The murine eye naturally undergoes post-natal changes in eye size. This dissertation quantifies longitudinal structural and functional changes in control mice (C57BL/6J (B6), D2-Gpnmb+/SjJ) and in DBA/2J (D2) mice, which spontaneously develop elevated intraocular pressure (IOP). IOP elevation results in abnormal eye elongation, retinal nerve fiber layer (RNFL) thickness thinning and retinal ganglion cell (RGC) dysfunction and demise resembling human glaucoma. I measured structural changes with Optical Coherence Tomography (OCT), and RGC function with Pattern Electroretinogram (PERG). I also developed and refined provocation approaches (IOP elevation with changes in body posture; metabolic load with flickering light) to probe susceptibility of RGC function in D2 mice prone to glaucoma. Finally, I developed a novel system for recording, simultaneously but independently, the PERG from both eyes using asynchronous visual stimuli and deconvolution analysis. Simultaneous PERG recording from each eye was hitherto impossible due to the interocular cross-talk of the PERG signal. Altogether, the combination of these measures (OCT, PERG) and provocative conditions may represent powerful tools for glaucoma research using mouse models.

Mammalian central nervous system (CNS) neurons lose their ability to regenerate their axons after injury during development. For example, optic nerve injury studies in hamsters have shown that optic nerve axons injured around the time of birth retain the ability to regenerate to their target, but this ability is lost during development (So et al., 1981). The development of an inhibitory CNS environment has been implicated in the inability of the adult CNS to regenerate, however there is also support for this loss being a result of changes in developmental programs intrinsic to the neurons themselves (Goldberg et al., 2002a; Goldberg, 2004). While some molecules have been identified as being involved in intrinsic mechanisms controlling axon growth, there is still much to be discovered. Using genes shown to be regulated in retinal ganglion cells (RGCs) during development (Wang et al., 2007), I performed an overexpression screen in embryonic primary neurons measuring changes in neurite growth. Of these genes, the most significant effect in neurite growth was seen with overexpression of Krüppel-like factor 4 (KLF4), resulting in a greater than 50% decrease in growth. KLF4 is a member of the KLF family of transcription factors which all possess a DNA binding domain containing 3 zinc finger motifs. Outside of the nervous system, KLF4 has been implicated in cancer (Black et al., 2001; Rowland and Peeper, 2006), mitotic growth arrest (Shields et al., 1996) and most recently in the induction of pluripotency (Yamanaka, 2008; Zhao and Daley, 2008). In the CNS, KLF4 has recently been implicated in increasing the sensitivity of cortical neurons to NMDA insult (Zhu et al, 2009), though no effect of KLF4 on neurite growth or regeneration has yet been described. I found that KLF4 overexpression in RGCs results in decreased neurite growth and neurite initiation. KLF4 overexpression also leads to decreases in polarity acquisition in hippocampal neurons, though even when they acquire polarity, they still display decreased neurite growth. Additionally, KLF4 knockout targeted to RGCs leads to an increased neurite growth ability and increased neurite initiation in vitro. In vivo, KLF4 knockout increases RGC axon regeneration after optic nerve injury. Interestingly, KLF4 is one of 17 members of the KLF family, known for their ability to act redundantly and competitively amongst family members for their binding sites. Therefore, we looked to see if other KLFs could affect neurite growth ability. 15 of 17 KLF family members are expressed in RGCs, and their overexpression results in differential effects on neurite growth in both cortical neurons and RGCs. Additionally, many of the family members are developmentally regulated in a manner that typically correlates with their ability to affect neurite growth. For example, KLF6 and -7, whose expression decreases during development, when overexpressed, increase neurite growth, whereas KLF9, whose expression increases developmentally, when overexpressed, decreases neurite growth. Surprisingly, there are multiple KLFs expressed in RGCs that are neurite growth-suppressors, and further study has revealed that the combination of KLF growth enhancers with KLF growth suppressors results in a suppressive or neutral phenotype (Moore et al., 2009), suggesting that to further enhance regeneration after injury in vivo, we will need to additionally remove the growth suppression from other KLF family members. Taken together, these data suggest that KLFs may play an important role in the intrinsic loss of axon growth and regeneration seen during development. Further characterization of downstream targets of KLF4 and other KLF family members may reveal specific neuronal gene targets that could mediate the phenotypic effects of these transcription factors. It is my hope that by determining the developmental programs that underlie the loss of intrinsic axon growth ability of CNS neurons, we may ultimately determine how to revert adult CNS neurons to their embryonic axon growth ability.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 180-205). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 180-205). Also available online.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (p. 113-121).

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

In order to understand the possible mechanisms in the glaucoma-related retinopathy, the role of the vasoconstrictor, endothelin-1 (ET-1), receptor for advanced glycation end-products (RAGE) as well as its ligand, Aβ in the degeneration of retinal ganglion cells (RGCs) were studied in experimental models. In addition, the relationship of ET-1, RAGE and Aβ for the RGC protective mechanism of Lycium Barbarum (LB) was also investigated. In the first part, ET-1 together with its receptors, ETA and ETB, were studied to understand their possible roles in chronic ocular hypertension (COH). The neuronal protective mechanism of LB was also determined by using a well established COH rat model. In normal rats, ET-1 and its receptors, ETA and ETB, were distributed in the retina, vasculature and optic nerve. Interestingly, ET-1 expression was up-regulated after COH. LB could decrease the expression of ET-1 and regulate its receptors (up-regulation of ETB and down-regulation of ETA in vasculature; up-regulation of ETA and down-regulation of ETB in RGCs) under the condition of COH. These data suggested that the RGC protective mechanism of LB might be related to its ability to regulate the biological effects of ET-1. To investigate the pathogenic effect of ET-1 in glaucoma, in the second part, we used transgenic mice with over-expression of ET-1 on endothelial cells (TET-1 mice). We found that beginning at 10-12 months, TET-1 mice showed a progressive retinal degeneration (loss of RGCs associated with neurons in the inner nuclear layer and outer nuclear layer of the retina) without elevation of the intraocular pressure (IOP). The data demonstrated that TET-1 mice may serve as a potential model to investigate the role of endothelial ET-1 in the pathogenesis of normal tension glaucoma and other degenerative retinopathy. To investigate whether LB plays a role on neuronal protection other than in COH, in the third part, we used an acute ocular hypertension (AOH)-induced ischemia mouse model. We found that LB could rescue RGCs under AOH insult, associating with blood vessel protection (decreasing the damage of blood-retinal-barriers and rescuing the survival of endothelial cells and pericytes) and inhibiting retinal gliosis. We also found the protective mechanism of LB was closely correlated with down-regulation of the expression of RAGE, ET-1, APP (amyloid precursor protein), AGE (advanced glycation end-product) as well as Aβ; therefore to reduce the damage effects of these RAGE-mediated reactions to the retinal neurons, blood vessels and glial cells involved in the ischemic insult. Taken together, the present study demonstrated that TET-1 mice may be a potential model for investigating the role of ET-1 in degenerative retinopathies, such as normal tension glaucoma. We also showed the neuronal protective mechanism of LB in vivo was associated with inhibiting the biological effect of ET-1 and down-regulating the damage signaling pathways mediated by the activation of RAGE and its ligands (AGE and Aβ). These results provided further understandings in the mechanism of the glaucoma-related retinopathy. In addition, LB could be a neuroprotective agent to the retina following both chronic and acute injuries. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy