Rosario Garrido

Arachidonic Acid‐Induced Oxidative Injury to Cultured Spinal Cord Neurons

Abstract : Spinal cord trauma can cause a marked release of free fatty acids, in particular, arachidonic acid (AA), from cell membranes. Free fatty acids, and AA by itself, may lead to secondary damage to spinal cord neurons. To study this hypothesis, cultured spinal cord neurons were exposed to increasing concentrations of AA (0.01‐10 μM). AA‐induced injury to spinal cord neurons was assessed by measurements of cellular oxidative stress, intracellular calcium levels, activation of nuclear factor‐κB (NF‐κB), and cell viability. AA treatment increased cell intracellular calcium concentrations and decreased cell viability. Oxidative stress increased significantly in neurons exposed to 1 and 10 μM AA. In addition, AA treatment activated NF‐κB and decreased levels of the inhibitory subunit, IκB. It is interesting that manganese superoxide dismutase protein levels and levels of intracellular total glutathione increased in neurons exposed to this fatty acid for 24 h, consistent with a compensatory response to increased oxidative stress. These results strongly support the hypothesis that free fatty acids contribute to the tissue injury observed following spinal cord trauma.

Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons.

Hydrolysis of membrane phospholipids of spinal cord neurons is one of the first events initiated in spinal cord trauma. In this process, free fatty acids, and in particular arachidonic acid, are released. Exposure of spinal cord neurons to free arachidonic acid can compromise cell survival and initiate apoptotic cell death. In order to determine potential mechanisms of apoptosis induced by arachidonic acid, activation of caspases ‐3, ‐8, and ‐9, as well as the release of cytochrome c into the cytoplasm were measured in cultured spinal cord neurons exposed to 10 µm of this fatty acid. In addition, because nicotine can exert a variety of neuroprotective effects, we hypothesized that it can prevent arachidonic acid induced apoptosis of spinal cord neurons. To study this hypothesis, spinal cord neurons were pretreated with nicotine (10 µm for 2 h) before arachidonic acid exposure and caspase activation as well as markers of apoptotic cell death were studied. Treatment of spinal cord neurons with arachidonic acid for up to 24 h significantly increased cytoplasmic levels of cytochrome c, induced caspase activation and induced DNA laddering, a hallmark of apoptotic cell death. Nicotine pretreatment markedly attenuated all these effects. In addition, antagonist studies suggest that the α7 nicotinic receptor is primarily responsible for these anti‐apoptotic effects of nicotine. These results indicate that nicotine can exert potent neuroprotective effects by inhibiting arachidonic acid induced apoptotic cascades of spinal cord neurons.

4-Hydroxynonenal Induces Oxidative Stress and Death of Cultured Spinal Cord Neurons

Abstract: Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid‐induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4‐hydroxynonenal (HNE). The levels of HNE‐protein conjugates in cultured spinal cord neurons increased in a dose‐dependent manner after a 24‐h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3‐h exposure to 10 μM HNE caused ∼80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose‐dependent decrease in MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N‐acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane‐released arachidonic acid to spinal cord neurons.

Nicotine attenuates arachidonic acid-induced neurotoxicity in cultured spinal cord neurons

Arachidonic acid release from cellular membranes due to spinal cord trauma may be one of the principal destructive events that can lead to progressive injury to spinal cord tissue. Exposure to arachidonic acid can compromise neuronal survival and viability. Because nicotine is known to be a neuroprotective agent, we propose that it can prevent arachidonic acid-induced neurotoxicity. To study this hypothesis, effects of nicotine on mitochondrial function, cellular energy content and apoptotic cell death were measured in cultured spinal cord neurons treated with arachidonic acid. Nicotine attenuated arachidonic acid-induced compromised cell viability and cellular ATP levels in spinal cord neurons. Nicotine exerted these protective effects when used at the concentration of 10 microM and only after a 2-h pre-treatment before a co-exposure to arachidonic acid. Antagonists of nicotinic receptors, such as alpha-bungarotoxin or mecamylamine, only partially reversed these neuroprotective effects of nicotine. In addition, nicotine prevented arachidonic acid-induced activation of caspase-3 activity and apoptotic cell death. These results indicate that nicotine pre-treatment can exert a protective effect against arachidonic acid-induced injury to spinal cord neurons.

Nicotine upregulates nerve growth factor expression and prevents apoptosis of cultured spinal cord neurons

Modulation of neurotrophic factor expression may constitute an important part of neuroprotective effects of nicotine. Therefore, the effects of nicotine on expression of nerve growth factor (NGF) and its receptor, tyrosine receptor kinase A (trkA), were studied in cultured spinal cord neurons treated with arachidonic acid. Because injury to spinal cord is associated with elevated levels of arachidonic acid, this cell culture system has been developed in our laboratory as an in vitro model of neuronal injury in spinal cord trauma. Treatment with nicotine markedly upregulated NGF mRNA and protein expression in spinal cord neurons. In addition, a 12h treatment with nicotine increased mRNA levels of trkA. Both nicotine and exogenous NGF inhibited arachidonic acid induced apoptosis of spinal cord neurons. However, the blockage of the trkA receptor prevented nicotine-mediated anti-apoptotic effects. The present results indicate that increased expression of NGF may be an important element of the neuroprotective effects of nicotine in injured spinal cord neurons.

Nicotine Attenuates Arachidonic Acid-Induced Overexpression of Nitric Oxide Synthase in Cultured Spinal Cord Neurons

Primary spinal cord trauma can initiate a cascade of pathophysiologic events which markedly contribute to the expansion and amplification of the primary insult. The detailed mechanisms of these secondary neurochemical reactions are largely unknown; however, they involve membrane lipid derangements with the release of free fatty acids, in particular, arachidonic acid (AA). AA can induce several injury effects on spinal cord neurons. We hypothesize that upregulation of nitric oxide synthase (NOS) is among the most important mechanisms of arachidonic-acid-induced neuronal dysfunction and that nicotine can attenuate this effect. To study these hypotheses, spinal cord neurons were exposed to AA and/or nicotine, and several markers of neuronal nitric oxide synthase (nNOS) metabolism were measured. In addition, cotreatments with either inhibitors of nicotinic receptors or inhibitors of specific NOS isoforms were employed. Treatment with AA markedly increased activity of nNOS, as well as mRNA and protein levels of this enzyme. Changes in nNOS expression were accompanied by an increase in cellular cGMP and medium nitrite levels. Pretreatment with nicotine decreased AA-induced overexpression of nNOS and elevation of nitrite levels. In addition, it appeared that these nicotine effects could be partially modulated both by the alpha7 nicotinic receptors or by nonreceptor mechanisms. Alternatively, the observed changes could also be mediated by an alternate nicotinic receptor mechanism which is not blocked by alpha-bungarotoxin or mecamylamine. Results of the present study indicate that exposure to AA can lead to induction of nNOS in cultured spinal cord neurons. In addition, nicotine can exert a neuroprotective effect by attenuation of AA-induced upregulation of nNOS metabolism. These data may have therapeutic implications for the treatment of acute spinal cord trauma.

Nicotine Attenuates Arachidonic Acid-Induced Apoptosis of Spinal Cord Neurons by Preventing Depletion of Neurotrophic Factors

Increased levels of free fatty acids and, in particular, arachidonic acid can lead to induction of apoptosis of spinal cord neurons. Because of the importance of neurotrophic factors in cell surviv...