Bernhard H.J. Juurlink

Low Glutathione and High Iron Govern the Susceptibility of Oligodendroglial Precursors to Oxidative Stress

Abstract: We have previously shown, using qualitative approaches, that oligodendroglial precursors are more readily damaged by free radicals than are astrocytes. In the present investigation we quantified the oxidative stress experienced by the cells using oxidation of dichlorofluorescin diacetate to dichlorofluorescein as a measure of oxidative stress; furthermore, we have delineated the physiological bases of the difference in susceptibility to oxidative stress found between oligodendroglial precursors and astrocytes. We demonstrate that (a) oligodendroglial precursors under normal culture conditions are under six times as much oxidative stress as astrocytes, (b) oxidative stress experienced by oligodendroglial precursors increases sixfold when exposed to 140 mW/m2 of blue light, whereas astrocytic oxidative stress only doubles, (c) astrocytes have a three times higher concentration of GSH than oligodendroglial precursors, (d) oligodendroglial precursors have >20 times higher iron content than do astrocytes, and (e) oxidative stress in oligodendroglial precursors can be prevented either by chelating intracellular free iron or by raising intracellular GSH levels to astrocytic values. We conclude that GSH plays a central role in preventing free radical‐mediated damage in glia.

Review of oxidative stress in brain and spinal cord injury: suggestions for pharmacological and nutritional management strategies.

Much of the damage that occurs in the central nervous system (CNS) following trauma is due to secondary effects of glutamate excitotoxicity, Ca2+ overload, and oxidative stress, three mechanisms that in a spiraling interactive cascade end in neuronal death. Oxidative stress activates mechanisms that result in a neutrophil-mediated inflammation that also causes secondary damage. Mechanisms of oxidative stress are reviewed, with particular attention paid to lipid peroxidation and the central role of reduced glutathione in scavenging peroxides. We suggest that decreasing oxidative stress will greatly reduce the amount of secondary damage due to trauma. Oxidative stress can be minimized by 1) maintaining reduced-glutathione levels through the administration of cysteine precursors such as N-acetylcysteine and 2) limiting neutrophil invasion by administering platelet-activating factor antagonists such as BN 52021. Aggressive nutritional support following CNS trauma can also contribute to maximizing antioxidant defenses. Furthermore, we suggest that flavonoids such as quercetin have the potential to be therapeutically effective because of their free radical quenching, iron chelating, and anti-inflammatory properties.

Nitric oxide donors enhance neurotrophin‐induced neurite outgrowth through a cGMP‐dependent mechanism

Nitric oxide (NO), a diffusible and unstable gas, has been implicated in inter‐ and intra‐cellular communication in the nervous system. NO also plays a role in neural development, plasticity and alterations of synaptic function such as long‐term potentiation and long‐term depression (Gally et al.: Proc NY Acad Sci, 87:354–355, 1990; Zhuo et al.: Science 260:1946–1950, 1993; Schuman and Madison.: Science 254:1503–1506, 1991; Bruhwyler et al.: Neurosci Biobehav Rev 17:373–384, 1993) some of which likely involve growth and remodelling of neurites. Some actions of NO are mediated directly by protein modification (e.g., nitrosylation) and others by activation of soluble guanylyl cyclase (soluble GC), which increases intracellular levels of guanosine 3′,5′‐cyclic monophosphate (cGMP). NO is synthesized by the enzyme nitric oxide synthase (NOS), which is induced by treatment of CNS neurons (Holtzman et al.: Neurobiol Disease 1:51–60, 1994) or pheochromocytoma PC12 cells (Hirsch et al.: Curr Biol 3:749–754, 1993) with NGF. NO has been proposed to mediate some of the effects of NGF on PC12 cells by inhibiting cell division (Peunova and Enikolopov: Nature 374:68–73, 1995). In addition, NO can substitute for NGF by delaying the death of trophic factor‐deprived PC12 cells through a mechanism that does not involve a cytostatic action (Farinelli et al.: J Neurosci 16:2325–2334, 1996). We investigated whether NO stimulated neurite outgrowth from hippocampal neurons and PC12 cells. Primary cultures of E17 mouse hippocampal neurons co‐cultured with neopallial astrocytes were exposed to the NO donors sodium nitrite (100 μM) or sodium nitroprusside (100 nM). After 48 hr, NO donor‐treated cultures contained a greater proportion of cells bearing neurites and neurites that were much longer than those found in control cultures. In cultures of PC12 cells, NO donors also enhanced the neuritogenic effects of NGF. The proportion of PC12 cells with neurites 48 hr after exposure to NO donors sodium nitrite (100 μM–10 mM) or sodium nitroprusside (100 nM–1 μM) plus 2.5S nerve growth factor (NGF) was approximately twice the proportion of cells with neurites in sister cultures grown in NGF alone. Neither of the NO donors elicited neurites from the PC12 cells in the absence of NGF. The effects of the NO donors were likely mediated by release of NO since their effects were antagonized by addition of hemoglobin, which avidly binds NO, to the culture medium. The enhancement by NO of NGF‐mediated neurite outgrowth in PC12 cells appeared to occur through a cGMP‐dependent mechanism. The NO donors stimulated a prompt increase in intracellular cGMP in PC12 cells. Moreover their action was mimicked by addition of the membrane‐permeant cGMP analogs 8‐Bromo‐cGMP (8‐Br‐cGMP) and para (chlorophenylthio)‐cGMP (pCPT‐cGMP) to the culture medium and by atrial natriuretic factor which stimulates particulate guanylyl cyclase. The neuritogenic activity of the NO donors was inhibited by LY83583 and methylene blue, inhibitors of guanylyl cyclase. These data imply that NO may act alone or with other growth factors to regulate synapse formation and maintenance by stimulating neurite outgrowth. J. Neurosci. Res. 47:427–439, 1997.

