Protein biomarkerek kísérleti modellekben, illetve terápiás szerepük súlyos agysérülések esetén = Protein biomarkers in experimental models and in the clinical care for severe traumatic brain injury

Jelen kutatási projekt időtartama alatt több irányadó megközelítést alkalmaztunk a protein biomarkerek jelenlétének, szerepének tanulmányozására, és a traumás agysérüléssel történő összefüggések keresésére. Humán szervezetben előforduló biomarkerek (főként a PACAP) mérése, szerepük feltárása valósult meg a PTE Idegsebészeti Klinikán nagy mintaszámban begyűjtött humán plazma és liquor mintákon, mely eredmények számos nemzetközi konferencián bemutatásra kerültek. A traumás agysérülés egyik típusát a diffúz axonkárosodást – a szöveti károsodás markereként használva – többféle módon tanulmányoztuk állatkísérletes koponyatrauma modellek alkalmazásával – eredményeinket szakfolyóiratokban megjelentetve. Ezen módszerek alkalmazásával többféle, jelenleg kísérleti stádiumban lévő neuroprotektív szert tanulmányoztunk, mely eredményeket szintén konferenciákon, tudományos közleményekben bemutattunk. Törekedtünk továbbá olyan összefoglaló közlemények megjelentetésére is, melyek hűen tükrözik a kutatási projekt témájában a tudomány akkori és jelenlegi állását. A pályázatban kitűzött főbb célok mellett számos más, a kutatási- és munkatervben megfogalmazott célokhoz kapcsolódó témában is értünk el sikeres – publikált – eredményeket a támogatás segítségével. Eredményeinket a kutatási projekt lezárulásáig 9 tudományos publikációban (IF: 22,821) és konferenciákon 6 idézhető absztraktban ismertettük, illetve tervezzük a még bemutatásra nem került eredmények közeljövőben történő megjelentetését. | During this research project we used several approaches to study the presence and role of relevant protein biomarkers, and in connection with it to search for correlations related to traumatic brain injury. Measurement and exploring the role of biomarkers that are present in human body (especially the PACAP) was realized on large number of human plasma and cerebrospinal fluid samples that were collected in Department of Neurosurgery, University of Pécs. These results were presented in many international conferences. Considering diffuse axonal injury as an important marker mechanism of the histological damage we examined it in small animal head injury models in different ways and published our results in Journals. Applying these various methods we studied neuroprotective agents – that are in experimental stage – which results was also presented in conferences and manuscripts. Our aim was also to publish review articles that reflect to the subject of this project and to summarize the former and current state of science. Besides the main objectives of relevant topics that were described in the research and work plan of the application other successful – published – results have been achieved with the help of the grant. Until the completion of this research project we published our results in nine peer reviewed manuscripts (IF: 22.821), and six citable abstracts in different conferences and we plan to present the remaining achievements in the near future

Traumatic axonal injury in the spinal cord evoked by traumatic brain injury

Although it is well known that traumatic brain injury (TBI) evokes traumatic axonal injury (TAI) within the brain, TBI-induced axonal damage in the spinal cord (SC) has been less extensively investigated. Detection of such axonal injury in the spinal cord would further the complexity of TBI while also challenging some functional neurobehavioral endpoints frequently used to assess recovery in various models of TBI. To assess TAI in the spinal cord associated with TBI, we analyzed the craniocervical junction (CCJ), cervico-thoracic (CT), and thoraco-lumber (ThL) spinal cord in a rodent model of impact acceleration of TBI of varying severities. Rats were transcardially fixed with aldehydes at 2, 6, and 24 h post-injury (n � 36); each group included on sham-injured rodent. Semi-serial vibratome sections were reacted with antibodies targeting TAI via alteration in cytoskeletal integrity or impaired axonal transport. Consistent with previous observations in this model, the CCJ contained numerous injured axons. Immunoreactive, damaged axonal profiles were also detected as caudal, as the ThL spinal cord displayed morphological characteristics entirely consistent with those described in the brainstem and the CCJ. Quantitative analyses demonstrated that the occurrence and extent of TAI is positively associated with the impact/energy of injury and negatively with the distance from the brainstem. These observations show that TBI can evoke TAI in regions remote from the injury site, including the spinal cord itself. This finding is relevant to shaken baby syndrome as well as during the analysis of data in functional recovery in various models of TBI

Cytotoxicity and the effect on the inflammation response of thyme oil and thymol: evaluation in human macrophage cells

The essential oil of Thymus vulgaris L. (Lamiaceae), thyme oil, shows a great variability of its composition with six main chemotypes recognized up to now: geraniol, linalool, g-terpineol, carvacrol, thymol, and trans-thujan-4-ol/terpinen-4-ol types. Due to this large chemical diversity, the subject of several investigations was to identify and determine their properties, including their potential effect on inflammation. In our previous microbiological study, this essential oil showed a significant antibacterial activity against bacteria of the respiratory tract [1].                The present research focuses on the evaluation of its cytotoxic and antiinflammatory effect in the case of the U937 human monocyte/macrophage cell line. Thyme oil composition was determined by GC/MS. Bürker chamber was used for cell counting and flow-cytometry to evaluate cellular toxicity (using 7-AAD). Then a qPCR method was used to determine the expression of TNFα mRNA.                The main component of the tested sample of thyme oil was thymol (38.7%) that showed a concentration-dependent cytotoxicity. Non-toxic dilutions showed preventive antiinflammatory potential

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

Problémás droghasználók viszonya a hazai kezelőrendszerhez

INST: L_11

Supravital microwave experiments support that the formation of “dark” neurons is propelled by phase transition in an intracellular gel system

Background: Based on circumstantial evidences, we (Gallyas, F., Farkas, O., Mázló, M., 2004. Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of “dark” (compacted) neurons. Biol. Cell 96, 313–324.) proposed that the formation of “dark” neurons (striking compaction of visibly normal ultrastructural elements accompanied with large-scale fluid excretion), which occur in many neurological diseases such as ischemia, proceeds with a non-enzymatic mechanism. Objective: To support this proposition, the present paper deals with the results of supravital experiments using microwave irradiation. Method: After transcardial glutaraldehyde fixation followed by decapitation, a pin was stuck into rat brains just before and just after they were warmed up to 80 °C and maintained at this temperature for various periods of time by controlled microwave irradiation. Results: Independently of the duration of irradiation, the pre-irradiation pin sticking produced numerous “dark” neurons in an approximating 500-μm-wide zone around its track whereas the post-irradiation pin sticking did the same only when the irradiation was shorter than 55 s. The excreted fluid was present in neighbouring astrocytic processes but not in the extracellular space. Conclusions: The formation of “dark” neurons is completed in less than 55 s under the circumstances of the experiment. As neurons are poor in readily consumable chemical energy in the absence of blood circulation, this rapid and massive fluid excretion cannot be explained by any enzyme-mediated membrane-related pump mechanism. An osmotic mechanism can also be discounted. In contrast, it is in conformity with the above mentioned non-enzymatic (physicochemical) phenomenon, the phase transition of a supracytoskeletal gel network storing free energy in the form of non-covalent interactions

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

Aldehyde Wxation is not necessary for the formation of “dark” neurons (Correspondence

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