David Pitt

Glutamate excitotoxicity in a model of multiple sclerosis

Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.

Multiple sclerosis: altered glutamate homeostasis in lesions correlates with oligodendrocyte and axonal damage.

Glutamate excitotoxicity, recently demonstrated in an animal model of multiple sclerosis (MS), is evoked by altered glutamate homeostasis. In the present study, we investigated the major regulating factors in glutamate excitotoxicity by immunohistochemistry in MS and control white matter with markers for glutamate production (glutaminase), glutamate transport (GLAST, GLT‐1 and EAAT‐1), glutamate metabolism (glutamate dehydrogenase [GDH] and glutamine synthetase [GS]), axonal damage (SMI 32) and CNS cell types. Active MS lesions showed high‐level glutaminase expression in macrophages and microglia in close proximity to dystrophic axons. Correlation between glutaminase expression and axonal damage was confirmed experimentally in animals. White matter from other inflammatory neurologic diseases displayed glutaminase reactivity, whereas normals and noninflammatory conditions showed none. All three glutamate transporters were expressed robustly, mainly on oligodendrocytes, in normal, control and MS white matter, except for GLT‐1, which showed low‐level expression around active MS lesions. GS and GDH were present in oligodendrocytes in normal and non‐MS white matter but were absent from both active and chronic silent MS lesions, suggesting lasting metabolic impediments. Thus, imbalanced glutamate homeostasis contributes to axonal and oligodendroglial pathology in MS. Manipulation of this imbalance may have therapeutic import.

Glutamate uptake by oligodendrocytes Implications for excitotoxicity in multiple sclerosis

Background: Excitotoxic damage is a common pathologic event in a number of neurologic diseases occurring after accumulation of excess extracellular glutamate in the CNS and subsequent overstimulation of glutamate receptors. In gray matter, astrocytes take up synaptically released glutamate and are thus key cells in maintaining glutamate homeostasis. In white matter, oligodendrocytes have been shown to express glutamate transporters, but their role in extracellular glutamate removal is unclear. Objective: To investigate whether cultured human fetal oligodendrocytes functionally express the main glutamate transporters EAAT-1 and EAAT-2. Methods: Cultures of fetal human oligodendrocytes were examined by immunocytochemistry and [3H]glutamate uptake, and the findings were correlated with glutamate transporter expression in normal and multiple sclerosis (MS) CNS tissue. Results: Both EAAT-1 and EAAT-2 were expressed by human oligodendrocytes in vitro. Incubation of oligodendrocytes with the proinflammatory cytokine tumor necrosis factor-α (TNFα) reduced EAAT-1 expression and inhibited glutamate uptake by >75%. Furthermore, in normal human white matter, oligodendrocytes were found to be the predominant cells to express EAAT-1 and EAAT-2, both at the mRNA and at the protein level. A small number of astrocytes in white matter expressed these receptors, more so EAAT-1 than EAAT-2. In MS white matter, oligodendrocytes lost expression of EAAT-1 and EAAT-2 receptors in the lesion vicinity. Conclusions: Oligodendrocytes appear to be predominant cells for glutamate clearance in human white matter. Glutamate receptor expression and glutamate removal were defective in MS white matter, possibly mediated by TNFα, changes that might underlie high extracellular glutamate and an increased risk for glutamate excitotoxicity.

B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes

In multiple sclerosis patients, B cells mature in the draining cervical lymph nodes before trafficking across the blood-brain barrier. B Cells Flip the Switch for MS B cells in multiple sclerosis (MS) patients may mature outside the central nervous system (CNS). Two complementary studies report that B cells found outside the CNS—in peripheral blood and draining cervical lymph nodes (CLNs)—share antigen specificity with intrathecal B cell repertoires. In patients with MS, immune cells attack the CNS; however, it remains unclear whether these cells mature in the CNS or traffic to the CNS as mature cells. Using paired tissues and high-throughput sequencing, Stern et al. found that clonally expanded B cells are found in both the CNS and CLNs but that founding members were more often found in the draining CLNs. Palanichamy et al. extend these findings by reporting a peripheral blood/CNS axis of mature B cells that have undergone class switch. These data support the therapeutic use of monoclonal antibodies that prevent lymphocytes from crossing the blood-brain barrier or induce peripheral B cell depletion in MS patients. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune-mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF), and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS-resident B cells encounter antigen and experience affinity maturation. Paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clones were more often found in the draining CLNs. More mature clonal members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier, with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or affect the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.