Health effects with consumption of the flax lignan secoisolariciresinol diglucoside.

Flaxseed is the richest source of the lignan secoisolariciresinol diglucoside (SDG). After ingestion, SDG is converted to secoisolariciresinol, which is further metabolised to the mammalian lignans enterodiol and enterolactone. A growing body of evidence suggests that SDG metabolites may provide health benefits due to their weak oestrogenic or anti-oestrogenic effects, antioxidant activity, ability to induce phase 2 proteins and/or inhibit the activity of certain enzymes, or by mechanisms yet unidentified. Human and animal studies identify the benefits of SDG consumption. SDG metabolites may protect against CVD and the metabolic syndrome by reducing lipid and glucose concentrations, lowering blood pressure, and decreasing oxidative stress and inflammation. Flax lignans may also reduce cancer risk by preventing pre-cancerous cellular changes and by reducing angiogenesis and metastasis. Thus, dietary SDG has the potential to decrease the incidence of several chronic diseases that result in significant morbidity and mortality in industrialised countries. The available literature, though, makes it difficult to clearly identify SDG health effects because of the wide variability in study methods. However, the current evidence suggests that a dose of at least 500 mg SDG/d for approximately 8 weeks is needed to observe positive effects on cardiovascular risk factors in human patients. Flaxseed and its lignan extracts appear to be safe for most adult populations, though animal studies suggest that pregnant women should limit their exposure. The present review discusses the potential health benefits of SDG in humans, with supporting evidence from animal studies, and offers suggestions for future research.

Response of Glial Cells to Ischemia: Roles of Reactive Oxygen Species and Glutathione

A review of reactive oxygen species (ROS) is followed by a discussion on the differential susceptibility of astrocytes and oligodendroglia to ischemia-related insults. Astrocytes can survive chronic hypoxia as well as long periods of simulated ischemia, i.e. hypoglycemia and anoxia. Oligodendroglia are preferentially injured over astrocytes by chronic hypoxia, reperfusion following ischemia, hypoglycemia or uncoupling of oxidative phosphorylation. Increasing the generation of ROS in mixed glial cultures by adding ROS generators results in preferential death of oligodendroglia. Oligodendroglia are more susceptible to oxidative stress because they have low glutathione contents while concomitantly having higher iron contents and are more dependent upon oxidative phosphorylation than are astrocytes. Glutathione plays a pivotal role in the ROS-scavenging strategies of the cell while iron plays a pivotal role in the generation of hydroxyl, peroxy and akoxy radicals. These in vitro findings delineate the physiological basis for the white matter damage seen in adults following prolonged periods of hypoperfusion and the damage seen in the oligodendroglial germinal zones resulting in periventricular leukomalacia seen following in utero hypoxia-ischemia.

Induction by Hydrocortisone of Glutamine Synthetase in Mouse Primary Astrocyte Cultures

Glutaminc synthetase activity was investigated in developing primary astroglial cultures established from newborn mouse cerebral hemispheres. Between the 2nd and 4th week of culture there was little change in activity under our standard culturing conditions; however, when hydrocortiwne (10 μM) was added to the cultures for 48 h, the enzyme activity increased two‐ to fourfold, depending upon the age of the culture, with maximum response in 2‐week‐old cultures. The addition of dibutyryl cyclic AMP (dRcAMP) to the culture medium caused morphological differentiation of the astroglial cells but eliminated the response of the cells to hydrocortisone. Culturing in elevated serum levels, which delays morphological differentiation and inhibits astroglial cytodifferentiation after exposure to dBcAMP, shifted the time of maximal response to hydrocortisone from 2 to 3 weeks and prevented the abolishment of glutamine synthetase induction by dBcAMP. The induction of glutamine synthetase by hydrocortisone was prevented by actinomycin D (0.5 μg/ml), indicating its dependence upon RNA and protein synthesis. The present work thus confirms reports in the literature that hydrocortisone induces glutamine synthetase in neural tissues, but differs from the findings of Moscona and co‐workers in the chick retina that intact tissues are required for the induction to occur.

Peroxide-scavenging deficit underlies oligodendrocyte susceptibility to oxidative stress.

