Biomarkerek szerepe a koponya/agysérülés súlyosságának, kimenetelének és a therápia hatékonyságának megítélésében = The role of biomarkers in the assessment of injury-severity, outcome and therapeutic efficacy in traumatic brain injury

A kutatás legfőbb eredményei az alábbiak: 1., Elsőként igazoltuk, hogy a súlyos kopnyasérültek liquorában a traumától eltelt idővel összefüggésben intakt spectrin és lebontási termékei szaporodnak fel; ezek megjelenése a koponya sérülésre specifikus jelenségnek tekinthető, ráadásul e lebontási termékek koncentrációja valószínűsíthetően összefüggést mutat a koponyasérülés súlyosságával és a kimenetellel is. E vizsgálatok a Munkacsoport Department of Defense (US) támogatású multicentrikus klinikai tanulmány előkészítésében és kivitelezésében történő részvételét eredményezték. 2., Patkány kísérletes traumás modellben végzett experimentális therápiás vizsgálatok során a diffúz axon károsodás gátlását igazoltuk posttraumás pituitary adenylate cyclase activating peptide adásával impakt akcelerációs és centralis folyadék percussiós modellen. 3., Elsőként írtuk le a különböző súlyosságú koponyasérülés hatására a gerincvelőben létrejövő diffúz axonális károsodás jelenségét. 4. A klinikai adatbázis feldolgozása során a morbiditási-mortalitási esélyeket meghatározó, az intézményi ellátás auditálására is lehetőséget nyújtó, a Marshall score és a Rotterdam score validálásán, továbbfejlesztésén alapuló CT-klasszifikációs rendszert dolgoztunk ki. A három éves kutatómunka során 20 közlemény, könyvfejezet és kongresszusi kivonat született, a peer reviewed közlemények kumulatív impakt faktora 17 feletti. | Major achievements of the project are the following: 1., We have provided the first evidence that traumatic brain injury in human leads to the accumulation of intact spectrin and its? breakdown products and that the appearance of these substances in the cerebrospinal fluid follows a well defined temporal pattern while also associated with injury severity and most probably with outcome, too. 2., Posttraumatic application of pituitary adenylate cyclase activating peptide resulted in the inhibition of diffuse axonal injury both in the impact acceleration- as well as the central fluid percussion model of diffuse traumatic brain injury in rats. 3., These studies provided the first description of diffuse axonal injury in the spinal cord evoked by/associated with diffuse traumatic brain injury of various severity. 4., We have developed a new scoring system based upon the first posttraumatic non-contrast skull CT scan that is capable of predicting outcome and represents an updated version and combination of the Rotterdam-score and the Marshall-classification. This three-years project led to the accumulation of 20 publications of various forms including peer reviewed articles with an impact factor of over 17

MSG-indukálta retinális degeneráció és a PACAP neuroprotektív hatásának vizsgálata = MSG-induced retinal degeneration and the neuroprotective effect of PACAP

A monosodium-glutamát (MSG) egy ismert ízfokozó, amelyet a kínai konyha nagy mennyiségben adagol ételeihez. E vegyület retinakárosító hatását, valamint annak kivédésének lehetséges módszereit vizsgáltuk patkányban. Újszülött patkányokba subcutan MSG-t injektáltunk. Három hét elteltével vizsgáltuk a retinákat. Az kísérletek egy részeben a MSG injekciókkal együtt neuroprotektív szereket, főleg PACAP-ot, alkalmaztunk a degeneratív hatások kivédésére. Munkánk során a MSG-modellt optimalizáltuk és kifejlesztettünk egy ischemiás modellt is, amikor 2 hetes állatokon bilaterális carotis communis lekötést alkalmaztunk és 1 hét elteltével vizsgáltuk a retinákat. Ebben a modellben is lehetőség nyílt a protektív ágensek alkalmazására. Mindezen modellekben morfológiai, morfometriai, immuncitokémiai, és biokémiai módszerekkel vizsgáltuk a sejt- és molekuláris szintű eseményeket. Főbb eredményeink a következőkben foglalhatók össze: 1. A MSG modellben elsősorban a belső retinális rétegek károsodnak. 2. Az ischemiás modellben minden sejtrétegben találtunk károsodott sejteket. 3. A PACAP intravitreálisan adagolva mindkét modellben protektív hatású. 4. A PACAP protektív hatását a proapoptikus faktorok gátlásával és az anti-apoptotikus faktorok serkentésén keresztül éri el. 5. A védő hatás következtében a retina rétegei és sejttípusai megtartottak maradnak, a javulás funkcionális vizsgáló módszerrel (elektroretinogram) is igazolható. 6. Védő hatást gyakorol az ingergazdag környezet is. | Monosodium-glutamát (MSG) is a well-known aromatizer which is widely used in the Chinese cuisine. The retinotoxic effects of this substance and the possible neuroprotective strategies against this compound were examined in rats. Newborn rats were injected subcutaneously with MSG and their retinas were examined after three weeks. In some experiments neuroprotective agents were simulatneously applied with MSG. During the project we optimized the MSG model and also developed an ischemic retina model, in which the common carotid arteries of 2-week-old rats were ligated and the retinas were processed one week after the ligation. Also, a screen for neurorotective agents was executed. The experimental results were obtained with morphological, morphometric, immunocytochemical and biochemical methods. The main results are as follows: 1. After MSG treatment the inner retinal layers were damaged. 2. In the ischemic modell we found damaged cells in all cellular layers of the retina. 3. PACAP is neuroprotective in both models. 4. This effect of PACAP is mediated through inhibition of proapoptotic and stimulation of anti-apopototic factors. 5, Due to this protective effect, the histological layers and the cells of the retina remain relatively intact. These results are also supported by functional examinations (electroretinogram). 6. Enriched environments proved to be retinoprotective too

