Allan MacKenzie-Graham

Differential expression, cytokine modulation, and specific functions of type-1 and type-2 tumor necrosis factor receptors in rat glia

Tumor necrosis factor alpha (TNF alpha) and lymphotoxin alpha (LT alpha) induce pleiotropic cellular effects through low-affinity 55 kDa type-1 receptors (TNFR1, CD120a) and high-affinity 75 kDa type-2 receptors (TNFR2, CD120b). Both cytokines have potent biological effects on glial cells and are strongly implicated in the pathology of central nervous system (CNS) demyelinating diseases. However, to date, neither constitutive nor cytokine-induced TNFR expression by glial cells have been definitively characterized. We therefore characterized TNF receptors at the molecular, protein, and functional levels in rat astrocytes, microglia, and oligodendrocytes. Northern blotting demonstrated that all three types of glia constitutively transcribed a single TNFR1 mRNA. IFN gamma increased transcript levels in all three types of glia, but TNF alpha increased levels only in oligodendrocytes Microglia constitutively transcribed three distinct TNFR2 mRNAs, levels of which were increased by either IFN gamma or TNF alpha. In contrast, astrocytes and oligodendrocytes constitutively transcribed nearly undetectable levels of TNFR2 mRNAs, and levels were not affected by IFN gamma, TNF alpha, or oligodendrocyte maturation. Immunocytochemical staining of glial cells corroborated Northern data by demonstrating that glia express a parallel pattern of TNFR proteins on their cell surfaces. In co-cultures of microglia plated atop irradiated astrocytes, human TNF alpha (which, on mouse cells, binds TNFR1 exclusively) induced microglial cell proliferation, whereas murine TNF alpha (which binds both TNFRs) did not. Collectively, the data show that microglia, a primary source of TNF alpha at CNS inflammatory sites, express both TNFR1 and TNFR2, whereas astrocytes and oligodendrocytes, whose embryological origin differs from that of microglia, predominantly express TNFR1. TNF alpha increases expression of TNFR1 by oligodendrocytes whereas it increases expression of TNFR2 by microglia. Microglia proliferation data suggest that signals transduced through TNFR2 directly or indirectly inhibit signals transduced through TNFR1. Different patterns of TNFR expression by glia at sites of CNS inflammation may be critical in determining whether TNF has activational, proliferative, or cytotoxic effects on these cells.

A multimodal, multidimensional atlas of the C57BL/6J mouse brain

Strains of mice, through breeding or the disruption of normal genetic pathways, are widely used to model human diseases. Atlases are an invaluable aid in understanding the impact of such manipulations by providing a standard for comparison. We have developed a digital atlas of the adult C57BL/6J mouse brain as a comprehensive framework for storing and accessing the myriad types of information about the mouse brain. Our implementation was constructed using several different imaging techniques: magnetic resonance microscopy, blockface imaging, classical histology and immunohistochemistry. Along with raw and annotated images, it contains database management systems and a set of tools for comparing information from different techniques. The framework allows facile correlation of results from different animals, investigators or laboratories by establishing a canonical representation of the mouse brain and providing the tools for the insertion of independent data into the same space as the atlas. This tool will aid in managing the increasingly complex and voluminous amounts of information about the mammalian brain. It provides a framework that encompasses genetic information in the context of anatomical imaging and holds tremendous promise for producing new insights into the relationship between genotype and phenotype. We describe a suite of tools that enables the independent entry of other types of data, facile retrieval of information and straightforward display of images. Thus, the atlas becomes a framework for managing complex genetic and epigenetic information about the mouse brain. The atlas and associated tools may be accessed at http://www.loni.ucla.edu/MAP.

Efficient, distributed and interactive neuroimaging data analysis using the LONI pipeline

The LONI Pipeline is a graphical environment for construction, validation and execution of advanced neuroimaging data analysis protocols (Rex et al., 2003). It enables automated data format conversion, allows Grid utilization, facilitates data provenance, and provides a significant library of computational tools. There are two main advantages of the LONI Pipeline over other graphical analysis workflow architectures. It is built as a distributed Grid computing environment and permits efficient tool integration, protocol validation and broad resource distribution. To integrate existing data and computational tools within the LONI Pipeline environment, no modification of the resources themselves is required. The LONI Pipeline provides several types of process submissions based on the underlying server hardware infrastructure. Only workflow instructions and references to data, executable scripts and binary instructions are stored within the LONI Pipeline environment. This makes it portable, computationally efficient, distributed and independent of the individual binary processes involved in pipeline data-analysis workflows. We have expanded the LONI Pipeline (V.4.2) to include server-to-server (peer-to-peer) communication and a 3-tier failover infrastructure (Grid hardware, Sun Grid Engine/Distributed Resource Management Application API middleware, and the Pipeline server). Additionally, the LONI Pipeline provides three layers of background-server executions for all users/sites/systems. These new LONI Pipeline features facilitate resource-interoperability, decentralized computing, construction and validation of efficient and robust neuroimaging data-analysis workflows. Using brain imaging data from the Alzheimers Disease Neuroimaging Initiative (Mueller et al., 2005), we demonstrate integration of disparate resources, graphical construction of complex neuroimaging analysis protocols and distributed parallel computing. The LONI Pipeline, its features, specifications, documentation and usage are available online (http://Pipeline.loni.ucla.edu).

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131‐kDa iNOS protein by Western blot analysis; protein was also detected in cells by single‐ and double‐label immunohistochemistry for iNOS and the oligodendrocyte‐specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin‐independent NO production in these in vitro cultures. J. Neurosci. Res. 48:372–384, 1997.

