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

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

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

Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.Peer reviewe

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis. Methodology/Principal Findings Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti- inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro- inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner. Conclusion/Significance Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases

Presence and Effects of Pituitary Adenylate Cyclase Activating Polypeptide Under Physiological and Pathological Conditions in the Stomach

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with widespread occurrence throughout the body including the gastrointestinal system. In the small and large intestine, effects of PACAP on cell proliferation, secretion, motility, gut immunology and blood flow, as well as its importance in bowel inflammatory reactions and cancer development have been shown and reviewed earlier. However, no current review is available on the actions of PACAP in the stomach in spite of numerous data published on the gastric presence and actions of the peptide. Therefore, the aim of the present review is to summarize currently available data on the distribution and effects of PACAP in the stomach. We review data on the localization of PACAP and its receptors in the stomach wall of various mammalian and non-mammalian species, we then give an overview on PACAP's effects on secretion of gastric acid and various hormones. Effects on cell proliferation, differentiation, blood flow and gastric motility are also reviewed. Finally, we outline PACAP's involvement and changes in various human pathological conditions

Pituitary Adenylate Cyclase-Activating Polypeptide—A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD)