests that cleavage of cystatin C may be an adaptive host response and may identify a subgroup of patients with MS.