Juan C. Leza

Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain.

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurodegenerative disorders. We have previously demonstrated that chronic stress induced an increase in nitric oxide (NO) production via an expression of inducible NO synthase (iNOS) in brain. Since it has been demonstrated that NO regulates mitochondrial function, we sought to study the susceptibility of the mitochondrial respiratory chain complexes to chronic restrain stress exposure in brain cortex. In adult male rats, stress (immobilization for six hours during 21 days) inhibits the activities of the first complexes of the mitochondrial respiratory chain (inhibition of 69% in complex I-III and of 67% in complex II-III), without affecting complex IV activity, ATP production and oxygen consumption. The mitochondrial marker citrate synthase is not significantly affected by stress after 21 days, indicating that at this time the mitochondrial structure is still intact. Moreover, the administration of the preferred inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) protects against the inhibition of the activity of complexes of the mitochondrial respiratory chain as well as prevents NOx− accumulation, lipid peroxidation and glutathione depletion induced by stress. These results suggest that a sustained overproduction of NO via iNOS is responsible, at least in part, of the inhibition of mitochondrial respiratory chain caused by stress and that this pathway also accounts for the oxidative stress found in this situation.

Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex

Abstract: Long‐term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium‐independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2‐ + NO3‐ (NOx‐) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx‐ accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Stress as a neuroinflammatory condition in brain: damaging and protective mechanisms.

Several neuropsychiatric diseases are related with stress (posttraumatic stress disorder, major depressive disorder, anxiety disorders, schizophrenia) and stress exposure modifies the onset and evolution of some neurological diseases (neurodegenerative diseases). It is accepted that brain inflammatory responses contribute to cell damage during these illnesses. Studies carried out with some stress protocols (physical, psychological or mixed) show a pro-inflammatory response in the brain and other systems mainly characterized by a complex release of several inflammatory mediators such as cytokines, prostanoids, free radicals and transcription factors. This review considers the current status of knowledge of stress-induced inflammation in the brain. Interestingly, anti-inflammatory pathways are also activated in brain in response to stress, constituting a possible endogenous mechanism of defence against excessive inflammation. The possibility of pharmacological modulation of these pathways to prevent the accumulation of pro-inflammatory mediators and subsequent brain damage in stress and in stress-related neuropsychological conditions is also reviewed. This dual response elicited by stress in brain, both pro- and anti-inflammatory deserves further attention in order to understand pathophysiological changes as well as possible new therapeutic approaches of stress-related neuropsychopathologies.

The Increase in TNF-α Levels Is Implicated in NF-κB Activation and Inducible Nitric Oxide Synthase Expression in Brain Cortex after Immobilization Stress ☆

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex after three weeks of repeated stress and that its overexpression accounts for the neurodegenerative changes found in this situation. Now we have found that a short duration of stress (immobilization for 6 h) also induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in iNOS expression, we have studied the role of the cytokine tumor necrosis factor-α (TNF-α) released in brain during stress. We have shown that there is an increase in soluble TNF-α levels after 1 h of stress in cortex and that this is preceded by an increase in TNF-α-convertase (TACE) activity in brain cortex as soon as 30 min after immobilization. Stress-induced increase in both TACE activity and TNF-α levels seems to be mediated by excitatory amino acids since they can be blocked by MK-801 (dizocilpine) (0.2 mg/kg i.p.), an antagonist of the N-methyl-D-aspartate subtype of glutamate receptor. In order to study the role of TACE and TNF-α in iNOS induction, a group of animals were i.p. injected with the preferred TACE inhibitor BB1101 (2 and 10 mg/kg). Indeed, BB1101 inhibited iNOS expression induced by six hours of stress. In addition, we studied the role of the transcription factor nuclear factor κB (NF-κB), which is required for iNOS expression. We have found that the administration of the TACE inhibitor BB1101 inhibited the stress-stimulated translocation of NF-κB to the nucleus. Taken together, these findings indicate that glutamate receptor activation induces TACE up-regulation and subsequent increase in TNF-α levels, and this account for stress-induced iNOS expression via NF-κB activation, supporting a possible neuroprotective role for specific TACE inhibitors in this situation.

