Antonio Belli

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.

S100B in neuropathologic states : The CRP of the brain?

In recent years there has been a proliferation of interest in the brain‐specific protein S100B, its many physiologic roles, and its behaviour in various neuropathologic conditions. Since the mid‐1960s, its wide variety of intracellular and extracellular activities has been elucidated, and it has also been implicated in an increasing number of central nervous system (CNS) disorders. S100B is part of a superfamily of proteins, some of which (including S100B) have been implicated as calcium‐dependent regulatory proteins that modulate the activity of effector proteins or cells. S100B is primarily an astrocytic protein. Within cells, it may have a role in signal transduction, and it is involved in calcium homeostasis. Information about the functional implication of S100B secretion by astrocytes into the extracellular space is scant but there is substantial evidence that secreted glial S100B exerts trophic or toxic effects depending on its concentration. This review summarises the historic development and current knowledge of S100B, including recent interesting findings relating S100B to a diversity of CNS pathologies such as traumatic brain injury, Alzheimers disease, Downs syndrome, schizophrenia, and Tourettes syndrome. These broad implications have led some workers to describe S100B as ‘the CRP (C‐reactive protein) of the brain.’ This review also examines S100Bs potential role as a neurologic screening tool, or biomarker of CNS injury, analogous to the role of CRP as a marker of systemic inflammation.

Pathogenesis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage: Putative mechanisms and novel approaches

Cerebral vasospasm is a potentially incapacitating or lethal complication in patients with aneurysmal subarachnoid hemorrhage (SAH). The development of effective preventative and therapeutic interventions has been largely hindered by the fact that the underlying pathogenic mechanisms of cerebral vasospasm remain poorly understood. However, intensive research during the last 3 decades has identified certain mechanisms that possibly play a role in its development. Experimental data suggest that calcium‐dependent and ‐independent vasoconstriction is taking place during vasospasm. It appears that the breakdown products of blood in the subarachnoid space are involved, through direct and/or indirect pathways, in the development of vasospasm after SAH. Free radicals reactions, an imbalance between vasoconstrictor and vasodilator substances (endothelium derived substances, e.g., nitric oxide, endothelin; arachidonic acid metabolites, e.g., prostaglandins, prostacyclin), inflammatory processes, an upheaval of neuronal mechanisms that regulate vascular tone, endothelial proliferation, and apoptosis have all been put forward as causative and/or pathogenic factors. Translational research in the field of vasospasm has traditionally aimed to identify agents/interventions in order to block the cascades initiated after SAH. The combination of novel approaches such as cerebral microdialysis, magnetic resonance spectroscopy, proteomics, and lipidomics could serve a dual purpose: elucidating the complex pathobiochemistry of vasospasm and providing clinicians with tools for early detection of this feared complication. The purpose of this Mini‐Review is to provide an overview of the pathogenesis of cerebral vasospasm and of novel approaches used in basic and translational research.

Metabolic failure precedes intracranial pressure rises in traumatic brain injury: a microdialysis study

SummaryBackground: Cerebral microdialysis (MD) is able to detect markers of tissue damage and cerebral ischaemia and can be used to monitor the biochemical changes subsequent to head injury. In this prospective, observational study we analysed the correlation between microdialysis markers of metabolic impairment and intracranial pressure (ICP) and investigated whether changes in biomarker concentration precede rises in ICP.Methods: MD and ICP monitoring was carried out in twenty-five patients with severe TBI in Neurointensive care. MD samples were analysed hourly for lactate:pyruvate (LP) ratio, glutamate and glycerol. Abnormal values of microdialysis variables in presence of normal ICP were used to calculate the risk of intracranial hypertension developing within the next 3u2009h.Findings: An LP ratio >25 and glycerol >100u2009µmol/L, but not glutamate >12u2009µmol/L, were associated with significantly higher risk of imminent intracranial hypertension (odds ratio: 9.8, CI 5.8–16.1; 2.2, CI 1.6–3.8; 1.7, CI 0.6–3, respectively). An abnormal LP ratio could predict an ICP rise above normal levels in 89% of cases, whereas glycerol and glutamate had a poorer predictive value.Conclusions: Changes in the compound concentrations in microdialysate are a useful tool to describe molecular events triggered by TBI. These changes can occur before the onset of intracranial hypertension, suggesting that biochemical impairment can be present before low cerebral perfusion pressure is detectable. This early warning could be exploited to expand the window for therapeutic intervention.

