Péter Bukovics

Multi-Modal Magnetic Resonance Imaging in the Acute and Sub-Acute Phase of Mild Traumatic Brain Injury: Can We See the Difference?

Advanced magnetic resonance imaging (MRI) methods were shown to be able to detect the subtle structural consequences of mild traumatic brain injury (mTBI). The objective of this study was to investigate the acute structural alterations and recovery after mTBI, using diffusion tensor imaging (DTI) to reveal axonal pathology, volumetric analysis, and susceptibility weighted imaging (SWI) to detect microhemorrhage. Fourteen patients with mTBI who had computed tomography with negative results underwent MRI within 3 days and 1 month after injury. High resolution T1-weighted imaging, DTI, and SWI, were performed at both time points. A control group of 14 matched volunteers were also examined following the same imaging protocol and time interval. Tract-Based Spatial Statistics (TBSS) were performed on DTI data to reveal group differences. T1-weighted images were fed into Freesurfer volumetric analysis. TBSS showed fractional anisotropy (FA) to be significantly (corrected p<0.05) lower, and mean diffusivity (MD) to be higher in the mTBI group in several white matter tracts (FA=40,737; MD=39,078 voxels) compared with controls at 72 hours after injury and still 1month later for FA. Longitudinal analysis revealed significant change (i.e., normalization) of FA and MD over 1 month dominantly in the left hemisphere (FA=3408; MD=7450 voxels). A significant (p<0.05) decrease in cortical volumes (mean 1%) and increase in ventricular volumes (mean 3.4%) appeared at 1 month after injury in the mTBI group. SWI did not reveal microhemorrhage in our patients. Our findings present dynamic micro- and macrostructural changes occurring in the acute to sub-acute phase in mTBI, in very mildly injured patients lacking microhemorrhage detectable by SWI. These results underscore the importance of strictly defined image acquisition time points when performing MRI studies on patients with mTBI.

Effect of PACAP in Central and Peripheral Nerve Injuries

Pituitary adenylate cyclase activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. In addition to its more classic role as a neuromodulator, PACAP functions as a neurotrophic factor. Several neurotrophic factors have been shown to play an important role in the endogenous response following both cerebral ischemia and traumatic brain injury and to be effective when given exogenously. A number of studies have shown the neuroprotective effect of PACAP in different models of ischemia, neurodegenerative diseases and retinal degeneration. The aim of this review is to summarize the findings on the neuroprotective potential of PACAP in models of different traumatic nerve injuries. Expression of endogenous PACAP and its specific PAC1 receptor is elevated in different parts of the central and peripheral nervous system after traumatic injuries. Some experiments demonstrate the protective effect of exogenous PACAP treatment in different traumatic brain injury models, in facial nerve and optic nerve trauma. The upregulation of endogenous PACAP and its receptors and the protective effect of exogenous PACAP after different central and peripheral nerve injuries show the important function of PACAP in neuronal regeneration indicating that PACAP may also be a promising therapeutic agent in injuries of the nervous system.

Rescuing neurons and glia: is inhibition of apoptosis useful?

Traumatic brain injury (TBI) represents a leading cause of death in western countries. Despite all research efforts we still lack any pharmacological agent that could effectively be utilized in the clinical treatment of TBI. Detailed unraveling of the pathobiological processes initiated by/operant in TBI is a prerequisite to the development of rational therapeutic interventions. In this review we provide a summary of those therapeutic interventions purported to inhibit the cell death (CD) cascades ignited in TBI. On noxious stimuli three major forms of CD, apoptosis, autophagia and necrosis may occur. Apoptosis can be induced either via the mitochondrial (intrinsic) or the receptor mediated (extrinsic) pathway; endoplasmic reticular stress is the third trigger of caspase-mediated apoptotic processes. Although, theoretically pan-caspase inhibition could be an efficient tool to limit apoptosis and thereby the extent of TBI, potential cross-talk between various avenues of CD suggests that more upstream events, particularly the preservation of the cellular energy homeostasis (cyclosporine-A, poly ADP ribose polymerase (PARP) inhibition, hypothermia treatment) may represent more efficient therapeutic targets hopefully also translated to the clinical care of the severely head injured.

