Christopher Bolton

Glutamate Receptors in Neuroinflammatory Demyelinating Disease

Multiple sclerosis (MS) is a chronic demyelinating disease of the human central nervous system (CNS). The condition predominantly affects young adults and is characterised by immunological and inflammatory changes in the periphery and CNS that contribute to neurovascular disruption, haemopoietic cell invasion of target tissues, and demyelination of nerve fibres which culminate in neurological deficits that relapse and remit or are progressive. The main features of MS can be reproduced in the inducible animal counterpart, experimental autoimmune encephalomyelitis (EAE). The search for new MS treatments invariably employs EAE to determine drug activity and provide a rationale for exploring clinical efficacy. The preclinical development of compounds for MS has generally followed a conventional, immunotherapeutic route. However, over the past decade, a group of compounds that suppress EAE but have no apparent immunomodulatory activity have emerged. These drugs interact with the N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-isoxazolepropionic acid (AMPA)/kainate family of glutamate receptors reported to control neurovascular permeability, inflammatory mediator synthesis, and resident glial cell functions including CNS myelination. The review considers the importance of the glutamate receptors in EAE and MS pathogenesis. The use of receptor antagonists to control EAE is also discussed together with the possibility of therapeutic application in demyelinating disease.

Changes in the cellular distribution of lipocortin-1 (Annexin-1) in C6 glioma cells after exposure to dexamethasone.

Glucocorticoid-induced changes in cellular levels of Lipocortin-1 (LC-1) (Annexin 1) in C6 glioma cells were determined by electrotransfer and immunoblotting techniques. Separate cell protein fractions were prepared to study the influence of the glucocorticoid steroid, dexamethasone, on LC-1 localisation. Cells were grown in steroid-depleted medium and exposed to dexamethasone (10(-8) and 10(-7) M) for 2, 6, and 16 hr. The glucocorticoid-dependent changes in cellular content of LC-1 were both dose- and time-related. Increases above control levels in intracellular and extracellular LC-1 content were detected with the greatest changes occurring at the cell surface. The glucocorticoid-dependent alteration in LC-1 distribution in C6 glioma cells was attenuated by the protein synthesis inhibitor, cycloheximide, indicating the involvement of de novo LC-1 synthesis. The significance of these results is discussed in relation to the current concept that some of the anti-inflammatory effect of glucocorticoids occurs through the action of extracellular LC-1.

Dexamethasone Induces an Increase in Intracellular and Membrane-Associated Lipocortin-1 (Annexin-1) in Rat Astrocyte Primary Cultures

Summary1. The antiinflammatory actions of glucocorticoid steroids are thought to occur through induction of the protein lipocortin-1 (LC-1; annexin-1). The purpose of the current study was to investigate whether astrocytic LC-1 content was increased in the presence of a synthetic glucocorticoid, dexamethasone.2. Steroid-induced changes in cellular levels of LC-1 in astrocytes were determined by electrotransfer and immunoblotting techniques. Separate cell fractions were investigated to study the influence of dexamethasone on astroglial LC-1 content. The effect of culture state on LC-1 expression was also examined.3. Intracellular LC-1 content was found to decrease after initiation of culture, with a substantial rise in both cell proliferation and LC-1 expression occurring after the replenishment of medium containing steroid-free serum. A further increase in intracellular LC-1 occurred upon incubation with dexamethasone. The glucocorticoid-induced change in intracellular LC-1 was a time-dependent event and coincided with an increase in membrane-associated LC-1.4. The findings in this study indicate that astrocytic LC-1 content is influenced by cell culture conditions and, in the presence of glucocorticoid steroids, the cellular localization of LC-1 is altered. This may indicate that LC-1 has functions at more than one cellular locality.

The detection and quantitation of inflammation in the central nervous system during experimental allergic encephalomyelitis using the radiopharmaceutical 99mTc-RP128.

RP128 is a novel agent which readily chelates 99mTc to form a radiopharmaceutical which binds in vivo to the tuftsin receptor located specifically on neutrophils and monocyte-macrophages, therefore removing the need for in vitro cell labelling prior to intravenous administration. We have assessed the ability of 99mTc-RP128 to detect central nervous system (CNS) inflammation in experimental allergic encephalomyelitis (EAE), an animal model of the human disease multiple sclerosis. The radiopharmaceutical was recorded at significantly increased levels in all EAE diseased CNS tissues, compared to normal and control samples, at 0.5, 1 and 3 h post-injection using a dual radioisotope technique to correct for non-extravasated tracer (P<0.05). Moreover, extravascular accumulation of the agent could be clearly demonstrated in inflammatory tissues with minimal loss of sensitivity when the secondary isotopic correction for blood volume was omitted. In addition, 99mTc-RP128 successfully monitored glucocorticoid suppression of inflammation (P<0.05), recording a typical dose-response to increasing steroid concentration. Clearly, 99mTc-RP128 can quantitatively detect CNS inflammation and assess responses to therapy indicating potential value as an imaging agent both clinically and as a research aid. Furthermore, the rapid in vivo labelling by 99mTc-RP128 of specific inflammatory cells combined with the ability to monitor the progress of anti-inflammatory therapeutics may recommend the agent for use in a variety of inflammatory conditions.

Neurovascular damage in experimental allergic encephalomyelitis: a target for pharmacological control

The blood-brain barrier (BBB) is composed of a continuous endothelial layer with pericytes and astrocytes in close proximity to offer homeostatic control to the neurovasculature. The human demyelinating disease multiple sclerosis and the animal counterpart experimental allergic encephalomyelitis (EAE) are characterized by enhanced permeability of the BBB facilitating oedema formation and recruitment of systemically derived inflammatory-type cells into target tissues to mediate eventual myelin loss and neuronal dysfunction. EAE is considered a useful model for examining the pathology which culminates in loss of BBB integrity and the disease is now proving valuable in assessing compounds for efficacy in limiting damage at neurovascular sites. The precise mechanisms culminating in EAE-induced BBB breakdown are unclear although several potentially disruptive mediators have been implicated and have been previously identified as potent effectors of cerebrovascular damage in non-disease related conditions of the central nervous system. The review considers evidence that common mechanisms may mediate cerebrovascular permeability changes irrespective of the initial insult and discusses therapeutic approaches for the control of BBB leakage in the demyelinating diseases.

Correlation between prostaglandin and thromboxane levels and clinical signs in cyclosporine A-treated guinea pigs with acute experimental allergic encephalomyelitis

Prostaglandin E, prostaglandin F2 alpha and thromboxane B2 levels were measured in the spinal cord and cerebellum of guinea pigs with acute experimental allergic encephalomyelitis treated with Cyclosporine A in a schedule which delayed the onset of the disease and completely suppressed it in 40% of the animals. A correlation was found between the eicosanoid levels in the central nervous system and clinical signs of the disease. Where lesions were completely suppressed the levels of eicosanoids were normal. The number of lesions found correlated well with the clinical state of the animals.