Mitchell R. Emerson

Statistical analysis of data from studies on experimental autoimmune encephalomyelitis

Research in multiple sclerosis often employs animal models of the disease, especially experimental autoimmune encephalomyelitis (EAE) in rodents. The statistical analysis procedures chosen for these studies are often suboptimal, either because of violations of the assumptions of the procedure or because the analysis selected is inappropriate for the research question. In this paper, we discuss the types of research questions frequently asked in EAE studies and suggest appropriate and useful research designs and statistical methods that will optimize the information contained within the data. We also discuss other troublesome issues such as missing data, atypical disease profiles, and power analysis.

Hypoxia preconditioning attenuates brain edema associated with kainic acid-induced status epilepticus in rats.

Kainic acid (KA)-induced seizures elicit edema associated with necrosis in susceptible brain regions (e.g., piriform cortex and hippocampal CA1 and CA3 regions). To test the hypothesis that hypoxia preconditioning protects against KA-induced edema formation, adult male rats were exposed to a 9% O2, 91% N2 atmosphere for 8 h. KA (14 mg/kg, i.p.) was administered 1, 3, 7, or 14 days later. Regional analysis of edema indicated that hypoxia exposure attenuated edema formation in piriform and frontal cortices and hippocampus when KA was given 1, 3, or 7 days later but not 14 days after hypoxia. Cycloheximide (2 mg/kg s.c.) given 1 h prior to hypoxia prevented the protective effect of hypoxia on KA-induced edema attenuation in the piriform cortex and hippocampus. Thus, hypoxic challenge induces a general adaptive response that protects against the seizure-associated pathophysiology, with no direct relationship to seizure intensity. This response may involve stress-related transcription factors and effector proteins.

Experimental allergic encephalomyelitis is exacerbated in mice deficient for 12/15-lipoxygenase or 5-lipoxygenase

12/15-Lipoxygenase (12/15-LO) produces 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) which are agonists for peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma agonists reduce clinical severity of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. In contrast, 5-lipoxygenase (5-LO) produces the generally proinflammatory leukotrienes (LTs) which would be expected to worsen EAE. We tested the hypotheses that EAE severity would be exacerbated in 12/15-LO-deficient mice and attenuated in 5-LO-deficient mice. 12/15-LO deficiency conferred a significantly worse disease course, and surprisingly, 5-LO deficiency also caused significantly more severe EAE compared to control mice. These data suggest that PPARgamma-regulated gene expression and that 5-LO production of certain LTs have the ability to diminish EAE. Continued analysis will provide insight into the endogenous LO-generated effectors that assist in tempering EAE.

Heme oxygenase-1 and NADPH cytochrome P450 reductase expression in experimental allergic encephalomyelitis: an expanded view of the stress response.

Abstract: Oxidative stress is implicated in the pathogenesis ofexperimental allergic encephalomyelitis (EAE), a model for multiple sclerosis.Heme oxygenase‐1 (HO‐1) is a heat shock protein induced by oxidative stress.HO‐1 metabolizes the pro‐oxidant heme to the antioxidant biliverdin and CO.HO‐1 requires electrons, donated by NADPH cytochrome P450 reductase(henceforth, reductase), for catalytic activity. EAE was induced with apeptide of proteolipid protein in SJL mice, and the expression of HO‐1 andreductase in the hindbrain was analyzed. HO‐1 protein levels weresignificantly increased in EAE animals compared with control mice. HO‐1expression was present in ameboid macrophages, reactive microglia, andastrocytes in white matter tracks. Bergmann glia and ameboid macrophages alsowere occasionally stained in the molecular layer of the cerebellum. UnlikeHO‐1, reductase protein levels decreased with disease severity. HO‐1 andreductase were associated with each other in endoplasmic reticulum micelles,suggesting that the decrease in reductase does not interfere with itsassociation with HO‐1. In cells that express HO‐1, the association ofreductase with HO‐1 should competitively inhibit the interaction of reductasewith cytochrome P450 isozymes and thereby limit free radical production as thelatter two enzymes act cooperatively to produce superoxide. The increase inHO‐1 together with the decrease in reductase may be part of a common defensemechanism attempting to minimize tissue damage in several neurologicalconditions.

Activation of histamine H2 receptors ameliorates experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis, is an autoimmune, demyelinating disease of the CNS. Pro-inflammatory cytokines (e.g. tumour necrosis factor-&agr; (TNF-&agr;) and interleukin (IL)-12) and reactive oxygen species are implicated in promoting EAE. Since histamine H2 receptor activation suppresses production of O2.−, TNF-&agr;, and IL-12 by inflammatory cells, we tested the hypothesis that dimaprit, an H2 agonist, would reduce the clinical severity and pathology of EAE. Dimaprit treatment significantly reduced clinical signs compared to vehicle in both C57BL/6 and iNOS deficient EAE mice. Furthermore, dimaprit significantly reduced CNS staining for lectin-positive macrophages and decreased extravasated albumin staining, an indicator of blood–brain barrier leakage. These data provide a rationale for exploring H2 receptor activation for therapeutic value in multiple sclerosis.