A hipofízis adenilát cikláz aktiváló polipeptid (PACAP) neuroprotektív hatásmechanizmusa in vitro és in vivo rendszerekben valamint az endogén PACAP vizsgálata = The mechanism of neuroprotection of pituitary adenylate cyclase activating polypeptide (PACAP) in in vivo and in vitro systems and the examination of endogenous PACAP

A kutatás legfőbb eredményei az alábbiak: 1. Igazoltuk, hogy a PACAP neuroprotektív és általános citoprotektív hatásainak hátterében az apoptosis, gyulladás és oxidatív stressz okozta károsodás elleni hatások állnak. Számos ebben résztvevő jelátviteli útvonalat, gyulladásos és oxidatív stressz markert írtunk le. 2. Igazoltuk a PACAP sejtvédő hatását különböző károsodás modellekben retinában, belső fülben, koponyatraumában, ischemiás szervi károsodásokban, különböző perifériás szervekben. 3. Az endogén PACAP jelenlétét kimutattuk emberi mintákból biológiai folyadékokban, mint vérplazma, anyatej, likvor és könny. A biológiai mintákban előforduló PACAP mérést standardizáltuk, és klinikai állapotok súlyosságával mértük össze számos pathológiás folyamatokban. 4. Leírtuk PACAP génhiányos egerek idegrendszeri fejlődését. Igazoltuk, hogy PACAP hiányában az idegrendszer és a perifériás szervek is káros behatásokra érzékenyebben reagálnak. A támogatás segítségével született közlemények összesített impakt faktora 197,3 (absztraktok nélkül). | Major findings of the present project are: 1. We have shown the involvement of antiapoptotic, antiinflammatory and antioxidant effects in the PACAP-induced neuroprotective and general cytoprotective actions. We have described several signal transduction pathways in the apoptotic process, inflammatory markers and oxidative stress markers upon PACAP treatment. 2. We have described the cytoprotective effects of PACAP in different lesion models in the retina, inner ear, traumatic brain injury, ischemic organ lesions, and in several peripheral organs. 3. We have shown the presence of endogenous PACAP in human biological samples, like blood plasma, breast milk, cerebrospinal fluid and tear fluid. We have standardized the PACAP measurements in biological fluids and we have drawn correlations between PACAP levels and clinical status in several pathological conditions. 4. We have described the nervous development of PACAP knockout mice. We have shown that the lack of PACAP leads to increased vulnerability to different stressors both in the nervous system and in the periphery. The impact factor of peer reviewed papers published with the support of the present grant is 197,3

Alzheimer’s Disease Mouse as a Model of Testis Degeneration

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with protective functions in the central nervous system and various peripheral organs. PACAP has the highest expression level in the testes, among the peripheral organs, and has a positive regulative role in spermatogenesis and in sperm motility. In the present study, we explored testicular degenerative alterations in a mouse model of Alzheimer’s disease (AD) (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) and demonstrated changes in PACAP-regulated signaling pathways. In addition, the effects of increased physical activity of AD (trained AD (TAD)) mice on testis were also followed. Reduced cell number and decreased thickness of basement membrane were detected in AD samples. These changes were compensated by physical activity. Expression of PACAP receptors and canonical signaling elements such as PKA, P-PKA, PP2A significantly decreased in AD mice, and altered Sox transcription factor expression was also detected. Via this signaling mechanism, physical activity compensated the negative effects of AD on the expression of type IV collagen. Our findings suggest that the testes of AD mice can be a good model of testis degeneration. Moreover, it can be an appropriate organ to follow the effects of various interventions such as physical activity on tissue regeneration and signaling alterations

A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate

Similar to other invertebrate and vertebrate animals, cAMP dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homologue of the vertebrate pituitary adenylate cyclase activating polypeptide (PACAP) and for the AC activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this strong food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a weak multi-trial food-reward conditioning paradigm, lip-touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multi-trial tactile conditioning accelerated the formation of transcription dependent memory.Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning

Posttraumatic Administration of Pituitary Adenylate Cyclase Activating Polypeptide in Central Fluid Percussion Injury in Rats

Several in vitro and in 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