Previous work showed that the susceptibility of oligodendroglial progenitors to oxidative stress is related to their low reduced‐glutathione (GSH) and high iron contents. This suggests that these cells have a poor ability to scavenge peroxides. All peroxides are scavenged by glutathione peroxidase. Glutathione peroxidase activity requires GSH as an electron donor resulting in the formation of oxidized‐glutathione. Cellular GSH content is dependent upon synthesis as well as reduction of oxidized‐glutathione. The objectives of the present study were to compare several parameters important in the ability to scavenge peroxides between astrocytes and oligodendroglia. Three stages of oligodendroglial differentiation were examined: the proliferative oligodendrocyte progenitor, the proliferative oligodendroblast, and the post‐mitotic oligodendrocyte.

Plasticity of Astrocytes in Primary Cultures: An Experimental Tool and a Reason for Methodological Caution

Astrocytes in primary cultures constitute an exceedingly useful preparation for studies of astroglial development and function. These cells, however, demonstrate a pronounced plasticity in their reactions to culturing conditions. Thus, species and spatiotemporal region of CNS chosen for source of cells, dissociation procedures used, cell density in culture, culture medium chosen, type and/or concentration of serum used (if any) and exposure to dibutyryl cyclic AMP (dBcAMP) may all markedly affect the epigenotype of the cells. This provides an experimental tool for studies of astroglial development and function, but is at the same time a reason for caution in the interpretation of data when such cultured cells are used as models of their in vivo counterparts. About 95% of the cells in cultures used by the authors are positive for astrocyte-specific markers (glial fibrillary acidic protein and glutamine synthetase), and the cells possess presumably astrocytic characteristics such as high potassium permeability. This latter characteristic may be drastically altered by minor changes in culturing conditions. It is likely that an overwhelming majority of the astrocytes in such cultures are protoplasmic in nature. Addition of dBcAMP to the culture medium results in a pronounced morphological and a more modest functional differentiation.

Redox regulation of nuclear factor kappa B: therapeutic potential for attenuating inflammatory responses.

Nuclear factor kappa B (NF‐κB) is a protein transcription factor that is required for maximal transcription of a wide array of pro‐inflammatory mediators that are involved in the pathogenesis of stroke. The purpose of this review article is to describe what is known about the molecular biology of NF NF‐κB and to review current understanding of the interaction between reactive oxygen species (ROS) in NF‐κB. ROS seem to play a duel role by participating in the NF‐κB activation cascade and by directly modulating DNA binding affinity. Exogenous and endogenous antioxidants are effective in blocking activation of NF‐κB and preventing the consequences of pro‐inflammatory gene expression. Phase II enzymes either directly or indirectly play a major in vivo role in minimizing oxidative stress by scavenging peroxides, peroxide breakdown products and dicarbonyls and in regeneration of lipid peroxidation chain‐breaker, vitamin E. Dietary phase II enzyme inducers have been demonstrated to increase phase II enzyme activities in a variety of tissues. These data, together, suggest that phase II enzyme inducers could have therapeutic value for ameliorating inflammatory conditions.

Mechanisms that Result in Damage During and Following Cerebral Ischemia

The destructive mechanisms associated with stroke are initiated by activation of glutamate receptors resulting in elevated intracellular Ca2+ and reactive oxygen species (ROS) formation. Three major approaches have been investigated to ameliorate ischemia-induced brain damage: (i) interfering with the excitatory action of glutamate; (ii) preventing intracellular accumulation of Ca2+; and (iii) preventing the destructive actions of reactive oxygen species (ROS). Interference with glutamate action can be achieved by: (i) facilitating mechanisms that maintain membrane potentials; (ii) blocking glutamate receptors; and (iii) inhibiting transmitter glutamate synthesis. Prevention of intracellular Ca2+ accumulation may be achieved by: (i) blocking Ca2+ channels; and (ii) facilitating endogenous Ca2+ homeostatic mechanisms. Destructive actions of ROS can be minimized by: (i) administration of ROS-scavenging drugs; (ii) upregulating endogenous ROS-scavenging mechanisms; and (iii) preventing leukocyte invasion of the affected brain tissue. Current therapies that have arisen out of animal experimentation have not met expectations due, mainly to actions of the drugs outside the lesion site. For future research, we suggest: (i) exploring the ability of compromised blood-brain barrier to specifically target therapeutic drugs to the site of lesion; (ii) preventing inflammation by preventing leukocyte infiltration; (iii) identifying signal transduction mechanisms that upregulate neuronal Ca2+ homeostatic mechanisms; and (iv) identifying means that will upregulate endogenous ROS-scavenging mechanisms. Past success in reducing the incidence of stroke has been due, to a great extent, to changes to lifestyle behavioural patterns. We predict that future success in decreasing the morbidity associated with stroke will, to a certain extent, also be due to long-term behavioural changes. It seems possible that simple dietary changes may enable the CNS to be better able to cope with ischemic insults by augmenting ROS-scavenging mechanisms, down-regulating pro-inflammatory responses and increasing Ca(2+)-homeostatic mechanisms.