Alain Beley

N-Acetylaspartate, a marker of both cellular dysfunction and neuronal loss: its relevance to studies of acute brain injury

To evaluate the contribution of cellular dysfunction and neuronal loss to brain N‐acetylaspartate (NAA) depletion, NAA was measured in brain tissue by HPLC and UV detection in rats subjected to cerebral injury, associated or not with cell death. When lesion was induced by intracarotid injection of microspheres, the fall in NAA was related to the degree of embolization and to the severity of brain oedema. When striatal lesion was induced by local injection of malonate, the larger the lesion volume, the higher the NAA depletion. However, reduction of brain oedema and striatal lesion by treatment with the lipophilic iron chelator dipyridyl (20 mg/kg, 1 h before and every 8 h after embolization) and the inducible nitric oxide synthase inhibitor aminoguanidine (100 mg/kg given 1 h before malonate and then every 9 h), respectively, failed to ameliorate the fall in NAA. Moreover, after systemic administration of 3‐nitropropionic acid, a marked reversible fall in NAA striatal content was observed despite the lack of tissue necrosis. Overall results show that cellular dysfunction can cause higher reductions in NAA level than neuronal loss, thus making of NAA quantification a potential tool for visualizing the penumbra area in stroke patients.

Microglial Involvement in Neuroplastic Changes Following Focal Brain Ischemia in Rats

The pathogenesis of ischemic stroke is a complex sequence of events including inflammatory reaction, for which the microglia appears to be a major cellular contributor. However, whether post-ischemic activation of microglial cells has beneficial or detrimental effects remains to be elucidated, in particular on long term brain plasticity events. The objective of our study was to determine, through modulation of post-stroke inflammatory response, to what extent microglial cells are involved in some specific events of neuronal plasticity, neurite outgrowth and synaptogenesis. Since microglia is a source of neurotrophic factors, the identification of the brain-derived neurophic factor (BDNF) as possible molecular actor involved in these events was also attempted. As a means of down-regulating the microglial response induced by ischemia, 3-aminobenzamide (3-AB, 90 mg/kg, i.p.) was used to inhibit the poly(ADP-ribose) polymerase-1 (PARP-1). Indeed, PARP-1 contributes to the activation of the transcription factor NF-kB, which is essential to the upregulation of proinflammatory genes, in particular responsible for microglial activation/proliferation. Experiments were conducted in rats subjected to photothrombotic ischemia which leads to a strong and early microglial cells activation/proliferation followed by an infiltration of macrophages within the cortical lesion, events evaluated at serial time points up to 1 month post-ictus by immunostaining for OX-42 and ED-1. Our most striking finding was that the decrease in acute microglial activation induced by 3-AB was associated with a long term down-regulation of two neuronal plasticity proteins expression, synaptophysin (marker of synaptogenesis) and GAP-43 (marker of neuritogenesis) as well as to a significant decrease in tissue BDNF production. Thus, our data argue in favour of a supportive role for microglia in brain neuroplasticity stimulation possibly through BDNF production, suggesting that a targeted protection of microglial cells could represent an innovative approach to potentiate post-stroke neuroregeneration.

N‐Acetylaspartate: a literature review of animal research on brain ischaemia

The present review examines and discusses the changes in N‐acetylaspartate (NAA) concentration in the ischaemic brain from the existing literature of animal research. By summarizing the current knowledge on NAA metabolic pathways and reviewing the data obtained in animal models of global and focal ischaemia, the following conclusions emerge from this compilation: (i) both magnetic resonance spectroscopy (MRS) and the absolute HPLC method of NAA quantification give converging information; (ii) decreases in brain NAA concentration in acute stroke can be considered as an index of neuronal loss or dysfunction, although NAA redistribution by glial cells and NAA trapping in cell debris restrict its use as a quantitative neuronal marker; (iii) further studies on NAA metabolism in pathologic brain are required before using NAA measurement in the chronic stage of ischaemia for evaluating neuroprotective strategies.

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARα-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARα independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

Stress response to hypoxia in gerbil brain: HO-1 and Mn SOD expression and glial activation

Hypoxic preconditioning has been shown to induce neuroprotection against a subsequent damaging insult. In order to study the underlying molecular and cellular mechanisms of hypoxic preconditioning, we investigated, in gerbil hippocampus, the effects in vivo of transient exposure to hypoxia (4% O(2) for 6 min followed by either 48 h or 7 days of reoxygenation) (i) on the induction of 72 kDa heat shock protein (HSP72), heme oxygenase-1 (HO-1) and manganese superoxide dismutase (Mn SOD) as assessed by Western immunoblotting and (ii) on the astroglial and microglial activation as detected by both immunohistochemistry and Western immunoblotting for GFAP, and histochemistry for isolectin B4, respectively. Our data show that, although hypoxia and subsequent reoxygenation led to neither neuronal damage nor HSP72 induction in gerbil hippocampus, it induced a progressive and sustained expression of HO-1 and Mn SOD. As expected from the absence of neuronal death, hypoxia was not associated with microglial activation but led to a significant astrocytic activation. These findings demonstrate that transient hypoxia enhances the antioxidative enzymatic defenses of the brain, which are susceptible to increased tolerance against a subsequent damaging insult.

Importance of iron location in iron-induced hydroxyl radical production by brain slices.

