Multiple receptor tyrosine kinases are expressed in adult rat retinal ganglion cells as revealed by single-cell degenerate primer polymerase chain reaction

BACKGROUND: To achieve a better understanding of the repertoire of receptor tyrosine kinases (RTKs) in adult retinal ganglion cells (RGCs) we performed polymerase chain reaction (PCR), using degenerate primers directed towards conserved sequences in the tyrosine kinase domain, on cDNA from isolated single RGCs univocally identified by retrograde tracing from the superior colliculi.RESULTS: All the PCR-amplified fragments of the expected sizes were sequenced, and 25% of them contained a tyrosine kinase domain. These were: Axl, Csf-1R, Eph A4, Pdgfrbeta, Ptk7, Ret, Ros, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3. Non-RTK sequences were Jak1 and 2. Retinal expression of Axl, Csf-1R, Pdgfrbeta, Ret, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3, as well as Jak1 and 2, was confirmed by PCR on total retina cDNA. Immunodetection of Csf-1R, Pdgfralpha/beta, Ret, Sky, TrkB, and Vegfr-2 on retrogradely traced retinas demonstrated that they were expressed by RGCs. Co-localization of Vegfr-2 and Csf-1R, of Vegfr-2 and TrkB, and of Csf-1R and Ret in retrogradely labelled RGCs was shown. The effect of optic nerve transection on the mRNA level of Pdgfrbeta, Csf-1R, Vegfr-2, Sky, and Axl, and of the Axl ligands Gas6 and ProteinS, was analysed. These analyses show transection-induced changes in Axl and ProteinS mRNA levels.CONCLUSIONS: The repertoire of RTKs expressed by RGCs is more extensive than previously anticipated. Several of the receptors found in this study, including Pdgfrbeta, Csf-1R, Vegfr-2, Sky, and Axl, and their ligands, have not previously been primarily associated with retinal ganglion cells

Experimental Injury to the Visual System : Molecular Studies of the Retina

Retinal ganglion cells play a crucial role in the relay of visual signals from the eye to the brain. This cell type is affected and eventually lost in the eye disease glaucoma, resulting in progressive and irreversible loss of vision. Studies of the molecular mechanisms leading to retinal ganglion cell death are important for the understanding of the disease and for designing future treatments. This thesis addresses and studies these molecular mechanisms, including alterations in gene expression after experimental retinal injuries. The effects of a neuroprotective drug, brimonidine, after transient retinal ischemia were also studied in order to help explain the mechanisms behind the protective properties of this drug. Several methods, including quantitative reverse transcriptase PCR, micro-arrays, western blot and immunohistochemistry, were used. The results showed that transient retinal ischemia triggers cell division in Müller cells and alters the gene expression of growth factors, their receptors, and intermediate filaments in the retina. Several genes related to the apoptosis process were less affected. Pre-treatment with brimonidine increased the levels of certain growth factors (BDNF, NT3, CNTF, FGF9) compared with vehicle. Brimonidine also had marked effects on genes related to progenitor cells, among them the recognized neural stem cell marker nestin. The increase in levels of nestin after ischemia was countered by brimonidine treatment. Moreover, retinal ganglion cell death following either optic nerve transection or optic nerve crush appears to involve the extrinsic apoptotic pathway although the gene expression response appears to differ between these injuries. The results obtained in this work contribute to an increased understanding of retinal injuries and highlight the importance of Müller cells in the endogenous defense against retinal injuries

