Barbara Tavazzi

Oxidant stress from nitric oxide synthase–3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load

Cardiac pressure load stimulates hypertrophy, often leading to chamber dilation and dysfunction. ROS contribute to this process. Here we show that uncoupling of nitric oxide synthase-3 (NOS3) plays a major role in pressure load-induced myocardial ROS and consequent chamber remodeling/hypertrophy. Chronic transverse aortic constriction (TAC; for 3 and 9 weeks) in control mice induced marked cardiac hypertrophy, dilation, and dysfunction. Mice lacking NOS3 displayed modest and concentric hypertrophy to TAC with preserved function. NOS3(-/-) TAC hearts developed less fibrosis, myocyte hypertrophy, and fetal gene re-expression (B-natriuretic peptide and alpha-skeletal actin). ROS, nitrotyrosine, and gelatinase (MMP-2 and MMP-9) zymogen activity markedly increased in control TAC, but not in NOS3(-/-) TAC, hearts. TAC induced NOS3 uncoupling in the heart, reflected by reduced NOS3 dimer and tetrahydrobiopterin (BH4), increased NOS3-dependent generation of ROS, and lowered Ca(2+)-dependent NOS activity. Cotreatment with BH4 prevented NOS3 uncoupling and inhibited ROS, resulting in concentric nondilated hypertrophy. Mice given the antioxidant tetrahydroneopterin as a control did not display changes in TAC response. Thus, pressure overload triggers NOS3 uncoupling as a prominent source of myocardial ROS that contribute to dilatory remodeling and cardiac dysfunction. Reversal of this process by BH4 suggests a potential treatment to ameliorate the pathophysiology of chronic pressure-induced hypertrophy.

Assessment of metabolic brain damage and recovery following mild traumatic brain injury: a multicentre, proton magnetic resonance spectroscopic study in concussed patients

Concussive head injury opens a temporary window of brain vulnerability due to the impairment of cellular energetic metabolism. As experimentally demonstrated, a second mild injury occurring during this period can lead to severe brain damage, a condition clinically described as the second impact syndrome. To corroborate the validity of proton magnetic resonance spectroscopy in monitoring cerebral metabolic changes following mild traumatic brain injury, apart from the magnetic field strength (1.5 or 3.0 T) and mode of acquisition, we undertook a multicentre prospective study in which a cohort of 40 athletes suffering from concussion and a group of 30 control healthy subjects were admitted. Athletes (aged 16-35 years) were recruited and examined at three different institutions between September 2007 and June 2009. They underwent assessment of brain metabolism at 3, 15, 22 and 30 days post-injury through proton magnetic resonance spectroscopy for the determination of N-acetylaspartate, creatine and choline-containing compounds. Values of these representative brain metabolites were compared with those observed in the group of non-injured controls. Comparison of spectroscopic data, obtained in controls using different field strength and/or mode of acquisition, did not show any difference in the brain metabolite ratios. Athletes with concussion exhibited the most significant alteration of metabolite ratios at Day 3 post-injury (N-acetylaspartate/creatine: -17.6%, N-acetylaspartate/choline: -21.4%; P < 0.001 with respect to controls). On average, metabolic disturbance gradually recovered, initially in a slow fashion and, following Day 15, more rapidly. At 30 days post-injury, all athletes showed complete recovery, having metabolite ratios returned to values detected in controls. Athletes self-declared symptom clearance between 3 and 15 days after concussion. Results indicate that N-acetylaspartate determination by proton magnetic resonance spectroscopy represents a non-invasive tool to accurately measure changes in cerebral energy metabolism occurring in mild traumatic brain injury. In particular, this metabolic evaluation may significantly improve, along with other clinical assessments, the management of athletes suffering from concussion. Further studies to verify the effects of a second concussive event occurring at different time points of the recovery curve of brain metabolism are needed.

Temporal window of metabolic brain vulnerability to concussion: A pilot 1H-magnetic resonance spectroscopic study in concussed athletes - Part III

OBJECTIVE In the present study, the occurrence of the temporal window of brain vulnerability was evaluated in concussed athletes by measuring N-acetylaspartate (NAA) using proton magnetic resonance (H-MR) spectroscopy. METHODS Thirteen nonprofessional athletes who had a sport-related concussive head injury were examined for NAA determination by means of H-MR spectroscopy at 3, 15, and 30 days postinjury. All athletes but three suspended their physical activity. Those who continued their training had a second concussive event and underwent further examination at 45 days from the initial injury. The single case of one professional boxer, who was studied before the match and 4, 7, 15, and 30 days after a knockout, is also presented. Before each magnetic resonance examination, patients were asked for symptoms of mild traumatic brain injury, including physical, cognitive, emotional, and sleep disturbances. Data for H-MR spectroscopy recorded in five normal, age-matched, control volunteers, who were previously screened to exclude previous head injuries, were used for comparison. Semiquantitative analysis of NAA relative to creatine (Cr)- and choline (Cho)-containing compounds was performed from proton spectra obtained with a 3-T magnetic resonance system. RESULTS Regarding the values of the NAA-to-Cr ratio (2.21 +/- 0.11) recorded in control patients, singly concussed athletes, at 3 days after the concussion, showed a decrease of 18.5% (1.80 +/- 0.04; P < 0.001). Only a modest 3% recovery was observed at 15 days (1.88 +/- 0.1; P < 0.001); at 30 days postinjury, the NAA-to-Cr ratio was 2.15 +/- 0.1, revealing full metabolic recovery with values not significantly different from those of control patients. These patients declared complete resolution of symptoms at the time of the 3-day study. The three patients who had a second concussive injury before the 15-day study showed an identical decrease of the NAA-to-Cr ratio at 3 days (1.78 +/- 0.08); however, at 15 days after the second injury, a further diminution of the NAA-to-Cr ratio occurred (1.72 +/- 0.07; P < 0.05 with respect to singly concussed athletes). At 30 days, the NAA-to-Cr ratio was 1.82 +/- 0.1, and at 45 days postinjury, the NAA-to-Cr ratio showed complete recovery (2.07 +/- 0.1; not significant with respect to control patients). This group of patients declared a complete resolution of symptoms at the time of the 30-day study. CONCLUSION Results of this pilot study carried out in a cohort of singly and doubly concussed athletes, examined by H-MR spectroscopy for their NAA cerebral content at different time points after concussive events, demonstrate that also in humans, concussion opens a temporal window of brain metabolic imbalance, the closure of which does not coincide with resolution of clinical symptoms. The recovery of brain metabolism is not linearly related to time. A second concussive event prolonged the time of NAA normalization by 15 days. Although needing confirmation in a larger group of patients, these results show that NAA measurement by H-MR spectroscopy is a valid tool in assessing the full cerebral metabolic recovery after concussion, thereby suggesting its use in helping to decide when to allow athletes to return to play after a mild traumatic brain injury.

N-Acetylaspartate reduction as a measure of injury severity and mitochondrial dysfunction following diffuse traumatic brain injury

N-Acetylaspartate (NAA) is considered a neuron-specific metabolite and its reduction a marker of neuronal loss. The objective of this study was to evaluate the time course of NAA changes in varying grades of traumatic brain injury (TBI), in concert with the disturbance of energy metabolites (ATP). Since NAA is synthesized by the mitochondria, it was hypothesized that changes in NAA would follow ATP. The impact acceleration model was used to produce three grades of TBI. Sprague-Dawley rats were divided into the following four groups: sham control (n = 12); moderate TBI (n = 36); severe TBI (n = 36); and severe TBI coupled with hypoxia-hypotension (n = 16). Animals were sacrificed at different time points ranging from 1 min to 120 h postinjury, and the brain was processed for high-performance liquid chromatography (HPLC) analysis of NAA and ATP. After moderate TBI, NAA reduced gradually by 35% at 6 h and 46% at 15 h, accompanied by a 57% and 45% reduction in ATP. A spontaneous recovery of NAA to 86% of baseline at 120 h was paralleled by a restoration in ATP. In severe TBI, NAA fell suddenly and did not recover, showing critical reduction (60%) at 48 h. ATP was reduced by 70% and also did not recover. Maximum NAA and ATP decrease occurred with secondary insult (80% and 90%, respectively, at 48 h). These data show that, at 48 h post diffuse TBI, reduction of NAA is graded according to the severity of insult. NAA recovers if the degree of injury is moderate and not accompanied by secondary insult. The highly similar time course and correlation between NAA and ATP supports the notion that NAA reduction is related to energetic impairment.