Increase of uric acid and purine compounds in biological fluids of multiple sclerosis patients

OBJECTIVESnIn this study, the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of multiple sclerosis (MS) patients were measured by HPLC and compared with corresponding values recorded in patients without MS (cerebrospinal fluid) and healthy subjects (serum).nnnDESIGN AND METHODSnAll samples were deproteinized with ultrafiltration (which ensures minimal sample manipulation and efficient protein removal) and then assayed for the synchronous HPLC separation of uric acid, hypoxanthine, xanthine, inosine, adenosine, guanosine and creatinine.nnnRESULTSnThe values of all compounds assayed were significantly higher in both biological fluids of MS patients with respect to values measured in controls. In particular, serum hypoxanthine, xanthine, uric acid and sum of oxypurines were, respectively, 3.17, 3.11, 1.23 and 1.27-fold higher in these patients than corresponding values recorded in controls (p<0.001).nnnCONCLUSIONSnDifferently from what previously reported, we here demonstrate that all purine compounds, including uric acid, are elevated in biological fluids of MS patients. Reinforced by the trend observed for creatinine, this corroborates the notion of sustained purine catabolism, possibly due to imbalance in ATP homeostasis, under these pathological conditions. These results cast doubt on the hypothesis that uric acid is depleted in MS because of increased oxidative stress, rather suggesting that this disease causes a generalized increase in purine catabolism. As observed in other pathological states, uric acid, purine compounds and creatinine, can be considered markers of metabolic energy imbalance rather than of reactive oxygen species, even in MS.

Operative and hardware complications of deep brain stimulation for movement disorders

The objective of this investigation was to present the operative and hardware complications encountered during follow-up of patients with in situ deep brain stimulators. The study took the form of a retrospective chart review on a series of consecutive patients who were treated successfully with insertion of deep brain stimulators at a single centre by a single surgeon between 1999 and 2005. During the study period, a total of 60 patients underwent 96 procedures for implantation of unilateral or bilateral DBS electrodes. The mean follow-up period was 43.7 months (range 6 – 78 months) from the time of the first procedure. No patients were lost to follow-up or died. Eighteen patients (30%) developed 28 adverse events, requiring 28 electrodes to be replaced. Seven patients developed two adverse events and two patients developed three adverse events. The rate of adverse events per electrode-year was 8%. We observed a higher proportion of early complications (<6 months postoperatively) in patients with Parkinsons disease, while dystonic patients had more late complications (>6 months postoperatively) and no early complications. Thirty per cent of our patients developed an adverse event that could potentially lead to revision of the implanted hardware. In patients with Parkinsons disease most of the complications tend to occur during the first 6 months postoperatively, while in dystonic patients most occur between 12 and 24 months postoperatively.

Transient alterations of creatine, creatine phosphate, N-acetylaspartate and high-energy phosphates after mild traumatic brain injury in the rat

In this study, the concentrations of creatine (Cr), creatine phosphate (CrP), N-acetylaspartate (NAA), ATP, ADP and phosphatidylcholine (PC) were measured at different time intervals after mild traumatic brain injury (mTBI) in whole brain homogenates of rats. Anaesthetized animals underwent to the closed-head impact acceleration “weight-drop” model (450xa0g delivered from 1xa0m heightxa0=xa0mild traumatic brain injury) and were killed at 2, 6, 24, 48 and 120xa0h after the insult (nxa0=xa06 for each time point). Sham-operated rats (nxa0=xa06) were used as controls. Compounds of interest were synchronously measured by HPLC in organic solvent deproteinized whole brain homogenates. A reversible decrease of all metabolites but PC was observed, with minimal values recorded at 24xa0h post-injury (minimum of CrPxa0=xa048xa0h after impact). In particular, Cr and NAA showed a decrease of 44.5 and 29.5%, respectively, at this time point. When measuring NAA in relation to other metabolites, as it is commonly carried out in “in vivo” 1H-magnetic resonance spectroscopy (1H-MRS), an increase in the NAA/Cr ratio and a decrease in the NAA/PC ratio was observed. Besides confirming a transient alteration of NAA homeostasis and ATP imbalance, our results clearly show significant changes in the cerebral concentration of Cr and CrP after mTBI. This suggests a careful use of the NAA/Cr ratio to measure NAA by 1H-MRS in conditions of altered cerebral energy metabolism. Viceversa, the NAA/PC ratio appears to be a better indicator of actual NAA levels during energy metabolism impairment. Furthermore, our data suggest that, under pathological conditions affecting the brain energetic, the Cr–CrP system is not a suitable tool to buffer possible ATP depletion in the brain, thus supporting the growing indications for alternative roles of cerebral Cr.