Imaging Cortical Lesions in Multiple Sclerosis With Ultra–High-Field Magnetic Resonance Imaging

OBJECTIVE To determine the sensitivity of T2*-weighted gradient-echo (T2*GRE) and inversion recovery turbo-field-echo (TFE) sequences for cortical multiple sclerosis lesions at 7 T. DESIGN, SETTING, AND PARTICIPANTS Autopsied brain tissue from individuals with multiple sclerosis was scanned with 3-dimensional T2*GRE and 3-dimensional inversion recovery white matter-attenuated TFE sequences at 7 T. Cortical lesions visible with either sequence were scored for each anatomical lesion type. Imaged brain tissue was then processed for immunohistochemical analysis, and cortical lesions were identified by labeling with antibody against myelin basic protein and CD68 for microglia. Magnetic resonance images were matched with corresponding histological sections and scored retrospectively to determine the sensitivity for each cortical lesion type. Main Outcome Measure Cortical lesion detection by 3-dimensional T2*GRE and white matter-attenuated TFE sequences. RESULTS The 3-dimensional T2*GRE and white matter-attenuated TFE sequences retrospectively detected 93% and 82% of all cortical lesions, respectively (with varying sensitivities for different lesion types). Lesion visibility was primarily determined by size as all undetected lesions were smaller than 1.1 mm at their smallest diameter. The T2*GRE images showed hypointense rings in some cortical lesions that corresponded with increased density of activated microglia. CONCLUSIONS Three-dimensional T2*GRE and white matter-attenuated TFE sequences at a 7-T field strength detect most cortical lesions in postmortem multiple sclerosis tissue. This study indicates the potential of T2*GRE and white matter-attenuated TFE sequences in ultra-high-field magnetic resonance imaging for cortical lesion detection in patients with multiple sclerosis.

Crowding norms at frontcountry sites: A visual approach to setting standards of quality

Indicators and standards of quality have emerged as key variables in defining and managing carrying capacity. Moreover, normative theory has been found useful when applied to the difficult task of setting standards of quality. However, the traditional numerical approach to measuring crowding and related norms has several potential shortcomings when applied to relatively high‐use areas such as frontcountry attraction sites. This study applies a visual approach to measuring crowding norms in addition to the traditional numerical approach. The study is focused on Delicate Arch, a primary visitor attraction in Arches National Park, Utah. Image capture technology was used to develop a series of 16 photographs illustrating a range of people at one time at Delicate Arch. A random sample of 473 visitors was asked to rate the acceptability of each photograph as part of a survey conducted in the summer and fall of 1993. A strong relationship was found between the number of people in the photographs and acceptabilit...

Experimental Autoimmune Encephalomyelitis (EAE) in CCR2−/− Mice: Susceptibility in Multiple Strains

Chemokines are low molecular weight cytokines which act as chemoattractants for infiltrating cells bearing appropriate receptors (CCR) to sites of inflammation. It has been proposed that CCR2 on monocytes is responsible for their recruitment into the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, and two previous reports have described resistance of CCR2(-/-) mice to EAE. The present study examined three different mouse strains with CCR2 deletions for susceptibility to EAE. Animals were studied up to 4 months post-sensitization and were examined by neuropathology, RNase protection assay, in situ hybridization, and in vitro assays. All three strains were found to be susceptible to EAE: C57BL/6 x J129 and Balb c strains, 100%; and C57BL/6, 67%. Unusual in CNS lesions of CCR2(-/-) mice was an overabundance of neutrophils versus monocytes in wild-type animals. An attempt of the immune system to develop compensatory mechanisms for the lack of CCR2 was evidenced by a corresponding increase in mRNA for other chemokines and CCR. Inasmuch as neutrophils replaced monocytes and led to demyelination, our findings support the concept that promiscuity of chemokines and CCR was able to surmount the deletion of CCR2, still resulting in full expression of this autoimmune disease.