Iron imbalance has been implicated in oxidative injury associated with many brain diseases. The present study investigated the importance of iron location in hydroxyl radical (.OH) generation and the link between .OH production evaluated by the salicylate method and lipid peroxidation monitored by thiobarbituric acid-reactive substances assay. Brain slices were exposed to increasing doses (2, 10 and 50 microM) of Fe(III) that was complexed either to a lipophilic (8-hydroxyquinoline, HQ) or to a hydrophilic (ammoniacal citrate) ligand. Both iron complexes resulted in an increased salicylate hydroxylation and lipid peroxidation, these effects being significantly more potent in presence of Fe(III)-HQ. Salicylate hydroxylation was linearly correlated to the intensity of TBARS formation but the slope of the curve was found to be higher with Fe(III)-HQ. The present results demonstrate that 1) cell-associated reactive iron is more prone than extracellular iron to induce .OH generation, 2) the level of lipid peroxidation depending on the site of .OH production, cannot be used as an index of the level of total .OH formation, 3) the salicylate method is a convenient method to detect .OH formed intracellularly, at least in vitro.

5‐Hydroxyindoleacetic Acid and Homovanillic Acid in Cerebrospinal Fluid of Children with Febrile Convulsions

Summary 5‐Hydroxyindoleacetic acid (5‐HIAA) and homovanillic acid (HVA) were measured by high‐performance liquid chromatography (HPLC) in lumbar cerebrospinal fluid (CSF) obtained from febrile children subdivided according to the presence or absence of convulsions. Lumbar puncture was made either early (mean time 2 h) or late (3–6 days) after the febrile convulsion. The level of 5‐HIAA was significantly decreased in children early and late after the febrile convulsion as compared with the convulsion‐free group, but the HVA level was reduced only early after the febrile convulsion. These results support the hypothesis that a decrease in CSF 5‐HIAA may be a biologic marker of susceptibility to convulsions and indicate that the transient decrease in HVA is a secondary phenomenon related to occurrence of convulsions.

Evidence of HIF-1 functional binding activity to caspase-3 promoter after photothrombotic cerebral ischemia

Hypoxia-inducible factor 1 alpha (HIF-1alpha) is a transcription factor that was suggested in vitro to promote cell death by modulation of proapoptotic genes. In this report, we tested the hypothesis of an in vivo proapoptotic role of HIF-1alpha after an ischemic insult. For this purpose, HIF-1alpha and procaspase-3 mRNA and protein expressions were examined in rat brain subjected to 12- and 24-h permanent focal ischemia and the presence of an HIF-1 binding activity to the caspase-3 gene promoter was explored. The results showed that HIF-1alpha and procaspase-3 expressions increased with a similar pattern in response to ischemia. In addition, caspase-3 activation was observed in cells that express HIF-1alpha. Moreover, electrophoretic mobility assay revealed a specific HIF-1 binding activity to the caspase-3 gene promoter. Altogether the present data provide strong arguments for a causative relationship between HIF-1alpha and caspase-3 inductions through a functional binding activity to the caspase-3 gene promoter.

Improved Method for Determination of Acetylcholine, Choline, and Other Biogenic Amines in a Single Brain Tissue Sample Using High Performance Liquid Chromatography and Electrochemical Detection

Abstract A simple method for determination of ACh, Ch, NA, DA, 5-HT and their related metabolites on the same brain tissue sample was developed by HPLC-ED. The electrochemical detection system is equipped with a platinum electrode for ACh and Ch detection, or a glassy carbon electrode for CA and 5-HT detection. ACh and Ch can be separated with bonded silica or polystyrene reverse phase columns, using a pH 7 mobile phase. They are converted to H2O2 by the passage of the effluent through an in line post column reactor with covalently bonded ACh esterase and Ch oxidase. This step ensures sensitivity, reliability and enzyme economy. Tissue preparation consists of formic acid/acetone extraction and purification by tetraphenyl boron exchange with high reproductible recoveries. The time necessary for the whole procedure is short, making it well adapted to large series. CA, 5-HT and related metabolites can be simply analysed on an aliquot of the tissue extract.

Effect of intracellular iron loading on lipid peroxidation of brain slices

The effect of artificially elevated cell iron content on oxygen-derived free radical production was assessed in brain slices by use of an iron ligand, 8-hydroxyquinoline (HQ). The iron complex Fe(3+)-HQ exhibited a high lipid solubility evidenced by n-octanol/water partition coefficient and was avidely taken up by brain slices. The catalytically active form of Fe3+ within the complex was evidenced by measuring the rate of ascorbate oxidation. Lipid peroxidation was assessed by measuring the thiobarbituric acid-reactive substances (TBARS) in brain homogenates or slices exposed to two doses of Fe(3+)-HQ (10 microM/20 microM, 100 microM/200 microM) or Fe(3+)-citrate (10 microM, 100 microM). Addition of the iron complexes to homogenates or slices resulted in a dose-dependent increase in lipid peroxidation. In homogenates, the effects were grossly similar with both complexes, whereas in slices the effects of Fe-HQ were significantly higher than those of Fe-citrate. Lipid peroxidation persisted in washed slices preexposed to Fe-HQ, but not in slices preexposed to the hydrophilic iron complex Fe-citrate. Fe-HQ-induced lipid peroxidation in slices was enhanced in the presence of H2O2, an effect that was not seen using Fe-citrate. Addition of Fe-HQ to brain homogenates in the presence of salicylic acid resulted in the production of 2,3-dihydroxybenzoic acid and the effect was potentiated in the presence of H2O2. This model of iron cell loading may be useful for evaluating the efficacy of antioxidant drugs.