Niclas Lindqvist

The growth factor response in ischemic rat retina and superior colliculus after brimonidine pre-treatment

The alpha-2-adrenergic receptor agonist brimonidine has been shown to increase survival of retinal ganglion cells following ischemic injury to the rat retina. Increased expression of growth factors has been suggested to be involved in this action. We investigated expressional changes of growth factors and their receptors following transient retinal ischemia induced by selective ligature of ophthalmic vessels in rats pre-treated with vehicle or 0.5% brimonidine. In addition, analysis of expression in retinal samples following unilateral administration of brimonidine to normal tissue was performed. Tissue samples of retina and superior colliculus were collected at time points between 6h and 14 days of retinal reperfusion. Analysis of mRNA levels of the ligands BDNF, NT3, CNTF, FGF1, FGF2, FGF9 and HGF; as well as the receptors TrkB, TrkC, p75(NTR), CNTFRalpha, FGFR1, FGFR3, FGFR4 and HGFR were performed using qRT-PCR. The cell specific markers Thy1 and GFAP were analysed. We report transiently increased retinal levels of BDNF, NT3, p75(NTR), FGFR1 and HGFR and decreased levels of FGF9, HGF, TrkB, TrkC, FGFR4 and Thy1 following ischemia. The decreases were counteracted by brimonidine. Brimonidine treatment gave an increase in BDNF, NT3 and CNTF levels compared to the vehicle treated group. In superior colliculus increased levels of growth factor mRNA were found. In conclusion, transient ischemia has a profound effect on gene expression in rat retina. Alterations can also be seen in the superior colliculus but are smaller. Brimonidine pre-treatment attenuates an acute injury-induced response by decreasing the expression of several genes, among them p75(NTR). Brimonidine also causes a prolonged increase of several growth factors as well as receptors in retina and superior colliculus compared to the ischemic situation. The increased expression of several growth factors represents a coordinated growth factor system response that differs from the ischemia-induced changes and is likely part of the neuroprotective activity that is elicited by BMD pre-treatment.

GDNF, Ret, GFRα1 and 2 in the adult rat retino-tectal system after optic nerve transection

In the present study, we have studied the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors Ret, GFRalpha1, and GFRalpha2 in the retino-tectal system before and after optic nerve transection. Using retrograde neuronal tracing in combination with in situ hybridization, we found that Ret and GFRalpha1 are expressed by 13-14% of the retinal ganglion cells (RGCs). These Ret-expressing RGCs could not be identified as belonging to any particular of the RG(A), RG(B), and RG(C) sub types. Ret is co-expressed with the brain-derived neurotrophic factor receptor TrkB in these RGCs. Optic nerve transection resulted in reduced Ret mRNA levels in retina, while the levels of GDNF, GFRalpha1, and 2 mRNA increased. Administration of GDNF protein supported the axotomized RGCs. Analysis of normal superior colliculus (SC) did not show any expression of GDNF mRNA, yet GDNF mRNA levels in SC increased after injury. Together, these findings identify a portion of RGCs as being possible targets for pharmacological treatment with GDNF in a direct mode of action. The absence of detectable GDNF mRNA in normal SC questions the role for GDNF as being a target-derived factor produced in the SC for adult RGCs. The results support a function for GDNF locally in the retina and as part of an injury-induced system that may act to enhance neuroprotective and neuroregenerative responses both to endogenous GDNF ligands and those administered exogenously.

Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching

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.

Overlapping and specific patterns of GDNF, c-ret and GFRα mRNA expression in the developing chicken retina

GDNF and the GDNF receptors, c-Ret, GFR alpha 1 and 2 mRNA is expressed in the developing chicken retina. GDNF labelling was mainly found in embryonic day 4-5 retina but weak labelling could also be found over scattered retinal cells at later stages. c-ret labelling was found over ganglion cells, amacrine and horizontal cells; the preferred GDNF receptor (GFR alpha 1) over amacrine and horizontal cells; and the less preferred GDNF receptor (GFR alpha 2) over ganglion cells, amacrine cells and photoreceptors.

Transactivation of EGF Receptors in Chicken Muller Cells by α2A-Adrenergic Receptors Stimulated by Brimonidine

PURPOSE Alpha2-adrenergic receptor agonists are used in glaucoma treatment and have been shown to have some neuroprotective effects. We performed this study to test the hypothesis that epidermal growth factor receptors on chicken Müller cells are transactivated by α2-adrenergic receptors and we focused on the extracellular signal-activated kinases 1/2 (ERK) pathway. METHODS Embryonic chicken retina and cultures of primary Müller cells were stimulated by α2-adrenergic receptor agonist brimonidine. Immunostaining, quantitative RT-PCR, and Western blot techniques, in combination with Src, epidermal growth factor receptor kinase, and matrix metalloproteinase inhibitors, were used for analysis of the cellular responses. RESULTS Our results showed that Müller cells express α2A-adrenergic receptors in vivo and in vitro and that brimonidine triggered a robust and transient phosphorylation of ERK1/2. This ERK-response was Src-kinase dependent, associated with tyrosine phosphorylation of epidermal growth factor receptors (phospho-Y1068, Y1173) and was mediated by matrix metalloproteinase activity on the Müller cells. CONCLUSIONS Müller cells express the α2A-adrenergic receptor, and brimonidine triggers both Src-kinase- and matrix metalloproteinase-mediated autocrine ligand-dependent activation of epidermal growth factor receptors on Müller cells. This response is consistent with transactivation of epidermal growth factor receptors by stimulation of α2-adrenergic receptors.

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

Abstract 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, Pdgfrβ, Ptk7, Ret, Ros, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3. Non-RTK sequences were Jak1 and 2. Retinal expression of Axl, Csf-1R, Pdgfrβ, 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, Pdgfrα/β, 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 Pdgfrβ, 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 Pdgfrβ, Csf-1R, Vegfr-2, Sky, and Axl, and their ligands, have not previously been primarily associated with retinal ganglion cells.