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ID naloge: 33 Letnik: 1998 Predmet: nevrologija
Vpliv vnetno-imunskega odziva na možganski presadek na plasticnost dopaminergicne inervacije striatuma pri podgani
Avtor: Uroš Prebil, Darja Polak
Mentor: doc. dr. Maja Bresjanac
Izhodišca: Popravljanje okvar osrednjega živcevja z uporabo tkivnih ali celicnih presadkov je že nekaj let prisotno tudi v klinicni medicini, sami mehanizmi delovanja presadkov pa so še vedno slabo raziskani in nezadostno razumljeni. Ena poglavitnih razlag pripisuje ucinkovitost presadkov predvsem njihovim troficnim ucinkom na prejemnikovo živcevje. Nekateri presadki lahko delujejo z izlocanjem troficnih snovi. Številni rezultati nedavno objavljenih študij pa kažejo na možnost, da lahko lokalni vnetno-imunski odziv na presaditev izzove troficne spremembe v parenhimu prejemnikovih možganov. Namen raziskave je bil preveriti vpliv vnetno-imunskega odziva na možgansko presaditev na spodbujeno plasticnost prejemnikovih nigrostriatnih dopaminergicnih povezav. Predpostavili smo, da bo do spodbujene plasticnosti dopaminergicnega oživcenja striatuma prišlo le, kadar bo presaditev izzvala lokalni vnetno-imunski odziv pri prejemniku. Dodatni namen je bil podrobneje raziskati mehanizme spodbujene plasticnosti in njen vpliv na prejemnikovo motoricno funkcijo.
Materiali in metode: Uporabili smo samce belih podgan, soj Wistar (n=48), ki smo jih razvrstili v tri poglavitne skupine. Z namenom stopnjevanja dražljaja za vnetni odziv, smo živalim v posameznih skupinah vbrizgali enega od sledecih "presadkov": injekcijo fiziološke raztopine ("lažni presadek"), kvinolinsko kislino ali suspenzijo celic goveje vranice. Raztopine smo injicirali v desni striatum. Skupini živali za vedenjske poskuse smo nevrotoksicno (6-hidroksidopamin) okvarili tudi desno substancijo nigro. Živali smo žrtvovali ob razlicnih casih po posegu. Z imunohistokemijo smo prikazali oznacevalce (1) vnetno-imunskega odziva, (2) dopaminergicnih nevronov, (3) brstecih aksonov in (4) troficnega odziva. Merili smo relativno opticno gostoto v imunohistokemicno oznacenem tretiranem in kontrolnem striatumu. S hibridizacijo in situ na mRNA za tirozin hidroksilazo in podganji dopaminski prenašalec smo ugotavljali morebitno spremenjeno vsebnost teh dopaminergicnih oznacevalcev v substanciji nigri. Motoricno vedenje živali smo testirali s standardno farmakološko-izzvano rotometrijo. Rezultate meritev smo ustrezno statisticno obdelali: (1) s parnim Studentovim t-testom, ali (2) analizo variance ter post hoc diskriminacijskim testom.
Rezultati: Pokazatelji vnetno-imunskega odziva so bili prisotni le v striatumu živali, ki smo jim vbrizgali kvinolinsko kislino, pri vseh casih opazovanja. Imunohistokemicno oznacevanje tirozin hidroksilaze je razkrilo mocno povecano plasticnost dopaminergicnih nevronov pri vseh živalih iz iste skupine. Pri merjenju relativne opticne gostote je bil ociten porast (92 +/- 13%) tega parametra na tretirani strani glede na kontrolno, kar je statisticno pomembna razlika (p<0,00092). Ojacan je bil tudi imunohistokemicni prikaz oznacevalcev brstecih aksonov. Vzporedno s tem smo prikazali , da v stritumu, injiciranem s kvinolinsko kislino, aktivirani astrociti izlocajo nevrotroficni dejavnik iz glije. Rezultati hibridizacije in situ so pokazali povecano oznacevanje mRNA za tirozin hidroksilazo in dopaminski transporter v ipsilateralni substanciji nigri. Nobene od teh sprememb nismo našli pri živalih iz drugih skupin. Pri farmakološko-izzvanem motoricnem vedenju ugotovljene razlike med posameznimi skupinami živali niso bile statisticno pomembne.