Cerebrospinal fluid ATP metabolites in multiple sclerosis

Increased axonal energy demand and mitochondrial failure have been suggested as possible causes for axonal degeneration and disability in multiple sclerosis. Our objective was to test whether ATP depletion precedes clinical, imaging and biomarker evidence for axonal degeneration in multiple sclerosis. The method consisted of a longitudinal study which included 21 patients with multiple sclerosis. High performance liquid chromatography was used to quantify biomarkers of the ATP metabolism (oxypurines and purines) from the cerebrospinal fluid at baseline. The Expanded Disability Status Scale, MRI brain imaging measures for brain atrophy (ventricular and parenchymal fractions), and cerebrospinal fluid biomarkers for axonal damage (phosphorylated and hyperphosphorylated neurofilaments) were quantified at baseline and 3-year follow-up. Central ATP depletion (sum of ATP metabolites >19.7 µmol/litre) was followed by more severe progression of disability if compared to normal ATP metabolites (median 1.5 versus 0, p< 0.05). Baseline ATP metabolite levels correlated with change of Expanded Disability Status Scale in the pooled cohort (r= 0.66, p= 0.001) and subgroups (relapsing—remitting patients: r= 0.79, p< 0.05 and secondary progressive/primary progressive patients: r= 0.69, p< 0.01). There was no relationship between central ATP metabolites and either biomarker or MRI evidence for axonal degeneration. The data suggests that an increased energy demand in multiple sclerosis may cause a quantifiable degree of central ATP depletion. We speculate that the observed clinical disability may be related to depolarisation associated conduction block.

Extracellular fluid S100B in the injured brain: a future surrogate marker of acute brain injury?

SummaryThe authors describe the measurement of S100B protein in brain extracellular fluid (ECF) of patients with acute brain injury (traumatic brain injury and subarachnoid haemorrhage) using the technique of microdialysis. To our knowledge, this is the first report of S100B measurement in the human brain. Acute Brain Injury (ABI) is a leading cause of death and disability and the need for a practical and sensitive biochemical marker for monitoring these patients is urgent. The calcium binding astrocyte protein, S100B, may be a candidate for this role. Previous serum studies have shown S100B to be a sensitive predictor of mortality and rise in intracranial pressure in ABI, but it has never before been measured directly within the brain. The ECF reflects the local biochemistry of the brain parenchyma, and the use of intracerebral microdialysis opens up the possibility of studying many novel surrogate markers of injury in the laboratory, in addition to the conventional markers it measures at the bedside (lactate, pyruvate, glucose, and glycerol). In this preliminary report of two cases, the authors demonstrate the quantification of S100B in ECF microdialysate, and investigate whether changes in hourly S100B profile can be related to secondary brain injury. It is shown that extracellular concentrations of S100B change markedly in response to secondary brain injury. Further investigation is required to determine whether extracellular S100B measurement in ABI could assist in patient management.

Free phenytoin concentration measurement in brain extracellular fluid: a pilot study

This article investigates the relationship between brain extracellular fluid free phenytoin concentration and plasma free phenytoin concentration in adults with acute brain injury. Daily cerebral microdialysate free phenytoin concentration was measured in eight adults with acute brain injury and compared with simultaneous measurement of plasma free phenytoin concentration. The group data revealed no significant correlation between microdialysate and plasma free phenytoin concentration (r = 0.34, p = 0.41). However, in two patients, with a sufficient number of samples for intra-individual analysis, there was a significant correlation between microdialysate and plasma free phenytoin concentration (r = 0.92, p < 0.001 and r = 0.88, p < 0.01). In vitro microdialysis relative recovery for phenytoin was 2.1%. In the context of acute brain injury, measurement of free plasma phenytoin concentration may not provide an accurate reflection of regional brain extracellular fluid free phenytoin concentration and may have limitations with respect to achieving reproducible brain extracellular fluid free phenytoin concentrations. This has implications for dosing regimens relying on plasma phenytoin levels.