Inducible nitric oxide synthase expression in brain cortex after acute restraint stress is regulated by nuclear factor κB-mediated mechanisms

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high‐output and long‐lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex during stress and that its overexpression accounts for the neurodegenerative changes seen after 3 weeks of repeated stress. Now we have found that acute stress (restraint for 6 h) increases the activity of a calcium‐independent NOS and induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in this induction, we studied the role of transcription nuclear factor κB (NF‐κB), which is required for iNOS synthesis. We have observed that an acute restraint stress session stimulates the translocation of the NF‐κB to the nucleus after 4 h and that the administration of the NF‐κB inhibitor pyrrolidine dithiocarbamate [PDTC, 75 and 150 mg/kg intraperitoneally (i.p.)] at the onset of stress inhibits the stress‐induced increase in iNOS expression. Since glutamate release and subsequent NMDA (N‐methyl‐d‐aspartate) receptor activation has been recognized as an early change after exposure to stressful stimuli, and glutamate has been shown to induce iNOS in brain via a NF‐κB‐dependent mechanism, we studied the possible role of excitatory amino acids in the induction of iNOS in our model. Pretreatment with the NMDA receptor antagonist dizocilpine (MK‐801, 0.1 and 0.3 mg/kg i.p.) inhibits the stress‐induced NF‐κB activation as well as the stress‐induced increase in iNOS expression. Taken together, these findings indicate that excitatory amino acids and subsequent activation of NF‐κB account for stress‐induced iNOS expression in cerebral cortex, and support a possible neuroprotective role for specific inhibitors in this situation.

Toll-Like Receptor 4 Is Involved in Subacute Stress–Induced Neuroinflammation and in the Worsening of Experimental Stroke

Background and Purpose— Psychological stress causes an inflammatory response in the brain and is able to exacerbate brain damage caused by experimental stroke. We previously reported that subacute immobilization stress in mice worsens stroke outcome through mechanisms that involve inflammatory mechanisms, such as accumulation of oxidative/nitrosative mediators and expression of inducible nitric oxide synthase and cyclooxygenase-2 in the brain. Some of these inflammatory mediators could be regulated by innate immunity, the activation of which takes place in the brain and produces an inflammatory response mediated by toll-like receptors (TLRs). Recently, we described the implications of TLR4 in ischemic injury, but the role of TLR4 in stress has not yet been examined. We therefore investigated whether inflammation produced by immobilization stress differs in mice that lack a functional TLR4 signaling pathway. Methods— We used an experimental paradigm consisting of the exposure of mice to repeated immobilization sessions (1 hour daily for 7 days) before permanent middle cerebral artery occlusion. Results— We found that TLR4-deficient mice subjected to subacute stress had a better behavioral condition compared with normal mice (C3H/HeN) and that this effect was associated with a minor inflammatory response (cyclooxygenase-2 and inducible nitric oxide synthase expression) and lipid peroxidation (malondialdehyde levels) in brain tissue. Furthermore, previous exposure to stress was followed by a smaller infarct volume after permanent middle cerebral artery occlusion in TLR4-deficient mice than in mice that express TLR4 normally. Conclusions— Our results indicate that TLR4 is involved in the inflammatory response after subacute stress and its exacerbating effect on stroke. These data implicate the effects of innate immunity on inflammation and damage in the brain after stroke.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARgamma, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFkappaB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-alpha also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-alpha activation and release, inhibitors of NFkappaB, specific inhibitors of iNOS and COX-2 activities and PPARgamma agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

In Vitro Ischemic Tolerance Involves Upregulation of Glutamate Transport Partly Mediated by the TACE/ADAM17-Tumor Necrosis Factor-α Pathway

