Is the Epidermal Growth Factor Receptor involved in visual system regenerative failure?

Morrison, Kevin Carlo (2011). Is the Epidermal Growth Factor Receptor involved in visual system regenerative failure? University of Birmingham. Ph.D.

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Abstract

Introduction: A study in 2005 found that Epidermal Growth Factor Kinase Inhibitors (EGFRki) could promote Retinal Ganglion Cell (RGC) axonal regeneration in vivo when delivered to the crushed Optic Nerve (ON). The axon regenerative effects of these EGFRki were attributed to their blockade of the Epidermal Growth Factor Receptor (EGFR), and EGFR activation was hence suggested to lead to growth cone collapse and failed RGC axon regeneration. Aims: To investigate the role of EGFR in RGC axonal regenerative failure, and to elucidate the mechanisms of action by which EGFRki promote RGC axonal regeneration. Methods: Immunohistochemistry and immunocytochemistry to visualize activated EGFR (pEGFR) Primary retinal cultures to examine the actions of EGFRki such as AG1478 on RGC in vitro. ELISA to examine the conditioned media from these cultures for NeuroTrophic Factors (NTF). Intravitreal (ivit) injections of EGFRki (PD168393) into Optic Nerve Crush (ONC) recipient rats to attempt to elicit in vivo regeneration. The implantation of PD168393-impregnated collagen matrices into ONC recipient rats to attempt to elicit in vivo regeneration. PCR on retinal lysates to detect NTF mRNA. Results: No pEGFR was detected on RGC axons, either in the retina or in the ON of any treatment or control group. pEGFR was detected on almost all ON and retinal glial types prior to injury and almost all glial types exhibited increased pEGFR levels post-ONC. A sub population (~30%) of RGC cell bodies were pEGFR+ but this proportion did not change between control and treatment groups. AG1478 was shown to disinhibit RGC in Nogo-P4 inhibited primary retinal cultures but ELISA on conditioned media for various NTF detected none, however PCR detected mRNA for several of these NTF in retinal lysates. Ivit PD168393 failed to elicit RGC survival or axonal regeneration in vivo. Intra ON implantation of PD168393 impregnated collagen matrices appeared to promote significant RGC axonal regeneration post-ONC, but did not affect RGC survival. Discussion: Several models explaining the in vitro regenerative and in vivo neuritogenic actions of EGFRki were developed. These included the abrogation of various harmful glially mediated processes, the stimulation of NTF release by local glia and the stimulation or blockade of several other non-EGFR dependent signalling cascades by EGFRki. Conclusions: The axogenic and neuritogenic actions of EGFRki in vitro and in vivo were confirmed. The identification of numerous other means by which EGFRki could indirectly promote RGC axonal regeneration, including by acting on targets other than EGFR allowed the construction of a combinatorial model of how EGFRki effect axonal regeneration, and the original hypothesis positing EGFR as an intra-axonal component of a growth cone collapsing signalling cascade was disgarded.

Type of Work:	Thesis (Doctorates > Ph.D.)
Award Type:	Doctorates > Ph.D.
Licence:
College/Faculty:	Colleges (2008 onwards) > College of Medical & Dental Sciences
School or Department:	School of Clinical and Experimental Medicine
Funders:	None/not applicable
Subjects:	R Medicine > RE Ophthalmology R Medicine > R Medicine (General)
URI:	http://etheses.bham.ac.uk/id/eprint/1700

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