Changes of PACAP level in cerebrospinal fluid and plasma of patients with severe traumatic brain injury

PACAP has well-known neuroprotective potential including traumatic brain injury (TBI). Its level is up-regulated following various insults of the CNS in animal models. A few studies have documented alterations of PACAP levels in human serum. The time course of post-ictal PACAP levels, for example, show correlation with migraine severity. Very little is known about the course of PACAP levels following CNS injury in humans and the presence of PACAP has not yet been detected in cerebrospinal fluid (CSF) of subjects with severe TBI (sTBI). The aim of the present study was to determine whether PACAP occurs in the CSF and plasma (Pl) of patients that suffered sTBI and to establish a time course of PACAP levels in the CSF and Pl. Thirty eight subjects with sTBI were enrolled with a Glasgow Coma Scale ≤8 on admission. Samples were taken daily, until the time of death or for maximum 10 days. Our results demonstrated that PACAP was detectable in the CSF, with higher concentrations in patients with TBI. PACAP concentrations markedly increased in both Pl and CSF in the majority of patients 24-48h after the injury stayed high thereafter. In cases of surviving patients, Pl and CSF levels displayed parallel patterns, which may imply the damage of the blood-brain barrier. However, in patients, who died within the first week, Pl levels were markedly higher than CSF levels, possibly indicating the prognostic value of high Pl PACAP levels.

Posttraumatic administration of pituitary adenylate cyclase activating polypeptide in central fluid percussion injury in rats

Severalin vitro andin vivo experiments have demonstrated the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in focal cerebral ischemia, Parkinson’s disease and traumatic brain injury (TBI). The aim of the present study was to analyze the effect of PACAP administration on diffuse axonal injury (DAI), an important contributor to morbidity and mortality associated with TBI, in a central fluid percussion (CFP) model of TBI. Rats were subjected to moderate (2 Atm) CFP injury. Thirty min after injury, 100 μg PACAP was administered intracerebroventricularly. DAI was assessed by immunohistochemical detection of β-amyloid precursor protein, indicating impaired axoplasmic transport, and RMO-14 antibody, representing foci of cytoskeletal alterations (neurofilament compaction), both considered classical markers of axonal damage. Analysis of damaged, immunoreactive axonal profiles revealed significant axonal protection in the PACAP-treated versus vehicletreated animals in the corticospinal tract, as far as traumatically induced disturbance of axoplasmic transport and cytoskeletal alteration were considered. Similarly to our former observations in an impact acceleration model of diffuse TBI, the present study demonstrated that PACAP also inhibits DAI in the CFP injury model. The finding indicates that PACAP and derivates can be considered potential candidates for further experimental studies, or purportedly for clinical trials in the therapy of TBI.

Calpain inhibition reduces axolemmal leakage in traumatic axonal injury.

Calcium-induced, calpain-mediated proteolysis (CMSP) has recently been implicated to the pathogenesis of diffuse (traumatic) axonal injury (TAI). Some studies suggested that subaxolemmal CMSP may contribute to axolemmal permeability (AP) alterations observed in TAI. Seeking direct evidence for this premise we investigated whether subaxolemmal CMSP may contribute to axolemmal permeability alterations (APA) and pre-injury calpain-inhibition could reduce AP in a rat model of TAI. Horseradish peroxidase (HRP, a tracer that accumulates in axons with APA) was administered one hour prior to injury into the lateral ventricle; 30 min preinjury a single tail vein bolus injection of 30 mg/kg MDL-28170 (a calpain inhibitor) or its vehicle was applied in Wistar rats exposed to impact acceleration brain injury. Histological detection of traumatically injured axonal segments accumulating HRP and statistical analysis revealed that pre-injury administration of the calpain inhibitor MDL-28170 significantly reduced the average length of HRP-labeled axonal segments. The axono-protective effect of pre-injury calpain inhibition recently demonstrated with classical immunohistochemical markers of TAI was further corroborated in this experiment; significant reduction of the length of labeled axons in the drug-treated rats implicate CMSP in the progression of altered AP in TAI.