A short ischemic event [ischemic preconditioning (IPC)] can result in a subsequent resistance to severe ischemic injury (ischemic tolerance). Although tumor necrosis factor-α (TNF-α) contributes to the brain damage found after cerebral ischemia, its expression and neuroprotective role in models of IPC have also been described. Regarding the role of TNF-α convertase (TACE/ADAM17), we have recently shown its upregulation in rat brain after IPC induced by transient middle cerebral artery occlusion and that subsequent TNF-α release accounts for at least part of the neuroprotection found in this model. We have now used an in vitro model of IPC using rat cortical cultures exposed to sublethal oxygen-glucose deprivation (OGD) to investigate TACE expression and activity after IPC and the subsequent mechanisms of ischemic tolerance. OGD-induced cell death was significantly reduced in cells exposed to IPC by sublethal OGD 24 hr before, an effect that was inhibited by the TACE inhibitor BB3103 (1 μm) and anti-TNF-α antibody (2 μg/ml) and that was mimicked by TNF-α (10 pg/ml) preincubation. Western blot analysis showed that TACE expression is increased after IPC. IPC caused TNF-α release, an effect that was blocked by the selective TACE inhibitor BB-3103. In addition, IPC diminished the increase in extracellular glutamate caused by OGD and increased cellular glutamate uptake and expression of EAAT2 and EAAT3 glutamate transporters; however, only EAAT3 upregulation was mediated by increased TNF-α. These data demonstrate that neuroprotection induced by IPC involves upregulation of glutamate uptake partly mediated by TACE overexpression.

Neuronal and inducible nitric oxide synthase and nitrotyrosine immunoreactivities in the cerebral cortex of the aging rat.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS‐immunoreactive multipolar neurons found in layers II–VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS‐positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II–VI of aged rat cortex. While no iNOS‐immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine‐positive cells outnumbered total NOS‐positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine‐positive cells. Western blots of brain extracts showed a several‐fold increase in both nNOS‐ and iNOS‐immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine‐containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation. Microsc. Res. Tech. 43:75–88, 1998.

Induction of Cyclooxygenase-2 Accounts for Restraint Stress-Induced Oxidative Status in Rat Brain

Cyclooxygenase (COX) is the rate-limiting enzyme in the metabolism of arachidonic acid into prostanoids. Although it is constitutively expressed in brain neurons, the inducible isoform (COX-2) is also upregulated in pathological conditions such as seizures, ischemia or some degenerative diseases. To assess whether COX-2 is regulated after stress, we have used adult male Wistar rats, some of which were immobilized during 6 h. An increase in PGE2 concentration occurs in brain cortex after 2–6 h of the onset of stress as well as an enhancement of COX-2 protein. Immunohistochemical studies indicate that COX-2 is expressed in the cortex and hippocampus after stress in cells with morphology of neurons. Administration of PDTC (150 mg/kg), an inhibitor of the transcription factor NF-κB or MK-801 (0.2 mg/kg), an N-methyl-D-aspartate receptor blocker, prevents both stress-induced increase in COX-2 activity and protein levels, suggesting an implication of these factors in the mechanism by which stress induces COX-2 in brain. To assess if COX-2 accounts for the oxidative status seen in brain after stress, a group of animals were i.p. injected with NS-398, a specific COX-2 inhibitor 1 h prior to the onset of stress. NS-398 (5 mg/kg) decreases stress-induced malondialdehyde accumulation in cortex as well as prevents the stress-induced oxidation of glutathione. Finally, NS-398 reduced Ca2+-independent inducible nitric oxide synthase (iNOS, NOS-2) activity and lowered the stress-induced accumulation of NO metabolite levels in cortex. These effects of NS-398 seem to be due to the specific inhibition of COX-2, since it has no effect on stress-induced corticosterone release, glutamate release, and NF-κB activation. These findings are discussed as possible damaging and/or adaptive roles for stress-induced COX-2 in the brain.