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Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
2002 (English)In: Brain Research Protocols, ISSN 1385-299X, E-ISSN 1872-809X, Vol. 10, no 2, 75-83 p.Article in journal (Refereed) Published
Abstract [en]

We describe a protocol for analysis of gene expression in single, acutely dissociated adult rat retinal ganglion cells using RT-PCR. Retrograde tracing of retinal ganglion cells from the superior colliculi was conducted using Fluorogold. Retinas were dissected and ganglion cells isolated using retinal layer separation (sandwiching). Single, fluorescently labelled retinal ganglion cells were aspirated using a micropipette and used for PCR. Two PCR protocols are described where single cell cDNA was analysed for TrkB and GAPDH or TrkB, TrkC, Ret, Met, ErbB2 and Beta-actin by multiplex-PCR. All five tyrosine kinase receptors were amplified from single retinal ganglion cells. The method will prove useful for the molecular characterization of adult retinal ganglion cells.

Place, publisher, year, edition, pages
2002. Vol. 10, no 2, 75-83 p.
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:uu:diva-170003DOI: 10.1016/S1385-299X(02)00184-8PubMedID: 12431706OAI: oai:DiVA.org:uu-170003DiVA: diva2:508148
Available from: 2012-03-07 Created: 2012-03-07 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Neurotrophic Factor Receptors in the Normal and Injured Visual System: Focus on Retinal Ganglion Cells
Open this publication in new window or tab >>Neurotrophic Factor Receptors in the Normal and Injured Visual System: Focus on Retinal Ganglion Cells
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The focus of this thesis is the life and death of adult retinal ganglion cells (RGCs). RGCs are neurons that convey visual information from the retina to higher centers in the brain. If the optic nerve is transected (ONT), adult RGCs die by a form of cell death called apoptosis, and a general hypothesis is that neurotrophic factors can support the survival of injured neurons.

With the intention to gain knowledge about systems that can be used to decrease RGC death after ONT, we have studied growth factor receptors belonging to the tyrosine kinase family of receptors (RTK), known to mediate important cell survival signals. We found that the RTK Ret and its coreceptor GFRα1 were expressed by RGCs, and to test the above-mentioned hypothesis, we intraocularly administered glial cell-line derived factor, which activates a Ret-GFRα1 complex, and found transiently mediated RGC survival after ONT.

To identify new, potential neurotrophic factor receptors expressed by RGCs, with the aim to improve RGC survival after ONT, we developed a method for the molecular analysis of acutely isolated RGCs. The method involves retrograde neuronal tracing, mechanical retinal layer-separation, and isolation of individual RGCs under UV-light for RT-PCR analysis. Using this method, in combination with degenerate PCR directed towards the tyrosine kinase domain, several RTKs were identified. Axl, Sky, VEGFR-2, VEGFR-3, CSF-1R, and PDGF-βR are expressed by adult RGCs, and considered to be receptors with potential neurotrophic activity. Other results have shown that RGCs may require depolarization or increase in intracellular cAMP levels in order to fully respond to exogenously added trophic factors. We found that melanocortin receptors (MCRs) were expressed by RGCs, and MCRs can mediate elevation of intracellular AMP. We observed that α-MSH induced neurite outgrowth from embryonic retinal cells, indicating that MCR ligands have direct effects on retinal cells. RTKs and their ligands may be involved in endogenous systems for neuronal repair within the visual system. BDNF, NT-3, FGF2, and HGFR all increased in the retina after ONT and may be a part of an activated system for neuronal repair locally within the retina.

Adult axotomized RGCs die by apoptosis, therefore we examined the regulation of apoptotic genes after ONT. Bim and Bax increased in the retina after ONT, and may promote death of axotomized RGCs, whereas the increase in Bcl-2 may contribute to limit RGC apoptosis after ONT.

All in all, this thesis provides insights into the expression and regulation of molecules involved in the death and survival of RGCs. The results have revealed a number of potential neurotrophic receptors expressed by RGCs, and both identified RTKs and MCRs will serve as new targets in therapeutic approaches aiming at counteraction of RGC death after injury.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 71 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1261
Keyword
Neurosciences, cell survival, gene expression, neurotrophic factor, receptor, retina, visual system, Neurovetenskap
National Category
Neurology
Research subject
Developmental Neurosciences
Identifiers
urn:nbn:se:uu:diva-3402 (URN)91-554-5629-4 (ISBN)
Public defence
2003-05-23, B21, BMC, Uppsala, 09:15
Opponent
Supervisors
Available from: 2003-04-29 Created: 2003-04-29 Last updated: 2013-09-19Bibliographically approved

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