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ID naloge: 77 Letnik: 2000 Predmet: patofiziologija
Acetilholinesteraza v elektricno draženi denervirani mišici EDL podgane
Avtor: Matjaž Sever, Andrej Bartolic
Mentor: doc. dr. Samo Ribaric, dr. med.
IZHODIŠCE. Temelj delovanja živcevja je sporocanje preko sinaps, zato so mehanizmi uravnavanja sinteze sinapticnih struktur in njihovega usmerjanja na mesto delovanja predmet mnogih raziskav. V teh se zaradi dostopnosti kot eksperimentalni model uporablja živcnomišicni stik, ki ga sestavlja med drugimi strukturami tudi acetilholinesteraza (AChE). Vloga AChE je kljucna za živcnomišicni prenos, ki ga prekinja s tem, da v sinapticni špranji razgrajuje nevrotransmiter acetilholin. Pri tem pa igrata pomembno vlogo njena aktivnost in razporeditev, ki sta odvisni od ekspresije gena. Slednja je odvisna od razlicnih dejavnikov in njihovega medsebojnega vpliva in to predvsem od elektromehanicne aktivnosti in lokalno delujocih živcnih troficnih dejavnikov. Denervacija mišice vodi zaradi odtegnitve omenjenih dejavnikov do znacilnih atroficnih sprememb mišice, upada aktivnosti AChE v živcnomišicnem stiku, kot tudi upada ravni AChE mRNA v celici.
NAMEN IN HIPOTEZA. V poskusih smo preucevali vpliv elektricnega draženja na raven AChE mRNA in aktivnost AChE v denervirani hitri mišici extensor digitorum longus (EDL). Na živalskem modelu smo to mišico dražili z razlicnimi vzorci draženja, ki so znacilni za hitre mišice. Primerjali smo ravni AChE mRNA in aktivnosti AChE v mišici pri razlicnih vzorcih draženja, da bi preverili naslednjo hipotezo:
Izbrani enostavni vzorec elektricnega draženja mišice EDL lahko delno prepreci spremembe ravni AChE mRNA in aktivnosti AChE, ki nastanejo po denervaciji.
RAZLAGA HIPOTEZE: Elektromehanicna aktivnost, povzrocena z živcem, je v mišici kljucna za uravnavanje presnove AChE. Zaradi odsotnosti te aktivnosti pride pri podgani po denervaciji v prvem tednu do padca ravni AChE mRNA in s tem aktivnosti AChE. Elektricno draženje vpliva na mnoge postdenervacijske spremembe mišice in jih do dolocene mere tudi prepreci. V literaturi je opisanih veliko razlicnih nacinov draženja, nikjer pa še ni ugotovljen najprimernejši vzorec neposrednega draženja, ki bi preprecil spremembe aktivnosti AChE in ravni AChE mRNA v mišici EDL. Z uporabo razlicnih vzorcev draženja mišice lahko pricakujemo, da bomo odkrili najprimernejšega, pri katerem bi prišlo do cim manjših sprememb glede na normalno oživceno mišico.
METODE. Poskuse smo izvedli na podganjih samicah soja Wistar. Poskusne živali smo razdelili v štiri skupine. V prvi skupini smo mišico EDL denervirali, nato pa elektricno dražili z razlicnimi vzorci, v drugi skupini smo mišico dražili posredno preko živca, v tretji skupini smo izvedli samo denervacijo in namestitev elektrod brez elektricnega draženja, v cetrti skupini pa smo le namestili elektrode brez denervacije in elektricnega draženja. Po štirih dneh smo mišice izolirali. Aktivnost AChE smo izmerili s spektrofotometrom, raven AChE mRNA pa z metodo Northern-blot. Rezultate smo statisticno obdelali s Kruskal-Wallisovim testom in neparnim Wilcoxonovim testom.
REZULTATI. Prav v vseh skupinah podgan je prišlo v denervirani mišici EDL do pomembnega upada aktivnosti AChE v primerjavi z normalno oživceno mišico druge zadnje okoncine. Razlicni vzorci elektricnega draženja so bili razlicno uspešni pri preprecevanju upada aktivnosti AChE v denervirani mišici. Najvišjo vrednost aktivnosti AChE, ki se je statisticno znacilno razlikovala od aktivnosti pri drugih skupinah denerviranih mišic (p<0,01), smo dosegli z vzorcem draženja s frekvenco 150 Hz, dolžino zaporedja dražljajev 0,2 s in trajanjem presledka med zaporedji dražljajev 15 min. Isti vzorec draženja je bil najbolj uspešen tudi pri preprecevanju upada ravni AChE mRNA.
