Luc Vachon

Hydroxylation of aromatic compounds as indices of hydroxyl radical production: A cautionary note revisited

While setting up an intracerebral microdialysis system to estimate the extent of oxidative stress induced by the neurotoxin, N-methylphenylpyridinium ion (MPP+), we encountered a problem in the use of hydroxybenzoic acids as traps of hydroxyl radicals. Using either 2-hydroxybenzoate (salicylate) or 4-hydroxybenzoate as trapping agents, we observed a nonspecific, that is, nontissue derived, production of hydroxyl radicals as measured by the hydroxylation products, 2,3- and 2,5-dihydroxybenzoate from 2-hydroxybenzoate and 3,4-dihydroxybenzoate from 4-hydroxybenzoate. This production of dihydroxybenzoates was 10 times that expected due to the administration of MPP+, thus making it impossible to interpret our results. Careful investigation of the various components of the microdialysis system indicated that contact of the microdialysate with metal surfaces resulted in dihydroxybenzoic acid formation. These results should serve as a reminder to perform stringent tests of the experimental system prior to experiments with biological tissues to evaluate the contribution of hydroxyl radical production from nonbiological sources. Therefore, along with the possibility of enzymatic production of dihydroxybenzoates, artefactual production by components of the experimental apparatus must be considered before assuming that one is measuring hydroxyl radical production by a biological system.

Development of noninvasive and quantitative methodologies for the assessment of chronic ulcers and scars in humans.

Chronic ulcers are a significant and common cause of morbidity and mortality worldwide. They disrupt the epidermis and dermis, resulting in a loss of barrier function. Keloids and hypertrophic scars (benign cutaneous tumors) represent an abnormal healing response. These fibroproliferative disorders are characterized by an overabundance of collagen and accumulation of extracellular matrix due to an imbalance between synthesis and degradation, culminating in excessive scarring. The objectives of this study were to evaluate and compare noninvasive biophysical methods for the measurement of outstanding quantitative parameters of scars and chronic ulcers, and to establish correlations between the parameters measured and the results of conventional subjective clinical evaluations. The development of new technologies, based on ultrasonography and laser Doppler, makes possible new dermatological evaluation methods. Fifteen patients (6 females and 9 males) with 15 chronic ulcers (4 diabetic ulcers, 10 venous ulcers and 1 pressure ulcer) and 30 patients (19 females and 11 males) with 30 scars (25 hypertrophic and 5 keloids) were included in this study. Clinical evaluation was performed by a dermatologist, an aesthetic surgeon and an endocrinologist. Biophysical measurements were used to assess local blood flow by laser Doppler flowmetry (Moor DRT4), thickness and echogenicity by high frequency ultrasonography (20 MHz, Dermascan C) and ulcer linear dimensions by image analysis. Our results show that blood flow within the ulcers and scars was higher than within normal skin. Also, skin thickness of chronic ulcers was decreased when compared to normal skin; the thickness of hypertrophic scars, but not of keloids, was increased in comparison to normal skin, and presented the possibility of measuring wound and scar surfaces with precision. In summary, this pilot study established the feasibility of measuring various biophysical parameters and adapted their potential utility to research on wounds.

Hydroxyl radical production in the cortex and striatum in a rat model of focal cerebral ischemia

BACKGROUND Increases in hydroxyl radical production have been used as evidence of oxidative stress in cerebral ischemia/ reperfusion. Ischemia can also induce increased dopamine release from the striatum that may contribute to hydroxyl radical formation. We have compared hydroxyl radical production in the cortex and striatum as an index of oxidative stress in a rat model of focal cerebral ischemia with cortical infarction. METHODS Using a three vessel occlusion model of focal cerebral ischemia combined with bilateral microdialysis, hydroxylation of 4-hydroxybenzoate (4HB) was continuously monitored in both hemispheres in either the lateral striatum or frontoparietal cortex. The ischemia protocol consisted of one hour equilibration, 30 min of three vessel occlusion, then release of the contralateral common carotid artery (CCA) for 2.5 h. RESULTS Induction of ischemia resulted in a 30-fold increase in dopamine release in the lateral striatum. Compared to the nonischemic striatum, the ratio of the hydroxylation product 3,4-dihydroxybenzoate (34DHB) to 4HB (trapping agent) in the ipsilateral striatum increased significantly 30 min after ischemia induction. In contrast, during the 30 min of three vessel occlusion there was no increase in the ratio in the cortex. Following the release of the contralateral CCA, the ratio from the ischemic cortex increased significantly compared to sham-operated animals. However, under all circumstances, the 34DHB/4HB ratio was greater in the striatum than in the cortex. CONCLUSION The increase in the 34DHB/4HB ratio in the lateral striatum coincides with the increased dopamine release suggesting a role for dopamine oxidation in the increased production of hydroxyl radicals. The significant increase in the ratio from the ischemic cortex compared to that from the sham-operated animals is consistent with increased oxidative stress induced by ischemia. However, the lower 34DHB/4HB ratio in the cortex which does not receive dopaminergic innervation compared to the striatum suggests a different mechanism for hydroxyl radical production. Such an alternate mechanism may represent a more toxic oxidative insult that contributes to infarction.