A biexponential DWI study in rat brain intracellular oedema

PURPOSE To examine the changes in MR parameters derived from diffusion weighted imaging (DWI) biexponential analysis in an in vivo intracellular brain oedema model, and to apply electron microscopy (EM) to shed more light on the morphological background of MR-related observations. MATERIALS AND METHODS Intracellular oedema was induced in ten male Wistar rats (380-450g) by way of water load, using a 20% body weight intraperitoneal injection of 140mmol/L dextrose solution. A 3T MRI instrument was used to perform serial DWI, and MR specroscopy (water signal) measurements. Following the MR examination the brains of the animals were analyzed for EM. RESULTS Following the water load induction, apparent diffusion coefficient (ADC) values started declining from 724±43μm(2)/s to 682±26μm(2)/s (p<0.0001). ADC-fast values dropped from 948±122 to 840±66μm(2)/s (p<0.001). ADC-slow showed a decrease from 226±66 to 191±74μm(2)/s (p<0.05). There was a shift from the slow to the fast component at 110min time point. The percentage of the fast component demonstrated moderate, yet significant increase from 76.56±7.79% to 81.2±7.47% (p<0.05). The water signal was increasing by 4.98±3.52% compared to the base line (p<0.01). The results of the E.M. revealed that water was detected intracellularly, within astrocytic preivascular end-feet and cell bodies. CONCLUSION The unexpected volume fraction changes (i.e. increase in fast component) detected in hypotonic oedema appear to be substantially different from those observed in stroke. It may suggest that ADC decrease in stroke, in contrast to general presumptions, cannot be explained only by water shift from extra to intracellular space (i.e. intracellular oedema).

Quantitative proton MRI and MRS of the rat brain with a 3 T clinical MR scanner

OBJECTIVE To demonstrate the capability of a clinical 3T human scanner in performing quantitative MR experiments in the rat brain. MATERIAL AND METHODS In vivo, measurements on eight Wistar rats were performed. Longitudinal relaxation time (T1) and transverse relaxation time (T2) measurements were set up at a spatial resolution of 0.3×0.3×1mm(3). Diffusion-weighted imaging was also applied and the evaluation included both mono- and biexponential approaches (b-value up to 6000s/mm(2)). Besides quantitative imaging, the rat brain was also scanned at a microscopic resolution of 130×130×130μm(3). Quantitative proton spectroscopy was also carried out on the rat brain with water as internal reference. RESULTS T1 and T2 for the rat brain cortex were 1272±85ms and 75±2ms, respectively. Diffusion-weighted imaging yielded accurate diffusion coefficient measurements at both low and high b-value ranges. The concentrations of MR visible metabolites were determined for the major resonances (i.e., N-acetyl-aspartate, choline and creatine) with acceptable accuracy. CONCLUSION The results suggest that quantitative imaging and spectroscopy can be carried out on small animals on high-field clinical scanners.

A Novel PARP Inhibitor L-2286 in a Rat Model of Impact Acceleration Head Injury: An Immunohistochemical and Behavioral Study

We examined the neuro/axono-protective potential of a novel poly (ADP-ribose) polymerase (PARP) inhibitor L-2286 in a rat impact acceleration brain injury model. Male Wistar rats (n = 70) weighing 300–350 grams were used to determine the most effective intracerebroventricular (i.c.v.) dose of L-2286 administered 30 min after injury, and to test the neuroprotective effect at two time points (immediately, and 30 min after injury). The neuroprotective effect of L-2286 was tested using immunohistochemical (amyloid precursor protein and mid-sized mouse anti-neurofilament clone RMO-14.9 antibody) and behavioral tests (beam-balance, open-field and elevated plus maze). At both time-points, a 100 μg/rat dose of i.c.v. L-2286 significantly (p < 0.05) reduced the density of damaged axons in the corticospinal tract and medial longitudinal fascicle compared to controls. In the behavioral tests, treatment 30 min post-injury improved motor function, while the level of anxiety was reduced in both treatment protocols.

