www.medenosrce.net/arhimed   arhimed(a-t>medenosrce.net   [22/04/2026 01:28:29] http://www.medenosrce.net/arhimed/poglej.asp?id=110 Vpliv drazljajev v tarcnem tkivu na dopaminergicne nevrone substance nigre pri podgani Avtor: Petja Fister in Matej Svetec Mentor: doc. dr. Mara Bresjanac, dr. med. IZHODIŠCE: Sodobna medicina ne razpolaga z ucinkovitimi postopki za zdravljenje nevrodegenerativnih bolezni in drugih okvar osrednjega živcevja. Najnovejši preizkusni postopki zdravljenja teh stanj vkljucujejo invazivne posege v osrednje živcevje, pri katerih prihaja do mehanske poškodbe, vnetnega odziva, ekscitotoksicne sekundarne okvare tkiva in aktivacije sinteze in izlocanja rastnih dejavnikov. Vplivi teh procesov na osrednje nevrone zaenkrat niso dobro raziskani. Uspešnost novih postopkov zdravljenja, ki posegajo v živcevje, bo v veliki meri odvisna od razumevanja in obvladovanja ucinkov mehanske ter ekscitotoksicne okvare, vnetja in zvecane dostopnosti rastnih dejavnikov na osrednje nevrone. NAMEN: Namen naše raziskave je bil v kratkotrajnem poskusu na podganjem nigrostriatnem dopaminergicnem (Da) sistemu in vivo opredeliti in kolicinsko ovrednotiti ucinke (1) mehanske okvare, (2) provnetnih dejavnikov, (3) ekscitotoksicne okvare in (4) vbrizganja rastnega dejavnika za Da nevrone, GDNF (iz angl. glial cell line derived neurotrofic factor). Kot kontrolo za neposredne škodljive ucinke na Da sistem smo dodali še (5) vbrizganje 6-hidroksidopamina (6-OHDA), toksina, ki selektivno okvari Da nevrone. Želeli smo preveriti ucinke navedenih dražljajev na število Da nevronov substance nigre (SN), na velikost teh celic in na gostoto Da oživcenja tarcnega podrocja, striatuma. Zanimalo nas je ali se ucinki dražljajev razlikujejo glede na mesto vbrizganja v nigrostriatni sistem. Na koncu smo preverjali tudi povezavo med spodbudnimi ucinki najmocnejših dražljajev na Da nevrone in vnetjem, ki so ga isti dražljaji izzvali v tkivu. HIPOTEZE I: Vbrizganje kvinolinske kisline v striatum podgane bo povzrocilo morfološke spremembe Da nevronov, ki se bodo kolicinsko pomembno razlikovale od ucinkov vbrizganja fiziološke raztopine v striatum. II: Ucinke kvinolinske kisline bomo lahko oponašali z neposrednim vbrizganjem dejavnikov vnetja ali samega GDNF v striatum. III: Dražljaji, ki izzovejo plasticne spremembe Da nevronov po vbrizganju v striatum, bodo povzrocili enake spremembe, ce jih dovedemo v neposredno bližino Da celic v SN. METODE: Mladim odraslim samcem podgan soja Wistar (n = 43) smo v globoki anesteziji sterotatkticno vbrizgali po 2 ml raztopin razlicnih snovi v Da tarcno tkivo, striatum, ali v neposredno bližino teles Da nevronov, 2 mm nad SN. Uporabili smo naslednje raztopine oziroma snovi: fizioloska raztopina (predvsem mehanski drazljaj), kvinolinska kislina (ekscitotoksin), mešanica lipopolisaharida in interlevkina-1b (nespecificni spodbujevalci vnetja) in rekombinantni podganji GDNF. Za primerjavo smo uporabili tudi 6-OHDA, ki selektivno okvari Da nevrone. Po 7 dneh smo iste živali v globoki anesteziji žrtvovali s transkardialnim prepiranjem, možgane izolirali in jih fiksirali. Na drsnem mikrotomu smo rezali 20 mm debele koronalne reze, ki smo jih imunohistokemicno oznacevali na znacilni Da encim, TH, in znacilni površinski antigen vnetnih celic v možganskem parenhimu, OX42. Na tako oznacenih vzorcih smo šteli Da nevrone v izbranem podrocju SN na strani dražljaja in merili velikost površine presekov Da nevronov. Kot kazalec gostote Da oživcenja striatuma smo merili relativno opticno gostoto imunohistokemicnega oznacevanja TH v striatumu in gostoto oznacevanja OX42 okoli mesta vbrizganja kot kazalec vnetnega odziva. Vrednosti na tretirani strani smo izrazili v odstotkih vrednosti nasprotne strani. Izmerjene vrednosti smo analizirali, da bi ugotovili statisticno znacilne razlike: primerjave med razlicnimi dražljaji pri enakem mestu vbrizganja smo opravili najprej z enosmerno analizo variance (ANOVA) in po potrebi z dodatnim post hoc diskriminacijskim testom za ugotavljanje znacilnosti razlik med posameznimi skupinami (LSD test, iz angl. least significant difference). Primerjave enakih dražljajev glede na mesto vbrizganja smo izpeljali s Studentovim t-testom. Nicelno predpostavko smo zavrnili s 5% tveganjem. Opravili smo tudi analizo korelacije med statisticno pomembnimi ucinki posameznih dražljajev. REZULTATI: Potrdili smo hipotezo I, ker smo po vbrizganju kvinolinske kisline v striatum opazili zvecanje gostote TH v striatumu ter zvecanje števila Da nevronov v istostranski SN, ki sta bila statisticno znacilno vecja od ucinkov fiziološke raztopine. Naši izsledki zavracajo hipotezo II, saj niti z neposrednim vbrizganjem dejavnikov vnetja niti z vbrizganjem GDNF v striatum nismo mogli izzvati enakih ucinkov kot s kvinolinsko kislino. Prav tako smo ovrgli hipotezo III, ker dražljaji v neposredni bližini teles Da nevronov niso imeli enakih ucinkov kot enaki dražljaji v striatumu. ZAKLJUCKI Nigrostriatni Da sistem odrasle podgane spreminja število Da nevronov in gostoto Da oživcenja striatuma. Nadaljni poskusi bi morali preuciti udeležene mehanizme, slediti ucinkom dlje casa, preveriti njihov morebitni vpliv na funkcijo okvarjenega Da sistema in opredeliti