Quantitative Susceptibility Mapping of Multiple Sclerosis Lesions at Various Ages

PURPOSE To assess multiple sclerosis (MS) lesions at various ages by using quantitative susceptibility mapping (QSM) and conventional magnetic resonance (MR) imaging. MATERIALS AND METHODS Retrospectively selected were 32 clinically confirmed MS patients (nine men and 23 women; 39.3 years ± 10.9) who underwent two MR examinations (interval, 0.43 years ± 0.16) with three-dimensional gradient-echo sequence from August 2011 to August 2012. To estimate the ages of MS lesions, MR examinations performed 0.3-10.6 years before study examinations were studied. Hyperintensity on T2-weighted images was used to define MS lesions. QSM images were reconstructed from gradient-echo data. Susceptibility of MS lesions and temporal rates of change were obtained from QSM images. Lesion susceptibilities were analyzed by t test with intracluster correlation adjustment and Bonferroni correction in multiple comparisons. RESULTS MR imaging of 32 patients depicted 598 MS lesions, of which 162 lesions (27.1%) in 23 patients were age measurable and six (1.0%) were only visible at QSM. The susceptibilities relative to normal-appearing white matter (NAWM) were 0.53 ppb ± 3.34 for acute enhanced lesions, 38.43 ppb ± 13.0 (positive; P < .01) for early to intermediately aged nonenhanced lesions, and 4.67 ppb ± 3.18 for chronic nonenhanced lesions. Temporal rates of susceptibility changes relative to cerebrospinal fluid were 12.49 ppb/month ± 3.15 for acute enhanced lesions, 1.27 ppb/month ± 2.31 for early to intermediately aged nonenhanced lesions, and -0.004 ppb/month ± 0 for chronic nonenhanced lesions. CONCLUSION Magnetic susceptibility of MS lesions increased rapidly as it changed from enhanced to nonenhanced, it attained a high susceptibility value relative to NAWM during its initial few years (approximately 4 years), and it gradually dissipated back to susceptibility similar to that of NAWM as it aged, which may provide new insight into pathophysiologic features of MS lesions. Online supplemental material is available for this article.

Glutamate excitotoxicity--a mechanism for axonal damage and oligodendrocyte death in Multiple Sclerosis?

Glutamate excitotoxicity mediated by the AMPA/kainate-type of glutamate receptors is known not only to damage neurons but also the myelin-producing cell of the central nervous system (CNS), the oligodendrocyte. In Multiple Sclerosis (MS), myelin, oligodendrocytes and axons are lost or damaged as a result of an inflammatory attack on the CNS. Activated immune cells produce glutamate in large quantities by deamidating glutamine via glutaminase. Thus, we hypothesized that during inflammation in MS, glutamate excitotoxicity may contribute to the lesion. This was addressed by treating mice sensitized to develop acute experimental autoimmune encephalomyelitis (EAE) with an AMPA/kainate antagonist, NBQX. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced axonal damage, as indicated by the levels of dephosphorylated neurofilament-H. Despite the clinical differences, NBQX-treatment had no effect on lesion size and did not reduce the degree of CNS inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect at the level of the immune system. In separate studies, infiltrating immune cells present in perivascular cuffs, commonly the site of entry for invading immune cells, were found to express glutaminase in abundance, supporting the production of glutamate in inflammatory lesions. Thus, glutamate excitotoxicity appears to be an important mechanism in autoimmune demyelination and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for MS.

Neuregulin and erbB receptor expression in normal and diseased human white matter.

Human white matter from non-neurologic cases, multiple sclerosis (MS) and other neurologic diseases (OND, inflammatory and non-inflammatory), was subjected to immunocytochemistry and Western blotting for expression of the neuregulin, glial growth factor-2 (GGF2), and its receptors, erbB2, erbB3 and erbB4. GGF2 has previously been shown to have mitogenic effects upon oligodendrocytes in vitro and an enhancing effect upon remyelination in animals with autoimmune demyelination. In all types of human white matter examined, expression of the ligand GGF2 and its three receptors was consistently found on oligodendrocytes, with higher levels being seen in cases of MS. Expression was also seen, albeit at lower levels, on astrocytes and microglial cells, the latter most commonly in MS and OND. In human lymph node tissue, some lymphocytes were positive for erbB2, erbB3 and erbB4. Western blots confirmed the presence of all three receptors in normal, MS and OND white matter. GGF2 and erbB receptor expression did not correlate with areas of remyelination and reactivity occurred throughout the tissue, with some increase in intensity at the edge of MS lesions. Examination of precursor oligodendrocyte immunoreactivity (with anti-PDGF-Ralpha and NG2), revealed widespread expression throughout both normal and diseased white matter. The presence of GGF2 and its receptors on oligodendrocytes and lymphocytes render this cell type a candidate for functional signaling via this pathway, perhaps in relationship to myelinating activity.