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From dopamine nerve fiber formation to astrocytes
Umeå University, Faculty of Medicine, Integrative Medical Biology. (Ingrid Strömberg)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Parkinson’s disease (PD) is a progressive neurodegenerative disease and characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the midbrain. The causes of the disease are still unknown. The most commonly used treatment is administration of L-DOPA, however, another possible treatment strategy is to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons. Clinical trials have demonstrated beneficial effects from transplantation, but one obstacle with the grafting trials has been the variable outcome, where limited graft reinnervation of the host brain is one important issue to solve. To improve and control the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration and dopamine nerve fiber formations at different time points and under varying conditions to study how to control nerve fiber formation. The early appearing DA nerve fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in the absence of glial cell bodies, are not persistent over time, and is called non-glial-associated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve fiber formation. Investigations of the interactions between the astrocytic migration and nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The results revealed decreased astrocytic migration, reduced glial-associated TH-positive outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths by either its absence or presence, respectively. The astrocytes synthesize proteoglycans. Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures. E18 VM and spinal cord cultures were evaluated to investigate how the different developmental stages influence astrocytes and the two nerve fiber formations after 14 DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial-associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. β-xyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18 VM cultures, while no effects was found in the spinal cord cultures. However, the neuronal migration found in control cultures was abolished in both VM and spinal cord cultures after both ChABC and β-xyloside. Neuroinflammation plays a critical role in the development of PD. Increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are observed in postmortem PD brains and the levels of TNFα receptors on circulating T-lymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα were studied on E14 VM cultures. The outgrowth of the non-glial-associated TH-positive nerve fibers was inhibited while it stimulated astrocytic migration and glial-associated TH-positive nerve fiber outgrowth at an early treatment time point. Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the TH-positive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis. CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve fiber outgrowth despite a normal astrocytic migration and the presence of glial-associated TH-positive nerve fiber outgrowth. For the first time, it was observed that the non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV. Taken together, there is an interaction between astrocytes and TH-positive nerve fiber formations. Both nerve fiber formations seem to have their task during the development of the DA system.

Place, publisher, year, edition, pages
Umeå: Integrativ medicinsk biologi , 2009. , 63 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1257
Keyword [en]
Dopamine, dopaminergic neurons, astroglia
National Category
Cell and Molecular Biology Cell and Molecular Biology
Research subject
URN: urn:nbn:se:umu:diva-20615ISBN: 978-91-7264-767-1OAI: diva2:209197
Public defence
2009-04-17, BiA 201, Biologihuset, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2009-03-30 Created: 2009-03-24 Last updated: 2009-03-30Bibliographically approved
List of papers
1. Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures
Open this publication in new window or tab >>Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures
2008 (English)In: International Journal of Developmental Neuroscience, ISSN 0736-5748, E-ISSN 1873-474X, Vol. 26, no 7, 683-691 p.Article in journal (Refereed) Published
Abstract [en]

Tyrosine hydroxylase-positive nerve fiber formation occurs in two diverse morphological patterns in rat fetal ventral mesencephalic slice cultures; one is non-glial-associated and the other is glial-associated. The aim of this study was to characterize the non-glial-associated nerve fibers and its relation to migration of astrocytes. Organotypic slice cultures were prepared from embryonic days 12, 14, and 18 rat fetuses and maintained for 5, 7 or 14 days in vitro. Inhibition of cell proliferation using cytosine beta-D-arabinofuranoside was conducted in embryonic day 14 ventral mesencephalic cultures. The treatment impaired astrocytic migration at 7 and 14 days in vitro. The reduced migration of astrocytes exerted a negative effect on the glial-associated tyrosine hydroxylase-positive nerve fibers, reducing the outgrowth from the tissue slice. The non-glial-associated outgrowth was, however, positively affected by reduced astrocytic migration, reaching distances around 3mm in 2 weeks, and remained for longer time in culture. Co-cultures of fetal ventral mesencephalon and frontal cortex revealed the cortex as a target for the non-glial-associated tyrosine hydroxylase-positive outgrowth. The age of the fetal tissue at plating affected the astrocytes such that older tissue increased the length of astrocyte migration. Younger tissue at plating promoted the presence of non-glial-associated outgrowth and long radial-glia-like processes, while older tissue promoted migration of neurons instead of formation of nerve fiber network. In conclusion, inhibition of astrocytic proliferation promotes the persistence of long-distance growing tyrosine hydroxylase-positive nerve fibers in ventral mesencephalic slices cultures. Furthermore, the long-distance growing nerve fibers target the frontal cortex and are absent in cultures derived from older tissue.

