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Regeneracija poškodovanega perifernega živca podgane brez celicne podpore distalno od mesta poškodbe
Avtor: Matevž Srpcic
Mentor: Prof. dr. Janez Sketelj
Somentor: mag. Fajko Bajrovic
Izhodišce. Pomen Schwannovih celic v perifernem živcu za regeneracijo aksonov po poškodbi še ni zadovoljivo raziskan. Rezultati dosedanjih raziskav na tem podrocju si delno nasprotujejo. Ugotovili so, da se aksoni takoj po aksonotmezi razmeroma hitro regenerirajo tudi skozi brezcelicni odsek distalno od mesta poškodbe.
Namen. Naš namen je bil preveriti naslednje hipoteze:
1. Zacetna hitra elongacija aksonov, ki se po aksonotmezi regenerirajo brez celicne podpore, se kasneje upocasni ali celo ustavi.
2. Ce dodatno zavremo še proliferacijo Schwannovih celic v proksimalnem krnu, je upocasnitev regeneracije po zacetnem obdobju relativne neodvisnosti od celicne podpore še bolj izražena.
3. Predhodno kolateralno brstenje, med katerim so aksoni izpostavljeni vplivu NGF, ugodno vpliva na regenerativno sposobnost aksonov, upocasnitev regeneracije med dolgotrajno izgubo celicne podpore distalno od poškodbe je manjša ali je celo ni.
Metode. Suralni živec podgane smo poškodovali z aksonotmezo. Celice v dolgem segmentu živca distalno od poškodbe smo unicili z zmrzovanjem. Proliferacijo Schwannovih celic v proksimalnem krnu smo zavrli z injekcijo citostatika mitomicina. Predhodno brstenje aksonov n. suralisa v koži smo sprožili s trajno prekinitvijo ostalih živcev zadnje okoncine. Hitrost regeneracije senzoricnih aksonov smo dolocali s testom všcipa živca. Število aksonov v poškodovanem živcu smo dolocali s štetjem aksonov v prerezih živca po imunohistokemicnem barvanju za prikaz nevrofilamentov oziroma barvanju mielinskih ovojnic.
Rezultati. Zacetna hitra rast senzoricnih aksonov skozi brezcelicni odsek se po 8 dneh ustavi. Ustavitvi sledi retrakcija aksonov in zmanjšanje števila aksonov distalno od poškodbe. Kasneje se rast zopet vzpostavi, a je zelo pocasna. Aplikacija mitomicina, ki prepreci proliferacijo Schwannovih celic v proksimalnem krnu, zacetne hitre rasti huje ne prizadene. Po ustavitvi rasti se aksoni umaknejo do mesta poškodbe, rast se v 6 tednih po poškodbi ne vzpostavi vec. Izpostavitev nevronov NGF med predhodnim kolateralnim brstenjem pospeši zacetno hitro rast v odsotnosti celicne podpore, odloži zastoj rasti in prepreci retrakcijo aksonov. Število aksonov distalno od poškodbe ostane visoko vsaj šest tednov.
Sklep. Schwannove celice v distalnem krnu regenerirajocim se aksonom zagotavljajo ugodno rastno podlago. Rastni dejavniki iz Schwannovih celic aksonom omogocajo elongacijo in preprecujejo retrakcijo tudi ce se rastna podlaga neugodno spremeni. Ob ohranjeni rastni podlagi rastni dejavniki najbrž zmerno pospešijo elongacijo regenerirajocih se senzoricnih aksonov.
[Abstract / English version]
The regeneration of an injured rat peripheral nerve devoid of cellular support in the distal stump
Author: Matevž Srpcic
Mentor: Prof. dr. Janez Sketelj
Co-mentor: mag. Fajko Bajrovic
Background. The role of Schwann cells during peripheral nerve regeneration is not well understood because of conflicting results provided by different experimental methods. Immediately after axonotmesis axons are able to regenerate fairly rapidly through an acellular nerve segment distal to the site of injury.
Objectives. We decided to examine the following hypotheses:
1. Rapid initial elongation of regenerating axons in crushed nerves observed even in absence of distal cell support is later slowed down or stops.
2. If, in addition, Schwann cell proliferation in the proximal nerve stump is prevented, reduction of axon elongation rate during prolonged absence of cell support becomes more severe.
3. Prior collateral sprouting which exposes neurons to higher concentrations of NGF has a stimulatory effect on axons regenerating in the absence of cell support, therefore axon regeneration rate is less affected.
Methods. Rat sural nerve was crushed and a long segment distal to the crush site frozen in order to kill the cells. Schwann cell proliferation in the proximal nerve stump was inhibited by mitomycin injection. Collateral sprouting of the sural nerve was triggered by transsection of adjacent nerves. Elongation rate of regenerating axons was monitored by the nerve pinch test. The number of axons in the injured nerves was determined in nerve transsections treated either by imunnohistochemical staining against neurofilament or myelin staining.
Results. Rapid initial rate of sensory axon growth through an acellular distal nerve segment is reduced 8 days after axonotmesis. During the next three weeks retraction of leading axons towards the site of the lesion takes place and the number of axons distally to the crush site is reduced. Axon growth is resumed thereafter but at a much lower rate. Mitomycin application which prevents Schwann cell proliferation in the proximal stump does not prevent the initial rapid axon elongation in the absence of cell support, but later the axons retract all the way to the crush site and no further growth occurs during 6 weeks. Exposure of regenerating axons to NGF during collateral sprouting enhances the initial elongation rate in the absence of cell support, delays cessation of growth and prevents axon retraction. The number of axons distally to the lesion site remains high.
Conclusions. Schwann cells in the distal nerve segment provide and maintain a favourable growth substratum for regenerating axons during a prolonged time period after nerve injury. Growth promoting substances secreted by Schwann cells seem to enable elongation or prevent retraction of axons even in the case of growth substratum deterioration. In the presence of good growth substratum these substances moderately enhance the elongation rate of regenerating sensory axons.