Time-course correlation of early toxic events in three models of striatal damage: modulation by proteases inhibition.

Metabolic alterations in the nervous system can be produced at early stages of toxicity and are linked with oxidative stress, energy depletion and death signaling. Proteases activation is responsible for triggering deadly cascades during cell damage in toxic models. In this study we evaluated the early time-course of toxic events (oxidative damage to lipids, mitochondrial dysfunction and LDH leakage, all at 1, 3 and 6h) in rat striatal slices exposed to quinolinic acid (QUIN, 100 microM) as an excitotoxic/pro-oxidant model, 3-nitropropionic acid (3-NP, 1mM) as an inhibitor of mitochondrial succinate dehydrogenase, and a combined model produced by the co-administration of these two toxins at subtoxic concentrations (21 and 166 microM for QUIN and 3-NP, respectively). In order to further characterize a possible causality of caspases or calpains on the toxic mechanisms produced in these models, the broad calpain inhibitor IC1 (50 microM), and the pan-caspase inhibitor Z-VAD (100 microM) were tested. Lipid peroxidation (LP) was increased at all times and in all models evaluated. Both IC1 and Z-VAD exerted significant protection against LP in all models and at all times evaluated. Mitochondrial dysfunction (MD) was consistently affected by all toxic models at 3 and 6h, but was mostly affected by 3-NP and QUIN at 1h. IC1 differentially protected the slices against 3-NP and QUIN at 1h and against QUIN at 3h, while Z-VAD exhibited positive actions against QUIN and 3-NP at all times tested, and against their combination at 3 and 6h. LDH leakage was enhanced at 1 and 3h in all toxic models, but this effect was evident only for 3-NP + QUIN and 3-NP at 6h. IC1 protected against LDH leakage at 1h in 3-NP + QUIN and 3-NP models, at 3h in all toxic models, and at 6h in 3-NP + QUIN and 3-NP models. In turn, Z-VAD protected at 1 and 6h in all models tested, and at 3h in the combined and QUIN models. Our results suggest differential chronologic and mechanistic patterns, depending on the toxic insult. Although LP, MD and membrane cell rupture are shared by the three models, the occurrence of each event seems to obey to a selective recruitment of damaging signals, including a differential activation of proteases in time. Proteases activation is likely to be an up-stream event influencing oxidative stress and mitochondrial dysfunction in these toxic models.

Protective effect of tert-butylhydroquinone on the quinolinic-acid-induced toxicity in rat striatal slices: role of the Nrf2-antioxidant response element pathway.

Tert-butylhydroquinone (tBHQ) is a xenobiotic with reported antioxidant properties. tBHQ has been shown to induce nuclear translocation of the transcription factor NF-E2-related factor 2 (Nrf2) to further activate the antioxidant response element (ARE). In turn, the Nrf2/ARE pathway is responsible for the induction of phase 2 antioxidant enzymes that detoxify oxidant promoters from different toxic insults. In this work, the antioxidant and protective actions of tBHQ were explored for the first time on different biomarkers of the neurotoxic model produced by the excitotoxic and pro-oxidant molecule quinolinic acid (QUIN) in rat striatal slices. For comparison purposes, 3-nitropropionic acid was used as reference model. Our results show that tBHQ (25 µM) prevented the QUIN-induced lipid peroxidation and mitochondrial dysfunction. In addition, tBHQ enhanced glutathione-S-transferase activity, partially recovering its depletion induced by QUIN treatment. Our results also demonstrated that tBHQ was able to induce nuclear accumulation of Nrf2 and further antioxidant protection: while QUIN alone decreased the nuclear Nrf2, a treatment with tBHQ preserved the nuclear levels Nrf2 in the presence of QUIN. Therefore, the tBHQ-mediated Nrf2/ARE induction constitutes a signaling-mediated antioxidant strategy and therapeutic tool to be tested in different neurotoxic models.

Antioxidant strategy to rescue synaptosomes from oxidative damage and energy failure in neurotoxic models in rats: protective role of S-allylcysteine.