Estriol combined with glatiramer acetate for women with relapsing-remitting multiple sclerosis: a randomised, placebo-controlled, phase 2 trial

BACKGROUND Relapses of multiple sclerosis decrease during pregnancy, when the hormone estriol is increased. Estriol treatment is anti-inflammatory and neuroprotective in preclinical studies. In a small single-arm study of people with multiple sclerosis estriol reduced gadolinium-enhancing lesions and was favourably immunomodulatory. We assessed whether estriol treatment reduces multiple sclerosis relapses in women. METHODS We did a randomised, double-blind, placebo-controlled phase 2 trial at 16 academic neurology centres in the USA, between June 28, 2007, and Jan 9, 2014. Women aged 18-50 years with relapsing-remitting multiple sclerosis were randomly assigned (1:1) with a random permuted block design to either daily oral estriol (8 mg) or placebo, each in combination with injectable glatiramer acetate 20 mg daily. Patients and all study personnel, except for pharmacists and statisticians, were masked to treatment assignment. The primary endpoint was annualised relapse rate after 24 months, with a significance level of p=0.10. Relapses were confirmed by an increase in Expanded Disability Status Scale score assessed by an independent physician. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00451204. FINDINGS We enrolled 164 patients: 83 were allocated to the estriol group and 81 were allocated to the placebo group. The annualised confirmed relapse rate was 0.25 relapses per year (95% CI 0.17-0.37) in the estriol group versus 0.37 relapses per year (0.25-0.53) in the placebo group (adjusted rate ratio 0.63, 95% CI 0.37-1.05; p=0.077). The proportion of patients with serious adverse events did not differ substantially between the estriol group and the placebo group (eight [10%] of 82 patients vs ten [13%] of 76 patients). Irregular menses were more common in the estriol group than in the placebo group (19 [23%] vs three [4%], p=0.0005), but vaginal infections were less common (one [1%] vs eight [11%], p=0.0117). There were no differences in breast fibrocystic disease, uterine fibroids, or endometrial lining thickness as assessed by clinical examination, mammogram, uterine ultrasound, or endometrial lining biopsy. INTERPRETATION Estriol plus glatiramer acetate met our criteria for reducing relapse rates, and treatment was well tolerated over 24 months. These results warrant further investigation in a phase 3 trial. FUNDING National Institutes of Health, National Multiple Sclerosis Society, Conrad N Hilton Foundation, Jack H Skirball Foundation, Sherak Family Foundation, and the California Community Foundation.

Neuroprotective effects of testosterone treatment in men with multiple sclerosis

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system. While current medication reduces relapses and inflammatory activity, it has only a modest effect on long-term disability and gray matter atrophy. Here, we have characterized the potential neuroprotective effects of testosterone on cerebral gray matter in a pilot clinical trial. Ten men with relapsing–remitting MS were included in this open-label phase II trial. Subjects were observed without treatment for 6 months, followed by testosterone treatment for another 12 months. Focal gray matter loss as a marker for neurodegeneration was assessed using voxel-based morphometry. During the non-treatment phase, significant voxel-wise gray matter decreases were widespread (p≤ 0.05 corrected). However, during testosterone treatment, gray matter loss was no longer evident. In fact, a significant gray matter increase in the right frontal cortex was observed (p≤ 0.05 corrected). These observations support the potential of testosterone treatment to stall (and perhaps even reverse) neurodegeneration associated with MS. Furthermore, they warrant the investigation of testosterones neuroprotective effects in larger, placebo controlled MS trials as well as in other neurodegenerative diseases. This is the first report of gray matter increase as the result of treatment in MS.

Provenance in neuroimaging

Provenance, the description of the history of a set of data, has grown more important with the proliferation of research consortia-related efforts in neuroimaging. Knowledge about the origin and history of an image is crucial for establishing data and results quality; detailed information about how it was processed, including the specific software routines and operating systems that were used, is necessary for proper interpretation, high fidelity replication and re-use. We have drafted a mechanism for describing provenance in a simple and easy to use environment, alleviating the burden of documentation from the user while still providing a rich description of an images provenance. This combination of ease of use and highly descriptive metadata should greatly facilitate the collection of provenance and subsequent sharing of data.

The informatics of a C57BL/6J mouse brain atlas.

The Mouse Atlas Project (MAP) aims to produce a framework for organizing and analyzing the large volumes of neuroscientific data produced by the proliferation of genetically modified animals. Atlases provide an invaluable aid in understanding the impact of genetic manipulation by providing a standard for comparison. We use a digital atlas as the hub of an informatics network, correlating imaging data, such as structural imaging and histology, with text-based data, such as nomenclature, connections, and references. We generated brain volumes using magnetic resonance microscopy (MRM), classical histology, and immunohistochemistry, and registered them into a common and defined coordinate system. Specially designed viewers were developed in order to visualize multiple datasets simultaneously and to coordinate between textual and image data. Researchers can navigate through the brain interchangeably, in either a text-based or image-based representation that automatically updates information as they move. The atlas also allows the independent entry of other types of data, the facile retrieval of information, and the straight-forward display of images. In conjunction with centralized servers, image and text data can be kept current and can decrease the burden on individual researchers’ computers. A comprehensive framework that encompasses many forms of information in the context of anatomic imaging holds tremendous promise for producing new insights. The atlas and associated tools can be found at http://www.loni.ucla.edu/MAP.

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.

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.