morebitne stranske ucinke. «» [Abstract / English version] The effects of stimuli from the target tissue on dopaminergic NEURONS of the rat substantia nigra Author: Petja Fister in Matej Svetec Mentor: doc. dr. Mara Bresjanac, dr. med. BACKGROUND: Contemporary medicine has no effective means for treatment of neurodegenerative diseases and other central nervous system disorders. The latest experimental treatment options for these conditions involve invasive procedures in the central nervous system, with accompanying mechanical injury, inflammatory response, excitotoxic secondary tissue damage and activation of trophic factor synthesis and release. Influences of these processes on the central neurons have not been sufficiently explored. Success of novel invasive treatment options will largely depend on understanding and control of the effects of mechanical and excitotoxic injury, inflammation and increased availability of trophic support to the central neurons. AIM The goal of this short-term in vivo study on the rat nigrostriatal dopaminergic (Da) system was to determine and quantify the effects of (1) a mechanical injury, (2) proinflammatory factors, (3) an excitotoxic injury and (4) an injection of a Da trophic factor, glial cell line derived neurotrofic factor, GDNF. In order to control for possible direct lethal effects of the above stimuli, an additional treatment group received an injection of (5) 6-hydroxydopamine (6-OHDA), a toxin which selectively kills Da neurons. We aimed to determine how the listed stimuli affect the number of Da neurons of the substantia nigra (SN), their cell size and the innervation density of their target region, the striatum. We wanted to see if the effects of the employed stimuli differ depending on the site of their injection into the nigrostriatal system. Finally, we also performed the correlation analysis of the stimulatory effects of the most potent stimuli on Da neurons and the inflammation they caused in the parenchyma. HYPOTHESES I: Intrastriatal injection of quinolinic acid will cause morphological changes of the rat Da neurons that will significantly differ in quantity from the effects of intrastriatal saline injection. II: Intrastriatal injections of either proinflammatory factors or GDNF will directly mimic the effects of quinolinic acid injection. III: The stimuli that cause plastic changes of the Da system upon intrastriatal injection will have the same effects when injected into the proximity of SN Da neurons. METHODS Young adult Wistar male rats (n = 42) were deeply anesthetized and stereotactically injected with 2 ml of a solution either into the striatum or into the proximity of Da neuron bodies, 2 mm above the SN. We used the following solutions/substances: saline (mainly a mechanical injury), quinolinic acid (excitotoxin), a mixture of lipopolysaccharide with interleukin-1b (nonspecific proinflammatory stimulus) and recombinant rat GDNF. A selective toxin for Da neurons, 6-OHDA, was used for comparison. After 7 days, the same rats were transcardially perfused in deep anesthesia; their brains were isolated and fixed. A sliding microtome was used to cut 20 mm thick coronal sections, which were immunohistochemically labeled for TH, a characteristic Da enzyme, and for OX42, a marker surface antigen on inflammatory cells in the brain parenchyma. We used the labeled sections to count Da neurons in a selected region of SN ipsilateral to the administered stimulus, and to measure the transected cell surface area. We also measured relative optic density of TH striatal labeling as an indicator of Da innervation and of OX42 labeling around the injection site as a measure of inflammatory response. Measured values on the treated side were expressed as % of the value from the contralateral side. The obtained values were analyzed for statistical significance: comparisons between groups of animals with stimuli applied at the same location were done by one-way analysis of variance (ANOVA) followed when appropriate by a post hoc (least significant difference, LSD) test to determine significant differences between specific groups. Student t-test was used for comparisons of equal stimuli effects at two different locations. The null hypothesis was rejected if p<0.05. We also performed analysis of correlation between plastic and inflammatory effects of the most potent stimuli RESULTS The intrastriatal quinolinic acid injection caused a statistically significant greater increase of striatal TH density and of ipsilateral SN number of Da neurons than the striatal saline injection, thus confirming hypothesis I. Hypothesis II was disproved because neither proinflammatory factors nor GDNF was able to mimic the effects of quinolinic acid. Hypothesis III was also rejected, because the stimuli injected intrastriatally did not have the same effects as when they were administered near the SN cell bodies. CONCLUSIONS: Adult rat nigrostriatal system responds to stimuli by changes in Da neuron numbers and in striatal innervation density. Further experiments would have to study the mechanisms involved, follow their long-term effects, test if they affect function in an injured Da system, and evaluate their possible side effects.