Temporal window of metabolic brain vulnerability to concussions: Mitochondrial-related impairment - Part I

OBJECTIVE In the present study, we investigate the existence of a temporal window of brain vulnerability in rats undergoing repeat mild traumatic brain injury (mTBI) delivered at increasing time intervals. METHODS Rats were subjected to two diffuse mTBIs (450 g/1 m height) with the second mTBI delivered after 1 (n = 6), 2 (n = 6), 3 (n = 6), 4 (n = 6), and 5 days (n = 6) and sacrificed 48 hours after the last impact. Sham-operated animals were used as controls (n = 6). Two further groups of six rats each received a second mTBI after 3 days and were sacrificed at 120 and 168 hours postinjury. Concentrations of adenine nucleotides, N-acetylated amino acids, oxypurines, nucleosides, free coenzyme A, acetyl CoA, and oxidized and reduced nicotinamide adenine dinucleotides, oxidized nicotinamide adenine dinucleotide phosphate, and reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate nicotinic coenzymes were measured in deproteinized cerebral tissue extracts (three right and three left hemispheres), whereas the gene expression of N-acetylaspartate acylase, the enzyme responsible for N-acetylaspartate (NAA) degradation, was evaluated in extracts of three left and three right hemispheres. RESULTS A decrease of adenosine triphosphate, adenosine triphosphate /adenosine diphosphate ratio, NAA, N-acetylaspartylglutamate, oxidized and reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, and acetyl CoA and increase of N-acetylaspartate acylase expression were related to the interval between impacts with maximal changes recorded when mTBIs were spaced by 3 days. In these animals, protracting the time of sacrifice after the second mTBI up to 1 week failed to show cerebral metabolic recovery, indicating that this type of damage is difficult to reverse. A metabolic pattern similar to controls was observed only in animals receiving mTBIs 5 days apart. CONCLUSION This study shows the existence of a temporal window of brain vulnerability after mTBI. A second concussive event falling within this time range had profound consequences on mitochondrial-related metabolism. Furthermore, because NAA recovery coincided with normalization of all other metabolites, it is conceivable to hypothesize that NAA measurement by 1H-NMR spectroscopy might be a valid tool in assessing full cerebral metabolic recovery in the clinical setting and with particular reference to sports medicine in establishing when to return mTBI-affected athletes to play. This study also shows, for the first time, the influence of TBI on acetyl-CoA, N-acetylaspartate acylase gene expression, and N-acetylaspartylglutamate, thus providing novel data on cerebral biochemical changes occurring in head injury.

Cerebral Oxidative Stress and Depression of Energy Metabolism Correlate with Severity of Diffuse Brain Injury in Rats

OBJECTIVE:The combined effect of traumatic brain injury (TBI) and secondary insult on biochemical changes of cerebral tissue is not well known. For this purpose, we studied the time-course changes of parameters reflecting ROS-mediated oxidative stress and modifications of cell energy metabolism determined in rats subjected to cerebral insult of increasing severity. METHODS:Rats were divided into four groups: 1) sham-operated, 2) subjected to 10 minutes of hypoxia and hypotension (HH), 3) subjected to severe diffuse TBI, and 4) subjected to severe diffuse TBI + HH. Rats were killed at different times after injury, and analyses of malondialdehyde, ascorbate, high-energy phosphates, nicotinic coenzymes, oxypurines, nucleosides, and N-acetylaspartate (NAA) were made by high-performance liquid chromatography on whole-brain tissue extracts. RESULTS:Data indicated a close relationship between degree of oxidative stress and severity of brain insult, as evidenced by the highest malondialdehyde values and lowest ascorbate levels in rats subjected to TBI + HH. Similarly, modifications of parameters related to cell energy metabolism were modulated by increasing severity of brain injury, as demonstrated by the lowest values of energy charge potential, nicotinic coenzymes, and NAA and the highest levels of oxypurines and nucleosides recorded in TBI + HH rats. Both the intensity of oxidative stress-mediated cerebral damage and perturbation of energy metabolism were minimally affected in rats subjected to HH only. CONCLUSION:These results showed that the severity of brain insult can be graded by measuring biochemical modifications, specifically, reactive oxygen species-mediated damage, energy metabolism depression, and NAA, thereby validating the rodent model of closed-head diffuse TBI coupled with HH and proposing NAA as a marker with diagnostic relevance to monitor the metabolic state of postinjured brain.

Temporal window of metabolic brain vulnerability to concussions: oxidative and nitrosative stresses - part II

OBJECTIVE In the present study, we investigated the occurrence of oxidative and nitrosative stresses in rats undergoing repeat mild traumatic brain injury (mTBI) delivered with increasing time intervals. METHODS Rats were subjected to two diffuse mTBIs (450 g/1 m height), with the second mTBI delivered after 1 (n = 6), 2 (n = 6), 3 (n = 6), 4 (n = 6), or 5 days (n = 6). The rats were sacrificed 48 hours after the last mTBI. Sham-operated animals were used as controls (n = 6). Concentrations of biochemical indices of oxidative stress (malondialdehyde, ascorbic acid, reduced and oxidized glutathione) and nitrosative stress (nitrite, nitrate) were synchronously measured by high-performance liquid chromatography in deproteinized tissue extracts (three right + three left hemispheres for each group of animals). RESULTS Increase of malondialdehyde, reduced/oxidized glutathione ratio, nitrite, nitrate, and decrease of ascorbic acid and glutathione were dependent on the interval between impacts with maximal changes recorded when mTBIs were spaced by 3 days. Biochemical markers of oxidative and nitrosative stresses were near control levels only in animals receiving mTBIs 5 days apart. CONCLUSION This study shows the remarkable negative contribution of reactive oxygen species overproduction and activation of inducible nitric oxide synthase in repeat mTBI. Because these effects were maximal when mTBIs were spaced by 3 days, it can be inferred that occurrence of a second mTBI within the temporal window of brain vulnerability not only causes profound derangement of mitochondrial functions, but also induces sustained oxidative and nitrosative stresses. Both phenomena certainly play a major role in the overall brain tissue damage occurring under these pathological conditions.

Myocardial release of malondialdehyde and purine compounds during coronary bypass surgery.

Free radicals and lipid peroxidation have been suggested to play an important role in the pathophysiology of myocardial reperfusion injury. The purpose of the present study was to monitor myocardial malondialdehyde (MDA) production as an index of lipid peroxidation during ischemiareperfusion sequences in patients undergoing elective coronary bypass grafting. There has been a lot of debate on the role of xanthine oxidase as a potential superoxide anion generator and thus lipid peroxidation in human myocardium. To evaluate the activity of xanthine oxidase pathway, we measured the changes in the transcardiac concentration differences in adenosine, inosine, hypoxanthine, xanthine, and uric acid. Methods and ResultsThe coronary sinus-aortic root differences (CS-Ao) of MDA, oxypurines, and nucleosides were measured by a recently developed ion-pairing high-performance liquid chromatographic (HPLC) method. Fifteen patients were included in the study, and 13 of them demonstrated a more than 10-fold increase in net myocardial production of MDA on intermittent reperfusion during the aortic crossclamp period. In 2 patients, MDA was not detectable in any of the CS or Ao samples. Before aortic cross-clamping, the CS-Ao concentration differences in adenosine, inosine, hypoxanthine, xanthine, and uric acid were 0.59±0.19, 0.23±0.05, 0.89±0.36, 0.58±0.32, and 11.4±4.9,μmol/L, respectively. After aortic cross-clamping, the sum of the transcardiac differences of these compounds increased up to 2.8-fold and then gradually decreased after declamping of the aorta. There was a weak positive correlation between transcardiac concentration differences of MDA and xanthine plus uric acid (r = .48, P < .01). The postoperative functional recovery or leakage of cardiac enzymes was not affected by the level of MDA net release during the aortic cross-clamp period, however. ConclusionsWe conclude that myocardial lipid peroxidation, estimated as MDA formation, is common during intermittent ischemia-reperfusion sequences in coronary bypass surgery, although some patients may be better protected. Xanthine oxidase appears to be operative in human myocardium, and free radicals generated in this reaction might also be involved in the observed lipid peroxidation process. Increased degradation of myocardial adenine nucleotides and concomitant lipid peroxidation may play a specific role in the development of reperfusion injury. In this study, however, more extensive lipid peroxidation was not associated with impaired functional recovery.

Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography

A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm x 4.6 mm 3 microns particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4 plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemic and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN3 and various concentrations of H2O2 are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid colorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxidative damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues.

The Pathophysiology of Concussion

Concussion is defined as a biomechanically induced brain injury characterized by the absence of gross anatomic lesions. Early and late clinical symptoms, including impairments of memory and attention, headache, and alteration of mental status, are the result of neuronal dysfunction mostly caused by functional rather than structural abnormalities. The mechanical insult initiates a complex cascade of metabolic events leading to perturbation of delicate neuronal homeostatic balances. Starting from neurotoxicity, energetic metabolism disturbance caused by the initial mitochondrial dysfunction seems to be the main biochemical explanation for most postconcussive signs and symptoms. Furthermore, concussed cells enter a peculiar state of vulnerability, and if a second concussion is sustained while they are in this state, they may be irreversibly damaged by the occurrence of swelling. This condition of concussion‐induced brain vulnerability is the basic pathophysiology of the second impact syndrome. N‐acetylaspartate, a brain‐specific compound representative of neuronal metabolic wellness, is proving a valid surrogate marker of the post‐traumatic biochemical damage, and its utility in monitoring the recovery of the aforementioned “functional” disturbance as a concussion marker is emerging, because it is easily detectable through proton magnetic resonance spectroscopy.