Aldehyde fixation is not necessary for the formation of „dark” neurons

Kherani and Auer [6] recently reported a study in Acta Neuropathologica relating to the mechanism of the formation of “dark” neurons. Their observations led them to suggest that (1) both in vivo and during the early postmortem period, glutamate release and transmembrane ion Xuxes cause some perturbation in a proportion of neurons, which become shrunken, hyperchromatic under the light microscope, and condensed, hyperelectron-dense at the ultrastructural level, but only while aldehyde Wxation is underway, (2) the shrinkage/condensation is executed by transmembrane ion Xuxes or by actin Wlaments, and (3) everything considered, the formation of “dark” neurons is a biotic process. This idea was logically deduced in part from the results of an experiment dealing with the formation of the artefactual “dark” neurons of Cammermeyer (i.e., neurons formed even in normal brain tissue when removed from the skull without previous transcardial aldehyde Wxation followed by a 24-h delay [1]), and in part from the earlier experience of neuropathologists that artefactual “dark” neurons are generally not encountered either in autopsy materials when the postmortem period is long or after nonaldehyde Wxation. Furthermore, they assumed that this idea also applies to the nonartefactual “dark” neurons produced in vivo by ischemia, hypoglycemia, or epilepsy. In a number of communications (reviewed by [3]), our team have postulated another mechanism, which unfortunately is not mentioned in their study. This mechanism postulates that, in each neuron, the intracellular spaces among the ultrastructural elements of conventional transmission electron microscopy are Wlled with a continuous trabecular gel consisting of proteins, ions and water; this gel stores noncovalent (mechanical) free energy, at the expense of which a volume-shrinking phase transition can spread throughout its whole soma-dendrite domain in response to a chemical or a physical noxa (for the existence and importance of such gels in cell biology, see [10]). The pivotal observations supporting this mechanism were as follows: (1) if initiated by physical (nonbiotic) noxae, the formation of “dark”-neurons took place even under conditions that were extremely unfavorable for enzyme-mediated (biotic) processes [4, 7, 9], (2) independently of the nature (physical or chemical) of the initiating noxa and of the circumstances (favorable or unfavorable for enzymatic processes) of the “dark”-neuron formation, the excess water appeared in neighboring astrocytic elements, but not in the surrounding extracellular space [2–9] (the “destination” of transmembrane ion Xuxes), and (3) in each in-vivo case we investigated [2, 5, 8], both the recovering and the dying “dark” neurons displayed ultrastructural signs proving indisputably that they had been compact and electron-dense even long before aldehyde Wxation. To obtain direct evidence against the role of aldehyde Wxation in the formation of the nonartefactual “dark” neurons, we produced “dark” granule neurons in the hippocampal dentate gyrus of four anesthetized living rats via a single condenser discharge, and in the neocortex of four other anesthetized living rats via a pin puncture head injury (for the methodological details and the animal care, see [2]). In each group, two rats were immediately Wxed by the transcardial perfusion of 500 ml of a 3:1 mixture of methanol and chloroform, while the cut-oV heads of the other rats were subjected to Wxation by microwave irradiation (continuous 750 W, controlled 80°C, 1 min). The brains were removed from the skull 1 day later [1] and processed (without aldehyde postWxation) for electron microscopy as usual. P. Bukovics · J. Pal · F. Gallyas (&) Department of Neurosurgery, University of Pecs, 7623 Ret utca 2, Pecs, Hungary e-mail: ferenc.gallyas.sen@aok.pte.hu