ZAKLJUCKI. Omenjeni rezultati so potrdili našo hipotezo. Namrec, med izbranimi enostavnimi vzorci elektricnega draženja obstaja vzorec, ki lahko delno prepreci upad ravni AChE mRNA in aktivnosti AChE po denervaciji. Glede na delno uspešnost uporabljenega enostavnega vzorca nas rezultati navajajo k sklepu, da pri vzdrževanju normalne ravni AChE mRNA in aktivnosti AChE sodelujejo še drugi mehanizmi. Ti so zelo verjetno kompleksnejši vzorci aktivacije mišice, locena aktivacija posameznih motoricnih enot v mišici z razlicnimi vzorci aktivacije in živcni troficni dejavniki, ki se sprošcajo iz perifernega živca.
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[Abstract / English version]
Attenuation of the fall of AChE activity with direct electrical stimulation of the denervated EDL muscle
Author: Matjaž Sever, Andrej Bartolic
Mentor: doc. dr. Samo Ribaric, dr. med.
BACKGROUND. The basis for the function of the nervous system is signalling among the nerve cells through the synapses. Normal signalling requires precise synthesis and localisation of synaptic structures within the cell. There is a lot of work done in this field and the most often used model for the research is the neuromuscular junction because of its accessability. The enzyme acetylcholinesterase (AChE) plays a crucial role in it by degrading acetylcholine and by that stopping the electrical signal among the nerve cells. The activity and localisation within the cell are very important for the proper function of the AChE. They depend on the expression of the AChE gene, which is further dependant on many factors and their interrelationships. Among them, the most important are electromechanical activity and local neural mediators. Denervation of the muscle causes a lack of the factors mentioned above and consequently leads to atrophic changes of the muscle, decrease of the AChE activity in neuromuscular junction and the level of AChE mRNA in the muscle cells.
AIM AND HYPOTHESIS. Our observations were based on the measurement of the AChE activity and the level of AChE mRNA in the fast rat extensor digitorum longus (EDL) muscle. We used different stimulation patterns on the animal model. The results were then compared in order to test the next hypothesis:
Change in the level of AChE mRNA and the activity of AChE in the EDL muscle after denervation can be partially prevented by a specific simple pattern of electrical muscle stimulation.
RATIONALE FOR HYPOTHESIS: Electromechanic activity caused by the nerve is crucial for the regulation of AChE metabolism. Denervation of the muscle causes a drop of AChE activity and level of AChE mRNA within the first week. Electrical stimulation can affect many postdenervational processes and can stop them to a certain extent. However, a stimulation pattern that would prevent the postdenervational changes of AChE activity and level of AChE mRNA in the EDL muscle has not been discovered yet. By using different patterns of stimulation we can expect to find the most appropriate one, that would cause the least change comparing to the normal innervated muscle.
METHODS. The experiments were performed on female Wistar rat strains. Animals were divided in four groups. In the first group of animals the EDL muscles were denervated and then stimulated with different electrical patterens. In the second group muscles were stimulated through the nerve. In the third group muscles were denervated and the electrodes were placed as usually, but there was no stimulation. And in the fourth group we only placed the electrodes on usual places without denervation and stimulation of the muscles. After four days of stimulation the isolation of the EDL muscles was performed. We measured the AChE activity in the muscles with a spectrophotometrical method and the level of AChE mRNA with the Northern-blot method. The results were statistically evaluated with Kruskal-Wallis test and unpaired Wilcoxon's test.
RESULTS. In all groups of denervated EDL muscles we measured a significant drop of the AChE activity comparing to the normally innervated muscle of the other leg. Different patterns of electrical stimulation had various success in preventing the postdenervational drop of AChE activity. However, the highest AChE activity was obtained with the stimulation pattern with frequency of impulses 150 Hz, duration of impulse trains 0.2 s and interval between trains of 15 min. This AChE activity was significantly different from the activities of other groups of denervated muscles (p<0,01). Similar results were obtained with the measurement of the level of AChE mRNA. The same pattern proved to be the most successful in preventing AChE mRNA level drop.
CONCLUSIONS. The above mentioned results proved our hypothesis. There does exist a simple stimulation pattern that can partially prevent the postdenervational decrease of AChE mRNA level and AChE activity. However, due to inability to completely prevent that decrease, we can conclude that there must be other mechanisms involved in maintenance of normal level of AChE mRNA and activity of AChE. These are most probably more complex muscle activation patterns, separate activation of motor units within the muscle with different activation patterns and local mediators from the peripheral nerve.
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