The functional preservation of nerve endings since the early stages of toxicity in a given damaging insult-either acute or chronic-by means of antioxidant and neuroprotective agents is a primary need to design therapeutic strategies for neurodegenerative disorders, with particular emphasis on those diseases with excitotoxic and depleted energy metabolism components. S-allylcysteine (SAC), a well-known antioxidant agent, was tested as a post-treatment in different in vitro and in vivo neurotoxic models. Quinolinic acid (QUIN) was used as a typical excitotoxic/pro-oxidant inducer, 3-nitropropionic acid (3-NP) was employed as a mitochondrial function inhibitor, and their combination (QUIN + 3-NP) was also evaluated in in vitro studies. For in vitro purposes, increasing concentrations of SAC (10-100 microM) were added to isolated brain synaptosomes at different times (1, 3 and 6 h) after the incubation with toxins (100 microM QUIN, 1 mM 3-NP or the combination of QUIN (21 microM) + 3-NP (166 microM). Thirty minutes later, lipid peroxidation (LP) and mitochondrial dysfunction (MD) were evaluated. For in vivo studies, SAC (100 mg/kg, i.p.) was given to QUIN- or 3-NP-striatally lesioned rats for 7 consecutive days (starting 120 min post-lesion). LP and MD were evaluated 7 days post-lesion in isolated striatal synaptosomes. Circling behavior was also assessed. Our results describe a differential pattern of protection achieved by SAC, mostly expressed in the 3-NP toxic model, in which nerve ending protection was found within the first hours (1 and 3) after the toxic insult started, supporting the concept that the ongoing oxidative damage and energy depletion can be treated during the first stages of neurotoxic events.

Early nerve ending rescue from oxidative damage and energy failure by L: -carnitine as post-treatment in two neurotoxic models in rat: recovery of antioxidant and reductive capacities.

Cell rescue is a primary need during acute and chronic insults to the central nervous system. Functional preservation during the early stages of toxicity in a given degenerative event may represent a significant amelioration of detrimental processes linked to neuronal cell loss. Excitotoxicity and depleted cellular energy are toxic events leading to cell death in several neurodegenerative disorders. In this work, the effects of the well-known antioxidant and energy precursor, L: -carnitine (L: -CAR), were tested as a post-treatment in two neurotoxic models under in vitro and in vivo conditions. The experimental models tested included: (1) a typical excitotoxic and pro-oxidant inducer, quinolinic acid (QUIN); and (2) a mitochondrial energy inhibitor, 3-nitropropionic acid (3-NP). For in vitro studies, increasing concentrations of L: -CAR (10-1,000 microM) were added to the isolated brain synaptosomes at different times (1, 3 and 6 h) after the incubation with toxins (100 microM QUIN and 1 mM 3-NP), and 30 min later, lipid peroxidation (LP) and mitochondrial dysfunction (MD) were evaluated. For in vivo purposes, L: -CAR (100 mg/kg, i.p.) was given to rats either as a single administration 120 min after the intrastriatal infusion of QUIN (240 nmol/microl) or 3-NP (500 nmol/microl), or for 7 consecutive days (starting 120 min post-lesion). LP and MD were evaluated 4 h and 7 days post-lesions in isolated striatal synaptosomes. Our results show that, despite some variations depending on the toxic model tested, the time of exposure, or the biomarker evaluated, nerve ending protection can be mostly achieved by L: -CAR within the first hours after the toxic insults started, suggesting that targeting the ongoing oxidative damage and/or energy depletion during the first stages of neurotoxic events is essential to rescue nerve endings.

Targeting oxidative/nitrergic stress ameliorates motor impairment, and attenuates synaptic mitochondrial dysfunction and lipid peroxidation in two models of Huntington's disease.

In this study, we reproduced two toxic models resembling some motor/kinetic deficits of Huntington's disease induced by bilateral intrastriatal injections of either quinolinic acid (QUIN, 120 nmol/microl per side) or 3-nitropropionic acid (3-NP, 250 nmol/microl per side) to rats. Motor skills (including total distance walked/traveled and total horizontal and vertical activities) were evaluated in a box-field system at 1 and 7 days post-lesion. In order to investigate whether these alterations were associated with the oxidative/nitrergic stress evoked by the nitrogen reactive species peroxynitrite (ONOO(-)) in the striatum, some rats were pretreated with the ONOO(-) decomposition catalyst iron porphyrinate (Fe(TPPS), 10 mg/kg, i.p.) 120 min prior to toxins infusion. With the aim to further characterize some possible mechanisms by which motor tasks were affected and/or preserved, biochemical analysis of peroxidative damage to lipids and mitochondrial dysfunction were both assessed in synaptic membranes isolated from the striata of QUIN-, 3-NP- and/or Fe(TPPS)-treated animals. Our results show that targeting oxidative/nitrergic stress by Fe(TPPS) in these toxic models results in amelioration of motor deficits linked to inhibition of peroxidative damage and recovery of mitochondrial function in synaptic membranes. Based on these findings, we hypothesize that the protection exerted by Fe(TPPS) on the biochemical markers analyzed reflects the possible preservation of the functional status of the nerve tissue by limiting the deleterious actions of ONOO(-), further accounting for partial recovery of integrative motor functions.