A global hypoxia preconditioning model: neuroprotection against seizure-induced specific gravity changes (edema) and brain damage in rats

Hypoxia preconditioning states that a sublethal hypoxia episode will afford neuroprotection against a second challenge in the near future. We describe and discuss a procedure for the development of global hypoxia preconditioning in adult male Wistar rats, using a mildly hypoxic (9% O(2), 91% N(2)) atmospheric exposure of 8 h. The persistence of neuroprotection was analyzed using a kainic acid (KA) model of brain injury. Rats were challenged with KA (14 mg/kg, i.p.) on 1-14 days post-hypoxia. The effects of hypoxia preconditioning on seizure score, weight loss, brain edema and histopathology were assessed. Brain edema, predominantly of vasogenic origin, was measured 24 h after KA administration using a reproducible and quantitative method based on the specific gravities of tissue samples. A density gradient column (1.0250-1.0650 g/cm(3)) comprised of kerosene and bromobenzene was used to assess the presence of edema in regions involved in seizure initiation and propagation that are normally extensively damaged (i.e., piriform cortex and hippocampus). Specific gravities of tissues were calculated through extrapolation with known NaCl standards. We found that hypoxia preconditioning prevented the formation of edema in these brain regions when KA challenge was given 1, 3, and 7, but not 14 days post-hypoxia exposure. Furthermore, neuroprotection was observed in animals that had robust seizures. The described procedure may be used to examine the neuroprotective mechanisms induced by global hypoxia preconditioning against many subsequent challenges reflecting a variety of experimental models of brain injury, and will provide a better understanding of the brain response to hypoxia and stress.

Cuprizone and piperonyl butoxide, proposed inhibitors of T-cell function, attenuate experimental allergic encephalomyelitis in SJL mice

Multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), are autoimmune demyelinating diseases with autoreactive T-cells acting as important mediators of pathogenesis. Cuprizone, a copper chelator, and piperonyl butoxide (PBO), a pesticide synergist, are implicated to inhibit T-cell activation and function. The purpose of this study was to assess whether either of these agents would suppress PLP-peptide-induced EAE in the SJL mouse. Indeed, treatment with cuprizone beginning 1 week prior to disease induction, and PBO administration from days 1 to 9 of EAE, significantly attenuated EAE clinical severity. Furthermore, both agents decreased blood CD4+/CD8+ ratios, and reduced signs of chronic graft vs. host disease (GVHD) indicating attenuation of an immune T-cell response. These results suggest that cuprizone and PBO suppress EAE and use of these agents will provide insights into the mechanisms of T-cell mediated diseases.

Apoferritin attenuates experimental allergic encephalomyelitis in SJL mice

Ferritin has been shown to attenuate iron-catalyzed oxidative damage in several experimental conditions. Since oxidative damage has been implicated in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), an animal model of MS, we tested the hypothesis that ferritin would act to attenuate disease. The experimental design was to increase plasma ferritin levels during the active stage of EAE by giving systemic injections of apoferritin and then compare disease activity between these mice and EAE mice administered vehicle. Additional mice received systemic injections of iron, which induces ferritin synthesis, in order to test the effects of exogenous iron on the disease course. Although plasma levels of ferritin were found to be elevated in both apoferritin and iron-injected EAE mice, only apoferritin treatment resulted in a reduction in disease activity compared to EAE mice given vehicle. The suppressive effects of apoferritin administration suggest that the increase in endogenous ferritin levels that have been previously observed in the cerebrospinal fluid of chronic progressive MS patients with active disease might be functioning to limit the severity and spread of tissue damage.

Aspirin and multiple sclerosis

Aspirin is widely used to lessen the risks of cardiovascular events. Some studies suggest that patients with multiple sclerosis have an increased risk for some cardiovascular events, for example, venous thromboembolism and perhaps ischemic strokes, raising the possibility that aspirin could lessen these increased risks in this population or subgroups (patients with limited mobility and/or antiphospholipid antibodies). However, aspirin causes a small increased risk of hemorrhagic stroke, which is a concern as it could potentially worsen a compromised blood-brain barrier. Aspirin has the potential to ameliorate the disease process in multiple sclerosis (for example, by limiting some components of inflammation), but aspirin also has the potential to inhibit mitochondrial complex I activity, which is already reduced in multiple sclerosis. In an experimental setting of a cerebral ischemic lesion, aspirin promoted the proliferation and/or differentiation of oligodendrocyte precursors, raising the possibility that aspirin could facilitate remyelination efforts in multiple sclerosis. Other actions by aspirin may lead to small improvements of some symptoms (for example, lessening fatigue). Here we consider potential benefits and risks of aspirin usage by patients with multiple sclerosis.

Metabolic pathways as possible therapeutic targets for progressive multiple sclerosis

Unlike relapsing remitting multiple sclerosis, there are very few therapeutic options for patients with progressive forms of multiple sclerosis. While immune mechanisms are key participants in the pathogenesis of relapsing remitting multiple sclerosis, the mechanisms underlying the development of progressive multiple sclerosis are less well understood. Putative mechanisms behind progressive multiple sclerosis have been put forth: insufficient energy production via mitochondrial dysfunction, activated microglia, iron accumulation, oxidative stress, activated astrocytes, Wallerian degeneration, apoptosis, etc. Furthermore, repair processes such as remyelination are incomplete. Experimental therapies that strive to improve metabolism within neurons and glia, e.g., oligodendrocytes, could act to counter inadequate energy supplies and/or support remyelination. Most experimental approaches have been examined as standalone interventions; however, it is apparent that the biochemical steps being targeted are part of larger pathways, which are further intertwined with other metabolic pathways. Thus, the potential benefits of a tested intervention, or of an established therapy, e.g., ocrelizumab, could be undermined by constraints on upstream and/or downstream steps. If correct, then this argues for a more comprehensive, multifaceted approach to therapy. Here we review experimental approaches to support neuronal and glial metabolism, and/or promote remyelination, which may have potential to lessen or delay progressive multiple sclerosis.