urn:nbn:se:umu:diva-19397 (URN)10.1016/j.ijdevneu.2008.07.014 (DOI)18718519 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2010-06-21Bibliographically approved
2. Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue culture
Open this publication in new window or tab >>Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue culture
(English)Manuscript (Other (popular science, discussion, etc.))
urn:nbn:se:umu:diva-20923 (URN)
Available from: 2009-03-30 Created: 2009-03-30 Last updated: 2010-06-24
3. Dual effects of TNFalpha on nerve fiber formation from ventral mesencephalic organotypic tissue cultures
Open this publication in new window or tab >>Dual effects of TNFalpha on nerve fiber formation from ventral mesencephalic organotypic tissue cultures
2008 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1215, 30-39 p.Article in journal (Refereed) Published
Abstract [en]

Tumor necrosis factor alpha (TNFalpha) is toxic to dopamine neurons and increased levels of TNFalpha are observed in Parkinson's disease. Dopamine nerve fiber outgrowth in organotypic cultures of fetal ventral mesencephalon occurs in two waves. The early appearing nerve fibers are formed in the absence of astroglia, while migrating astrocytes guide the late appearing dopamine nerve fibers. TNFalpha (40 ng/ml) was added to the medium of organotypic ventral mesencephalic tissue cultures between days 4-7 and 11-14. The cultures were evaluated at days 7 or 19 to study the effects of TNFalpha on both types of nerve fiber formation. Tyrosine hydroxylase (TH)-immunohistochemistry demonstrated that the number of cultures showing non-glial-guided TH-positive outgrowth was reduced compared to controls, when TNFalpha was added at day 4. By contrast, the glial-guided TH-positive nerve fiber outgrowth and the astrocytic migration reached significantly longer distances by early TNFalpha treatment. Ki67-immunohistochemistry revealed that TNFalpha did not affect proliferation of astrocytes. Treatment with TNFalpha and antibodies against TNFalpha receptor 1 between days 4 and 7 revealed that the non-glial-guided TH-positive outgrowth reappeared. TNFalpha treatment between days 11 and 14 triggered neither the TH-positive glial-guided outgrowth, nor promoted the astrocytic migration to reach longer distances. The number of microglia was significantly increased after the late but not early TNFalpha treatment. In conclusion, TNFalpha is toxic for the non-glial dopaminergic nerve fiber outgrowth but stimulates the glial-guided outgrowth and the migration of astrocytes at an early time point. TNFalpha increased the number of microglia in VM tissue cultures after late but not after early treatment.

urn:nbn:se:umu:diva-19394 (URN)10.1016/j.brainres.2008.03.070 (DOI)18482714 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2010-06-24Bibliographically approved
4. CD47 - Sirpα interactions affects long-distance growing nerve fibers from cultured ventral mesencephalic dopamine neurons
Open this publication in new window or tab >>CD47 - Sirpα interactions affects long-distance growing nerve fibers from cultured ventral mesencephalic dopamine neurons
(English)Manuscript (Other (popular science, discussion, etc.))
urn:nbn:se:umu:diva-20924 (URN)
Available from: 2009-03-30 Created: 2009-03-30 Last updated: 2010-06-24

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