Dehydroascorbic acid normalizes several markers of oxidative stress and inflammation in acute hyperglycemic focal cerebral ischemia in the rat.

We investigated the effect of dehydroascorbic acid (DHA), the oxidized form of vitamin C which is a superoxide scavenger, on manganese superoxide dismutase (MnSOD), copper-zinc SOD (CuZnSOD), cyclooxygenase-2 (COX-2) and interleukin-1beta (IL-1beta) expression in a rat model of focal cerebral ischemia under normo- and hyperglycemic conditions. Edema formation was also assessed. MnSOD, CuZnSOD, COX-2 and IL-1beta mRNA and protein expression were studied 3 h post-ischemia. No changes were observed in MnSOD and CuZnSOD mRNA expression among the groups. COX-2 and IL-1beta mRNA expression were upregulated by ischemia but were not influenced by the glycemic state. At the protein level, hyperglycemic cerebral ischemia increased MnSOD and CuZnSOD [Bémeur, C., Ste-Marie, L., Desjardins, P., Butterworth, R.F., Vachon, L., Montgomery, J., Hazell, A.S., 2004a. Expression of superoxide dismutase in hyperglycemic focal cerebral ischemia in the rat. Neurochem. Int. 45, 1167-1174] and IL-1beta expression compared to normoglycemic ischemia. COX-2 protein expression was also significantly higher following hyperglycemic ischemia compared to hyperglycemic shams. DHA administration did not change the pattern of COX-2 or IL-1beta mRNA expression, but normalized the increased protein expression following hyperglycemic ischemia. DHA administration also normalized MnSOD and CuZnSOD protein expression to the levels observed in normoglycemic ischemic animals. Edema formation was significantly reduced by DHA administration in hyperglycemic ischemic animals. The DHA-induced post-transcriptional normalization of MnSOD, CuZnSOD, COX-2 and IL-1beta levels and the decreased edema formation suggest that hyperglycemia accelerates superoxide formation and the inflammatory response, thus contributing to early damage in hyperglycemic stroke and strategies to scavenge superoxide should be an important therapeutic avenue.

Decreased β-actin mRNA expression in hyperglycemic focal cerebral ischemia in the rat

Abstract β-Actin is often used as a housekeeping gene when performing reverse transcription–polymerase chain reaction (RT–PCR) analysis for cerebral ischemia models. In the present study, we tested two different control genes used for RT–PCR experiments, β-actin and porphobilinogen deaminase (PBG-D), in a rat model of focal cerebral ischemia under normo- or hyperglycemic conditions. A three-vessel occlusion model with permanent middle cerebral artery occlusion was used in the rat. β-Actin mRNA expression was decreased in hyperglycemic ischemic rats compared to normoglycemic ischemic animals 3 h post-ischemia. β-Actin protein content was unchanged. As for PBG-D, its mRNA expression remained constant throughout the groups. Our data thus show that, following focal cerebral ischemia in hyperglycemic conditions, β-actin is an unsuitable housekeeping gene whereas PBG-D is more appropriate. This study clearly demonstrates the importance of selecting a stable housekeeping gene when performing RT–PCR experiments.

Expression of superoxide dismutase in hyperglycemic focal cerebral ischemia in the rat

This study investigated the possibility that hyperglycemia induces early expression of various superoxide dismutases (SOD) and nitric oxide synthases (NOS) following focal cerebral ischemia in the rat. MnSOD, CuZnSOD, nNOS and eNOS mRNA and protein expression were examined 3 h after permanent middle cerebral artery occlusion under acute hyperglycemic or normoglycemic conditions. 2,3,5-triphenyltetrazolium chloride (TTC) treatment post-mortem revealed a significant area at risk of infarction following ischemia in hyperglycemic compared to normoglycemic rats. Although no changes in MnSOD, CuZnSOD, nNOS and eNOS mRNA expression were detected, Western blots of ischemic cortex revealed an increase in MnSOD and CuZnSOD protein expression in hyperglycemic compared to normoglycemic rats. Pre-treatment of hyperglycemic rats with the NOS inhibitors L-nitroarginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) or dehydroascorbic acid (DHA), a superoxide scavenger, significantly reduced the TTC delineated zone. The hyperglycemia-induced post-transcriptional upregulation of MnSOD and CuZnSOD levels suggest a response to increased superoxide production which, in the presence of increased nitric oxide production, may play a major role in the increased risk of damage following hyperglycemic stroke.