Zakljucki: Raziskava je potrdila izhodišcno hipotezo, da vnetno-imunski odziv na presaditev v striatum spodbudi plasticnost dopaminergicnih nevronov. Pri tem pojavu smo našli spremenjeno vsebnost dopaminergicnih markerjev v nigrostriatnih vlaknih in tudi brstenje vlaken. Za tak ucinek je najverjetneje odgovoren troficni vpliv aktivirane astroglije na dopaminergicne nevrone. Sklepamo, da ucinki kvinolinske kisline v striatumu ponujajo dober, ponovljiv model plasticnosti osrednjega živcevja, na katerem bi bilo mogoce podrobneje raziskati mehanizme tega pojava.
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[Abstract / English version]
The influence of the inflammatory-immune response to grafting on plasticity of dopaminergic nigrostriatal connections in the rat
Author: Uroš Prebil, Darja Polak
Mentor: doc. dr. Maja Bresjanac
Introduction: Cell and tissue transplantation for functional repair of injured central nervous system has entered the stage of clinical testing over a decade ago, but mechanisms of transplantation effects are still insufficiently understood. A major explanation ascribes transplantation-induced functional recovery to a trophic effect of grafting. This effect could be due to the release of trophic factors from grafted cells. However, numerous recent studies point to the possibility that the recipient inflammatory-immune response to grafting may induce trophic changes in the recipient nervous system parenchyma. The aim of the study was to test the role of a local inflammatory-immune response to intrastriatal grafting in graft-induced plasticity of the recipient nigrostriatal dopaminergic connections. It was hypothesized that a local inflammatory-immune response to intrastriatal grafting is a prerequisite for the recipient striatal dopaminergic innervation to display signs of plasticity. A further aim was to explore the mechanisms of induction of the plastic response to grafting, and its effects on the recipient motor function.
Materials and methods: White male Wistar rats (ntotal = 48) were used in the study. They were divided into three main treatment groups. In order to produce a graded inflammatory stimulus, three different types of "transplants" were used: a sham transplant (saline injection), quinolinic acid injection and bovine spleen cell transplant. The injections were delivered into the right striatum of deeply anesthetized rats. A group of rats in the behavioral part of the study also received a toxic injury (6-hydroxydopamine) of the right substantia nigra. The animals were sacrificed at different time points after surgery. Immunohistochemistry to select markers of the (1) inflammatory-immune response, (2) dopaminergic neurons, (3) axonal sprouting and (4) trophic factors were used to determine the effects of various procedures on these parameters. Measurements of relative optical density were performed on treated and control immunohistochemically labeled striata. In situ hybridization to tyrosine hydroxylase and rat dopamine transporter mRNAs was used to determine possible changes in dopaminergic markers' synthesis in the nigral neurons. Standard drug-induced rotometry was used to determine the animals' motor behavior after. Statistical analysis of the obtained data included (1) a paired Student t-test, and (2) analysis of variance, followed where appropriate by a post hoc discriminatory test.
Results: Indicators of inflammatory-immune response to grafting were evident only in the quinolinic acid-injected striata at all time points. In all these animals a pronounced increase in dopaminergic fiber plasticity was found in immunohistochemical labeling of tyrosine hydroxylase. Measurements of relative optical density demonstrated a striking increase (92 +/- 13%) in this parameter on the treated side relative to the control, which was found to be a very significant difference, statistically (p<0,00092). In addition, immunohistochemistry revealed an increased labeling of axonal sprouting markers. In parallel, reactive astrocytes in the quinolinic acid-injected striatum were found to express glial-derived neurotrophic factor. In situ hybridization results demonstrated an increased labeling of tyrosine hydroxylase and dopamine transporter mRNAs in the ipsilateral substantia nigra. None of these changes were found in other treatment groups. Drug-induced rotometry revealed differences between the treatment groups, which were not found to be statistically significant.
Conclusions: The study confirmed the initial hypothesis, that the inflammatory-immune response to striatal grafting is a prerequisite for dopaminergic fiber plasticity. The latter was found to involve changes in dopaminergic marker content of the nigrostriatal fibers, as well as actual fiber sprouting. The efect seems to be mediated by the increased availability of trofic support to the dopaminergic neurons by the activated astroglia. We conclude that the quinolinic acid-injected striatum offers a good, reproducible model of central nervous system plasticity. It may be valuable in the future studies of the plastic phenomena in neurons.
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