Estrogen treatment prevents gray matter atrophy in experimental autoimmune encephalomyelitis.

Gray matter atrophy is an important correlate to clinical disability in multiple sclerosis (MS), and many treatment trials include atrophy as an outcome measure. Atrophy has been shown to occur in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The clinical severity of EAE is reduced in estrogen-reated mice, but it remains unknown whether estrogen treatment can reduce gray matter atrophy in EAE. In this study, mice with EAE were treated with either estrogen receptor (ER)-α ligand or ER-ß ligand, and diffusion tensor images (DTI) were collected and neuropathology was performed. DTI showed atrophy in the cerebellar gray matter of vehicle-treated EAE mice compared with healthy controls but not in ER-α or ER-ß ligand-treated EAE mice. Neuropathology demonstrated that Purkinje cell numbers were decreased in vehicle-treated EAE mice, whereas neither ER ligand-treated EAE groups showed a decrease. This is the first report of a neuroprotective therapy in EAE that unambiguously prevents gray matter atrophy while sparing a major neuronal cell type. Fractional anisotropy (FA) in the cerebellar white matter was decreased in vehicle- and ER-ß ligand-treated but not in ER-α ligand-treated EAE mice. Inflammatory cell infiltration was increased in vehicle- and ER-ß ligand-treated but not in ER-α ligand-treated EAE mice. Myelin staining was decreased in vehicle-treated EAE mice and was spared in both ER ligand-treated groups. This is consistent with decreased FA as a potential biomarker for inflammation rather than myelination or axonal damage in the cerebellum in EAE.

Reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the CNS.

Factors that regulate leukocyte entry and spread through CNS parenchyma during different types of CNS insults are incompletely understood. Reactive astrocytes have been implicated in restricting the spread of leukocytes from damaged into healthy parenchyma during the acute and local innate inflammatory events that follow CNS trauma, but the roles of reactive astrocytes during the chronic and widespread CNS inflammation associated with adaptive or acquired immune responses are uncertain. Here, we investigated the effects of transgenically targeted ablation of proliferating, scar-forming reactive astrocytes on the acquired immune inflammation associated with experimental autoimmune encephalitis (EAE). In wild-type mice with EAE, we found that reactive astrocytes densely surrounded perivascular clusters of leukocytes in a manner reminiscent of astrocyte scar formation after CNS trauma. Transgenically targeted ablation of proliferating astrocytes disrupted formation of these perivascular scars and was associated with a pronounced and significant increase in leukocyte entry into CNS parenchyma, including immunohistochemically identified macrophages, T lymphocytes and neutrophils. This exacerbated inflammation was associated with a substantially more severe and rapidly fulminant clinical course. These findings provide experimental evidence that reactive astrocytes form scar-like perivascular barriers that restrict the influx of leukocytes into CNS parenchyma and protect CNS function during peripherally initiated, acquired immune inflammatory responses in the CNS. The findings suggest that loss or disruption of astrocyte functions may underlie or exacerbate the inflammation and pathologies associated with autoimmune diseases of the CNS, including multiple sclerosis.

Estrogen treatment decreases matrix metalloproteinase (MMP)-9 in autoimmune demyelinating disease through estrogen receptor alpha (ERalpha).

Matrix metalloproteinases (MMPs) have a crucial function in migration of inflammatory cells into the central nervous system (CNS). Levels of MMP-9 are elevated in multiple sclerosis (MS) and predict the occurrence of new active lesions on magnetic resonance imaging (MRI). This translational study aims to determine whether in vivo treatment with the pregnancy hormone estriol affects MMP-9 levels from immune cells in patients with MS and mice with experimental autoimmune encephalomyelitis (EAE). Peripheral blood mononuclear cells (PBMCs) collected from three female MS patients treated with estriol and splenocytes from EAE mice treated with estriol, estrogen receptor (ER) alpha ligand, ERbeta ligand or vehicle were stimulated ex vivo and analyzed for levels of MMP-9. Markers of CNS infiltration were assessed using MRI in patients and immunohistochemistry in mice. Supernatants from PBMCs obtained during estriol treatment in female MS patients showed significantly decreased MMP-9 compared with pretreatment. Decreases in MMP-9 coincided with a decrease in enhancing lesion volume on MRI. Estriol treatment of mice with EAE reduced MMP-9 in supernatants from autoantigen-stimulated splenocytes, coinciding with decreased CNS infiltration by T cells and monocytes. Experiments with selective ER ligands showed that this effect was mediated through ERalpha. In conclusion, estriol acting through ERalpha to reduce MMP-9 from immune cells is one mechanism potentially underlying the estriol-mediated reduction in enhancing lesions in MS and inflammatory lesions in EAE.

Purkinje cell loss in experimental autoimmune encephalomyelitis.

Gray matter atrophy observed by brain MRI is an important correlate to clinical disability and disease duration in multiple sclerosis. The objective of this study was to link brain atrophy visualized by neuroimaging to its underlying neuropathology using the MS model, experimental autoimmune encephalomyelitis (EAE). Volumetric changes in brains of EAE mice, as well as matched healthy normal controls, were quantified by collecting post-mortem high-resolution T2-weighted magnetic resonance microscopy and actively stained magnetic resonance histology images. Anatomical delineations demonstrated a significant decrease in the volume of the whole cerebellum, cerebellar cortex, and molecular layer of the cerebellar cortex in EAE as compared to normal controls. The pro-apoptotic marker caspase-3 was detected in Purkinje cells and a significant decrease in Purkinje cell number was found in EAE. Cross modality and temporal correlations revealed a significant association between Purkinje cell loss on neuropathology and atrophy of the molecular layer of the cerebellar cortex by neuroimaging. These results demonstrate the power of using combined population atlasing and neuropathology approaches to discern novel insights underlying gray matter atrophy in animal models of neurodegenerative disease.

Differential neuroprotective and antiinflammatory effects of estrogen receptor (ER)alpha and ERbeta ligand treatment.

Treatment with either estradiol or an estrogen receptor (ER)alpha ligand has been shown to be both antiinflammatory and neuroprotective in a variety of neurological disease models, but whether neuroprotective effects could be observed in the absence of an antiinflammatory effect has remained unknown. Here, we have contrasted effects of treatment with an ERalpha vs. an ERbeta ligand in experimental autoimmune encephalomyelitis, the multiple sclerosis model with a known pathogenic role for both inflammation and neurodegeneration. Clinically, ERalpha ligand treatment abrogated disease at the onset and throughout the disease course. In contrast, ERbeta ligand treatment had no effect at disease onset but promoted recovery during the chronic phase of the disease. ERalpha ligand treatment was antiinflammatory in the systemic immune system, whereas ERbeta ligand treatment was not. Also, ERalpha ligand treatment reduced CNS inflammation, whereas ERbeta ligand treatment did not. Interestingly, treatment with either the ERalpha or the ERbeta ligand was neuroprotective, as evidenced by reduced demyelination and preservation of axon numbers in white matter, as well as decreased neuronal abnormalities in gray matter. Thus, by using the ERbeta selective ligand, we have dissociated the antiinflammatory effect from the neuroprotective effect of estrogen treatment and have shown that neuroprotective effects of estrogen treatment do not necessarily depend on antiinflammatory properties. Together, these findings suggest that ERbeta ligand treatment should be explored as a potential neuroprotective strategy in multiple sclerosis and other neurodegenerative diseases, particularly because estrogen-related toxicities such as breast and uterine cancer are mediated through ERalpha.

Cerebellar cortical atrophy in experimental autoimmune encephalomyelitis.

Brain atrophy measured by MRI is an important correlate with clinical disability and disease duration in multiple sclerosis (MS). Unfortunately, neuropathologic mechanisms which lead to this grey matter atrophy remain unknown. The objective of this study was to determine whether brain atrophy occurs in the mouse model, experimental autoimmune encephalomyelitis (EAE). Postmortem high-resolution T2-weighted magnetic resonance microscopy (MRM) images from 32 mouse brains (21 EAE and 11 control) were collected. A minimum deformation atlas was constructed and a deformable atlas approach was used to quantify volumetric changes in neuroanatomical structures. A significant decrease in the mean cerebellar cortex volume in mice with late EAE (48-56 days after disease induction) as compared to normal strain, gender, and age-matched controls was observed. There was a direct correlation between cerebellar cortical atrophy and disease duration. At an early time point in disease, 15 days after disease induction, cerebellar white matter lesions were detected by both histology and MRM. These data demonstrate that myelin-specific autoimmune responses can lead to grey matter atrophy in an otherwise normal CNS. The model described herein can now be used to investigate neuropathologic mechanisms that lead to the development of gray matter atrophy in this setting.

Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis.

Multiple sclerosis is an inflammatory, neurodegenerative disease for which experimental autoimmune encephalomyelitis (EAE) is a model. Treatments with estrogens have been shown to decrease the severity of EAE through anti-inflammatory mechanisms. Here we investigated whether treatment with an estrogen receptor alpha (ERalpha) ligand could recapitulate the estrogen-mediated protection in clinical EAE. We then went on to examine both anti-inflammatory and neuroprotective mechanisms. EAE was induced in wild-type, ERalpha-, or ERbeta-deficient mice, and each was treated with the highly selective ERalpha agonist, propyl pyrazole triol, to determine the effect on clinical outcomes, as well as on inflammatory and neurodegenerative changes. ERalpha ligand treatment ameliorated clinical disease in both wild-type and ERbeta knock-out mice, but not in ERalpha knock-out mice, thereby demonstrating that the ERalpha ligand maintained ERalpha selectivity in vivo during disease. ERalpha ligand treatment also induced favorable changes in autoantigen-specific cytokine production in the peripheral immune system [decreased TNFalpha, interferon-gamma, and interleukin-6, with increased interleukin-5] and decreased CNS white matter inflammation and demyelination. Interestingly, decreased neuronal staining [NeuN+ (neuronal-specific nuclear protein)/beta3-tubulin+/Nissl], accompanied by increased immunolabeling of microglial/monocyte (Mac 3+) cells surrounding these abnormal neurons, was observed in gray matter of spinal cords of EAE mice at the earliest stage of clinical disease, 1-2 d after the onset of clinical signs. Treatment with either estradiol or the ERalpha ligand significantly reduced this gray matter pathology. In conclusion, treatment with an ERalpha ligand is highly selective in vivo, mediating both anti-inflammatory and neuroprotective effects in EAE.