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Sproženje kolateralnega brstenja senzoricnih aksonov pri podgani
Avtor: Maja Pavcnik, Uroš Kovacic
Mentor: prof. dr. Janez Sketelj
Somentor: mag. Fajko Bajrovic, dr. med.
IZHODIŠCE. Kolateralno brstenje v perifernem živcevju je razvejeno izrašcanje poganjkov iz nepoškodovanih aksonov v sosednja podrocja denerviranega tarcnega tkiva. Pri sproženju kolateralnega brstenja aksonov verjetno hkrati delujejo dejavniki iz degeneriranih živcnih poti, denerviranega tarcnega tkiva in morebitni transnevronski mehanizmi.
NAMEN. Preverili bomo hipotezi, da:
a) za sprožitev kolateralnega brstenja prisotnost praznih, degeneriranih živcnih poti ne zadostuje, potrebno je denervirano tarcno tkivo (ali transnevronska spodbuda),
b) poleg dejavnikov denerviranega tarcnega tkiva lahko sproži kolateralno brstenje tudi interakcija med poškodovanimi in nepoškodovanimi nevroni v spinalnem gangliju (transnevronska spodbuda),
c) bližina denerviranih živcnih poti doloca mesto, kjer aksonski brsti izrašcajo iz maticnih aksonov.
METODE. Za proucevanje kolateralnega brstenja smo pri podganah uporabili model, pri katerem je na nepoškodovan n. suralis od strani prišit odsek drugega živca ("end to side" anastomoza). V skupini A smo na nepoškodovani n. suralis od strani prišili odsek n. peroneusa iz nasprotne noge, tako da na strani anastomoze ni bil poškodovan noben živec. V skupini B smo naredili enako, poleg tega pa smo na strani anastomoze prerezali še dorzo-kutane živce (DKZ) iz spinalnih segmentov L4-L6 in tako poškodovali nevrone v spinalnem gangliju n. suralisa, koža v okolici koncicev n. suralisa pa ni bila denervirana. V skupini C smo izrezali odsek n. peroneusa in ga od strani prišili na nepoškodovani n. suralis na isti nogi. S tem in s prerezanjem n. safenusa smo denervirali tudi kožo v okolici inervacijskega podrocja n. suralisa. Vrašcanje nociceptivnih aksonov v odseke živcev, od strani prišitih na n. suralis, smo ugotavljali s testom všcipa živca in s štetjem mieliniziranih aksonov na precnih rezih teh odsekov. Mielinizirana vlakna smo šteli tudi v n. suralisu tako poskusnih kot tudi kontrolnih netretiranih živali.
REZULTATI. S testom všcipa živca pri živalih v skupini A po 8 tednih nikjer nismo ugotovili brstenja aksonov n. suralisa v od strani prišite odseke n. peroneusa. Nasprotno pa smo brstenje ugotovili v vseh odsekih pri živalih v skupini C in pri štirih od petih odsekov v skupini B. Histološko smo v prišitih odsekih živca v skupini A našli le posamezne mielinizirane aksone, kar je bilo statisticno znacilno manj kot v skupinah B in C (p < 0.05). Število aksonov pri skupini C pa je bilo statisticno znacilno vecje kot pri skupini B (p < 0.02). Število mieliniziranih živcnih vlaken nad in pod mestom anastomoze v n. suralisih, ki so brsteli, se statisticno ni znacilno razlikovalo od števila vlaken v živcih, ki niso brsteli. Tudi glede površin precnih presekov mieliniziranih aksonov nismo našli pomembne razlike, kar kaže, da proksimalno in distalno od anastomoze v brstecem n. suralisu ni bilo aksonskih brstov.
ZAKLJUCKI. Rezultati podpirajo naše hipoteze. Prvic, za sprožitev kolateralnega brstenja prisotnost praznih, degeneriranih živcnih poti ne zadostuje, ampak je potrebno denervirano tarcno tkivo (ali transnevronska spodbuda). Drugic, transnevronska spodbuda pomembno prispeva kot sprožilni dejavnik kolateralnega brstenja. Vprašanje relativnega pomena transnevronske spodbude za sprožitev kolateralnega brstenja v primerjavi s spodbudo iz denerviranega tarcnega tkiva ostaja odprto. Tretjic, bližina denerviranih živcnih poti verjetno doloca mesto, kjer aksonski brsti izrašcajo iz maticnih aksonov
[Abstract / English version]
Induction of collateral sprouting of sensory axons in the rat
Author: Maja Pavcnik, Uroš Kovacic
Mentor: prof. dr. Janez Sketelj
Co-mentor: mag. Fajko Bajrovic, dr. med.
BACKGROUND. Collateral sprouting of axons in the peripheral nervous system is a branching outgrowth of new axon terminals from noninjured axons into adjacent denervated regions of a target tissue. Initiation of collateral sprouting is probably triggered by an interaction between the factors released by degenerated neural pathways or denervated target tissue, and putative transneuronal mechanisms.
OBJECTIVE. We will investigate the following hypotheses:
a) the presence of degenerated neural pathways is not sufficient to induce collateral sprouting. Denervated target tissue or transneuronal mechanisms are necessary for induction of sprouting;
b) interaction between the injured and non-injured neurons within a dorsal root ganglion (transneuronal mechanism) is an important stimulus for initiation of collateral sprouting,
c) the sprouts arise only from parts of axons adjacent to denervated perineurial sheats.
METHODS. Experiments were preformed on the hind limb of the rat. Sprouting of axons along the neurilemmal tubes of Schwann cells was investigated by an end-to-side anastomosis of a peroneal nerve segment to the intact sural nerve. In group A the peroneal nerve segment from the contralateral limb was attached to the sural nerve. None of the nerves on the side of anastomosis were injured. In group B the same procedure was made as in group A. In addition, dorsal-cutaneous nerves (DCN) from spinal segments L4-L6 were cut. Injured neurons were therefore present in the dorsal root ganglia from which axons of the sural nerve arise. The skin of the limb was not denervated. In group C an end-to-side anastomosis of the peroneal nerve segment from ipsilateral limb was made on the sural nerve. On the same limb, saphenus nerve was cut and ligated. The skin around the terminal innervation field of the sural nerve was therefore denervated. Growth of sensory axon sprouts into anastomosed nerve segments was monitored by the nerve pinch-test. In addition, the number of myelinated axons in the anastomosed nerve cross-sections was counted. Myelinated axons were counted also in samples of sural nerve of treated and untreated animals. RESULTS. Eight weeks after surgery nerve pinch test showed no sprouting of n. suralis into anastomosed nerve segments of peroneal nerve in all animals of group A. In contrast, we detected sprouting in all animals of group C and in four out of five animals of group B. We found only few myelinated fibres in anastomosed peroneal nerves in group A. This was statistically significantly less than in groups B and C (p < 0.05). The number of myelinated fibres in anastomosed peroneal nerve was statistically significantly higher in group B than in group C (p < 0.02). Regarding the number of myelinated axons in the sural nerve proximal and distal to the anastomosis, the difference between sprouting and non-sprouting nerves was not statistically significant. Because we also did not find any important difference in cross-section areas of myelinated axons at these sites between the sprouting and non-sprouting sural nerves, we conclude that the sprouting sural nerve did not contain axonal sprouts proximally or distally to the anastomosis site.
CONCLUSIONS. The results corroborate our hypotheses. First, Schwann cell tubes are not sufficient to induce collateral sprouting. Denervated target tissue or transneuronal mechanisms are needed for induction. Second, induction of axonal sprouting of intact neurons by the presence of injured nerve cells within the same dorsal root ganglion (transneuronal mechanism) is an important stimulus for initiation of collateral sprouting. The question of relative contribution of transneuronal mechanisms for induction of collateral sprouting in comparison with denervated target tissue still needs to be resolved. Third, axonal sprouts arise only from parts of axons close to denervated perineurial sheaths.