A Metabolic Signature of Mitochondrial Dysfunction Revealed through a Monogenic Form of Leigh Syndrome

SUMMARY A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD+ biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.

Local striatal infusion of MPP+ does not result in increased hydroxylation after systemic administration of 4-hydroxybenzoate

In vivo bilateral microdialysis in the rat striatum was used to investigate hydroxyl radical formation under basal conditions and after intrastriatal administration of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). After a short equilibration period, 4-hydroxybenzoate (4HBZ), which scavenges hydroxyl radicals to produce 3,4-dihydroxybenzoate (34DHB), was injected intraperitoneally 15 min before infusion of MPP+. To evaluate the enzymatic contribution to hydroxyl radical formation, two other series of microdialyses were performed following administration of monoamine oxidase B inhibitors, either 1-deprenyl (selegiline) or MDL 72,974A [(E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride]. Microdialysate samples were analyzed by high-performance liquid chromatography for catecholamines, 3,4-dihydroxyphenylacetate (DOPAC), homovanillate (HVA), along with the hydroxyl radical adduct, 34DHB and its precursor, 4HBZ. MPP+ administration resulted in a massive release of dopamine along with a decrease in DOPAC and HVA in all three groups. A striking effect in all three groups was noted in which MPP+ resulted in a decrease in interstitial 4HBZ to < 50% of the non-MPP+ -treated side. In absolute terms, the amount of 34DHB produced was low but similar in all three groups, even after unilateral MPP+ infusion. When 34DHB was normalized to 4HBZ release to account for differences in precursor availability, there were no significant differences in the 34DHB/4HBZ ratios either with or without MAO inhibitor treatment or after local MPP+ infusion. Systemic 4HBZ administration appears to result predominantly in intra-cellular sampling of hydroxyl radicals which produces different results from local infusion of trapping agents such as salicylate.

In vivo hydroxylation of the neurotoxin, 1-methyl-4-phenylpyridinium, and the effect of monoamine oxidase inhibitors : Electrospray-MS analysis of intra-striatal microdialysates

The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) has been shown to increase hydroxyl radical formation in the striatum. The production of hydroxyl radicals correlates with the MPP(+)-driven dopamine release which presumably leads to increased metabolism via monoamine oxidase or increased dopamine autoxidation. Both processes result in enhanced production of hydrogen peroxide, which in the presence of iron(II) ions decomposes to the hydroxyl radical. Monoamine oxidase inhibitors decrease the production of hydroxyl radicals as measured by salicylate and 4-hydroxybenzoate trapping. As both MPP+ and monoamine oxidase inhibitors, such as deprenyl and MDL-72,974A, possess aromatic rings, hydroxyl radical adduct formation was investigated in vitro in defined Fenton systems and also in vivo using intra-striatal microdialysis to infuse MPP+ to rats pretreated systemically with either deprenyl or MDL-72,974A. Electrospray mass spectrometric analysis, using full-scan, fragment ion and constant neutral loss spectra, demonstrated ring hydroxylation of all three compounds in the Fenton systems. Spectral comparison of microdialysis samples with spectra from the Fenton reactions indicated the in vivo hydroxyl radical adduct attachment to MPP+, deprenyl and possibly MDL-72,974A.

Patients’ perception of their involvement in shared treatment decision making: Key factors in the treatment of inflammatory bowel disease

OBJECTIVES This study aims to characterize the relationships between the quality of the information given by the physician, the involvement of the patient in shared decision making (SDM), and outcomes in terms of satisfaction and anxiety pertaining to the treatment of inflammatory bowel disease (IBD). METHODS A Web survey was conducted among 200 Canadian patients affected with IBD. The theoretical model of SDM was adjusted using path analysis. SAS software was used for all statistical analyses. RESULTS The quality of the knowledge transfer between the physician and the patient is significantly associated with the components of SDM: information comprehension, patient involvement and decision certainty about the chosen treatment. In return, patient involvement in SDM is significantly associated with higher satisfaction and, as a result, lower anxiety as regards treatment selection. CONCLUSIONS This study demonstrates the importance of involving patients in shared treatment decision making in the context of IBD. PRACTICE IMPLICATIONS Understanding shared decision making may motivate patients to be more active in understanding the relevant information for treatment selection, as it is related to their level of satisfaction, anxiety and adherence to treatment. This relationship should encourage physicians to promote shared decision making.