Experimental Injury to the Visual System : Molecular Studies of the Retina

Retinal ganglion cells play a crucial role in the relay of visual signals from the eye to the brain. This cell type is affected and eventually lost in the eye disease glaucoma, resulting in progressive and irreversible loss of vision. Studies of the molecular mechanisms leading to retinal ganglion cell death are important for the understanding of the disease and for designing future treatments. This thesis addresses and studies these molecular mechanisms, including alterations in gene expression after experimental retinal injuries. The effects of a neuroprotective drug, brimonidine, after transient retinal ischemia were also studied in order to help explain the mechanisms behind the protective properties of this drug. Several methods, including quantitative reverse transcriptase PCR, micro-arrays, western blot and immunohistochemistry, were used. The results showed that transient retinal ischemia triggers cell division in Müller cells and alters the gene expression of growth factors, their receptors, and intermediate filaments in the retina. Several genes related to the apoptosis process were less affected. Pre-treatment with brimonidine increased the levels of certain growth factors (BDNF, NT3, CNTF, FGF9) compared with vehicle. Brimonidine also had marked effects on genes related to progenitor cells, among them the recognized neural stem cell marker nestin. The increase in levels of nestin after ischemia was countered by brimonidine treatment. Moreover, retinal ganglion cell death following either optic nerve transection or optic nerve crush appears to involve the extrinsic apoptotic pathway although the gene expression response appears to differ between these injuries. The results obtained in this work contribute to an increased understanding of retinal injuries and highlight the importance of Müller cells in the endogenous defense against retinal injuries

Emotions in engineering education : Towards a research agenda

This Work-in-Progress research paper describes preliminary work on a research agenda for emotions in engineering education. Emotions play an important role for teaching and learning in engineering education, but research on the topic is scarce. To spur research in this area, the authors participate in an international collaboration that aims to map existing research, identify questions that are under-researched, and outline important questions for future research on emotions in engineering education. In this paper, we describe preliminary work that has been done in preparation of an international symposium during which a first draft of the research agenda on emotions in engineering education will be developed. At FIE 2020, we will present both this preparatory work and the agenda itself

Time course regulation of cell death–related proteins in naïve and optic nerve injured retinas

Western blot time course analyses showing the regulation of tumor necrosis factor receptor superfamily member 1a (TNFR1a), tumor necrosis factor receptor type 1, associated death domain (TRADD), total Caspase 3, active Caspase 3, and c-fos in naïve, intraorbital nerve transection (IONT)-, and intraorbital nerve crush (IONC)-injured retinas. Graphs show quantification of protein signals (n=4 animals per lesion and time point, western blots were replicated three times). The signal from injured retinas is referred to the naïve signal, which was arbitrarily considered 100%. To verify the amount of loaded protein, western blots were incubated with β-actin (an example is shown). Error bars show the standard error of the mean (SEM) for each experiment.<p><b>Copyright information:</b></p><p>Taken from "Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush"</p><p></p><p>Molecular Vision 2008;14():1050-1063.</p><p>Published online 03 Jun 2008</p><p>PMCID:PMC2426719.</p><p></p

Expression pattern of cell death–related proteins in naïve and optic nerve injured retinas

Immunohistofluorescence analyses for tumor necrosis factor receptor superfamily member 1a (TNFR1a), tumor necrosis factor receptor type 1, associated death domain (TRADD), Caspase 3, and c-fos to fluorogold-traced retinas in naïve, intraorbital nerve transection (IONT) and intraorbital nerve crush (IONC) injured retinas. Expression pattern of TNFR1a (red signal) in naïve retinas (, right), IONT-injured retinas (, right) and IONC-injured retinas (, right). Left images are the corresponding fluorogold (FG) images (blue signal). Expression pattern of TRADD (red signal) in naïve retinas (, right), IONT-injured retinas (, right) and IONC-injured retinas (, right). Left images are the corresponding fluorogold (FG) images (blue signal). Expression pattern of Caspase 3 (red signal) in naïve retinas (, right), IONT-injured retinas (, right) and IONC-injured retinas (, right). Left images are the corresponding fluorogold (FG) images (blue signal). Expression pattern of c-fos (red signal) in naïve retinas (, right), IONT-injured retinas (, right) and IONC-injured retinas (, right). Left images are the corresponding fluorogold (FG) images (blue signal). Magnifications in squares show the co-localization of a given protein with FG-labeled retinal ganglion cells (RGCs). Arrows point to RGC, arrowheads indicate the outer nuclear layer and asterisks mark the nerve layer.<p><b>Copyright information:</b></p><p>Taken from "Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush"</p><p></p><p>Molecular Vision 2008;14():1050-1063.</p><p>Published online 03 Jun 2008</p><p>PMCID:PMC2426719.</p><p></p