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Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue cultures
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).ORCID iD: 0000-0001-7693-4764
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
2014 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1564, 22-32 p.Article in journal (Refereed) Published
Abstract [en]

Chondroitin sulfate proteoglycans (CSPGs) promote nerve growth during development, and inhibit axonal growth in the adult CNS after injury. Chondroitinase ABC (ChABC) and methyl-umbelliferyl-β-d-xyloside (β-xyloside), two enzymes that degrade CSPGs, promote regeneration after injury, however, they demonstrate opposing results in tissue culture. To elucidate the effect of the two enzymes, organotypic tissue cultures, treated with ChABC or β-xyloside, were employed to monitor nerve fiber outgrowth and astrocytic migration. Rat ventral mesencephalon (VM) and spinal cord (SC) from embryonic day (E) 14 and E18 were treated early, from the plating day for 14 days in vitro, or late where treatment was initiated after being cultured for 14 days. In the early treatment of E14 VM and SC cultures, astrocytic migration and nerve fiber outgrowth were hampered using both enzymes. Early treatment of E18 cultures reduced the astrocytic migration, while nerve growth was promoted by β-xyloside, but not by ChABC. In the late treated cultures of both E14 and E18 cultures, no differences in distances that astrocytes migrated or nerve fiber growth were observed. However, in β-xyloside-treated cultures, the confluency of astrocytic monolayer was disrupted. In E18 cultures both early and late treatments, neuronal migration was present in control cultures, which was preserved using ChABC but not β-xyloside. In conclusion, ChABC and β-xyloside had similar effects and hampered nerve fiber growth and astrocytic migration in E14 cultures. In E18 cultures nerve fiber growth was stimulated and neuronal migration was hampered after β-xyloside treatment while ChABC treatment did not exert these effects.

Place, publisher, year, edition, pages
Elsevier, 2014. Vol. 1564, 22-32 p.
Keyword [en]
CSPGs; Spinal cord; Ventral mesencephalon; ChABC; β-xyloside
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:umu:diva-87978DOI: 10.1016/j.brainres.2014.03.043ISI: 000336703900003OAI: oai:DiVA.org:umu-87978DiVA: diva2:712817
Available from: 2014-04-16 Created: 2014-04-16 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Interaction between nerve fiber formation and astrocytes
Open this publication in new window or tab >>Interaction between nerve fiber formation and astrocytes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Parkinson’s disease, the second most common neurodegenerative disorder,is characterized by loss of nigrostriatal dopaminergic neurons. To date,there is no defined cause and cure for the disease. An ideal treatmentstrategy is to replace the lost neurons by transplanting fetal dopaminergicneurons to the brain of parkinsonian patients. Clinical trials have beenperformed and the outcome was variable where one significant obstaclewas the limited graft reinnervation of the host brain. To study this issue,organotypic tissue culture can be utilized to monitor dopaminergic nervefiber outgrowth in vitro and their association with astrocytes. Using thisculture technique, dopaminergic nerve fibers appear in twomorphologically and temporally different types. The early appearing nervefibers are formed in the absence of astrocytes, reach long distances, andare called non-glial-associated tyrosine hydroxylase (TH) -positive nervefibers. After a few days, the second sequence of nerve fibers, the glialassociatedTH-positive nerve fibers, are formed, and their growth arelimited to the presence of astrocytes, that migrate and form a monolayersurrounding the plated tissue. The aim of this thesis was to study theinteraction between nerve fiber formation and astrocytes with a specialfocus on the long-distance growing nerve fibers. Ventral mesencephalic(VM) organotypic slice cultures from embryonic day (E) 12, E14, and E18were incubated for 14, 21, 28, and 35 days in vitro (DIV). The resultsrevealed that the two morphologically different processes were found incultures from the younger stages, while no non-glial-associated growthwas found in cultures of tissue from E18. Instead neurons had migratedonto the migrating astrocytes. Astrocytes migrated longer distances intissue from older stages, and the migration reached a plateau at 21 DIV.Co-cultures of E14 VM tissue pieces and cell suspension of matureastrocytes promoted migration of neurons, as seen in E18 cultures. Thus,9the maturity of the astrocytes was an important factor for nerve fiberoutgrowth. Hence, targeting molecules secreted by astrocytes might bebeneficial for regeneration. Chondroitin sulfate proteoglycan (CSPG), amember of proteoglycan family, is produced by the astrocytes and has adual role of being permissive during development and inhibitory afterbrain injury in adult brain. Cultures were treated with chondroitinase ABC(ChABC) or methyl-umbelliferyl-β-D-xyloside (β-xyloside) in twodifferent protocols, early and late treatments. The results from the earlytreated cultures showed that both compounds inhibited the outgrowth ofnerve fibers and astrocytic migration in cultures from E14 tissue, while β-xyloside but not ChABC promoted the non-glial-associated growth incultures derived from E18 fetuses. In addition, β-xyloside but not ChABCinhibited neuronal migration in E18 cultures. Taken together, β-xylosideappeared more effective than ChABC in promoting nerve fiber growth.Another potential candidate, integrin-associated protein CD47, was studiedbecause of its role in synaptogenesis, which is important for nerve fibergrowth. Cultures from E14 CD47 knockout (CD47-/-) mice were plated andcompared to their wildtypes. CD47-/- cultures displayed a massive and longnon-glial-associated TH-positive nerve fiber outgrowth despite theirnormal astrocytic migration. Blocking either signal regulatory protein-α(SIRPα) or thrombospondin-1 (TSP-1), which bind to CD47, had nogrowth promoting effect. In conclusion, to promote nerve growth, youngertissue can grow for longer distances than older tissue, and inhibiting CSPGproduction promotes nerve growth in older tissue, while gene deletion ofCD47 makes the astrocytes permissive for a robust nerve fiber growth.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2014. 56 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1650
Keyword
dopamine, nerve fiber outgrowth, astrocytes, ventral mesencephalon, spinal cord, CSPG, CD47
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-88366 (URN)978-91-7601-057-0 (ISBN)
Public defence
2014-05-28, BiA201, Biologihuset, Umeå Universitet, Umeå, 09:00 (English)
Opponent
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Available from: 2014-05-07 Created: 2014-05-05 Last updated: 2014-05-07Bibliographically approved

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Hashemian, SanazMarschinke, Franziskaaf Bjerkén, SaraStrömberg, Ingrid
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