Optic nerve section (ONS) is an experimental model for damage of the optic nerve associated with diseases such as glaucoma and optic neuritis. Damage to the optic nerve causes loss of retinal ganglion cells that are attached, once the cells are damaged, they are not typically replaced. Recently, Fischer and Reh (2003) found that Müller glia have the potential to adopt phenotypes and functional capabilities similar to those of retinal progenitors, a potential source of retinal regeneration. In the chick, when the specific retinal cells are targeted for damage by chemotoxins, there is widespread apoptosis but also mitotically active cells that label with retinal progenitor markers. Fischer and Reh (2002) also discovered that the combination of growth factors FGF2 and insulin is capable of stimulating the regenerative response of the Müller glia to retinal progenitor cells in chick eyes. This study was conducted to analyse damage to the ganglion cells by optic nerve section in chicks to determine the effect of age on the cell death timeline, the proliferative qualities of the retina and to see if injections of growth factors had the ability to increase the proliferation. Histological methods were used to analyse cellular changes and ultrasound to monitor eye growth. Apoptotic activity preceded retinal thinning and ganglion cell loss, indicating that ONS-related cell death is mediated at least in part by apoptotic mechanisms and age did not affect the time course, although, age did affect the eye growth changes, which may be attributed to the plasticity of the younger eyes. ONS damage elicited proliferative activity in the retina as did growth factor injections alone. The combination of ONS damage and growth factor injections increased the proliferative activity and the overall total number of cells in the ganglion cell layer. These findings can potentially lead to the development of therapeutic strategies for the preservation or restoration of retinal cells in diseased eyes.

The crystalline lens is a unique cellular organ that performs metabolic processes while maintaining optical functionality. Mitochondria play a vital role in providing the cell with the energy necessary for these metabolic processes and have recently been shown to be more metabolically active than previously thought. To test the hypothesis that mitochondrial function directly influences the optical function of the lens, bovine lenses were treated with 50 μM, 200 μM, 600 μM and 1000 μM menadione, a mitochondrial specific toxin that renders the mitochondria inactive, and the Back Vertex Distance (BVD) variability was observed over 216 hours. Confocal micrographs of secondary fibre cells’ mitochondria were also analyzed for 50 μM, 200 μM, and 600 μM menadione treatment over 48 hours. Increase in BVD variability (± s.e.m.) was observed within 24 hours from 0.28 ± 0.021 to 1.83 ± 0.75 for the 600 μM treated lenses. Confocal micrograph analysis showed a trend toward a decrease in the average length of mitochondria from 7.9 ± 0.8 to 3.7 ± 0.9 over for 200 μM treated lenses and from 5.9 ± 1.0 to 3.6 ± 0.6 for the 600 μM treated lenses over 48 hours. These data show that indeed menadione has a detrimental effect on mitochondria as a function of both time and concentration and this change in mitochondria precedes changes in BVD variability directly linking mitochondrial function to optical function.

The crystalline lens is a unique cellular organ that performs metabolic processes while maintaining optical functionality. Mitochondria play a vital role in providing the cell with the energy necessary for these metabolic processes and have recently been shown to be more metabolically active than previously thought. To test the hypothesis that mitochondrial function directly influences the optical function of the lens, bovine lenses were treated with 50 μM, 200 μM, 600 μM and 1000 μM menadione, a mitochondrial specific toxin that renders the mitochondria inactive, and the Back Vertex Distance (BVD) variability was observed over 216 hours. Confocal micrographs of secondary fibre cells’ mitochondria were also analyzed for 50 μM, 200 μM, and 600 μM menadione treatment over 48 hours. Increase in BVD variability (± s.e.m.) was observed within 24 hours from 0.28 ± 0.021 to 1.83 ± 0.75 for the 600 μM treated lenses. Confocal micrograph analysis showed a trend toward a decrease in the average length of mitochondria from 7.9 ± 0.8 to 3.7 ± 0.9 over for 200 μM treated lenses and from 5.9 ± 1.0 to 3.6 ± 0.6 for the 600 μM treated lenses over 48 hours. These data show that indeed menadione has a detrimental effect on mitochondria as a function of both time and concentration and this change in mitochondria precedes changes in BVD variability directly linking mitochondrial function to optical function.

Optic nerve section (ONS) is an experimental model for damage of the optic nerve associated with diseases such as glaucoma and optic neuritis. Damage to the optic nerve causes loss of retinal ganglion cells that are attached, once the cells are damaged, they are not typically replaced. Recently, Fischer and Reh (2003) found that Müller glia have the potential to adopt phenotypes and functional capabilities similar to those of retinal progenitors, a potential source of retinal regeneration. In the chick, when the specific retinal cells are targeted for damage by chemotoxins, there is widespread apoptosis but also mitotically active cells that label with retinal progenitor markers. Fischer and Reh (2002) also discovered that the combination of growth factors FGF2 and insulin is capable of stimulating the regenerative response of the Müller glia to retinal progenitor cells in chick eyes. This study was conducted to analyse damage to the ganglion cells by optic nerve section in chicks to determine the effect of age on the cell death timeline, the proliferative qualities of the retina and to see if injections of growth factors had the ability to increase the proliferation. Histological methods were used to analyse cellular changes and ultrasound to monitor eye growth. Apoptotic activity preceded retinal thinning and ganglion cell loss, indicating that ONS-related cell death is mediated at least in part by apoptotic mechanisms and age did not affect the time course, although, age did affect the eye growth changes, which may be attributed to the plasticity of the younger eyes. ONS damage elicited proliferative activity in the retina as did growth factor injections alone. The combination of ONS damage and growth factor injections increased the proliferative activity and the overall total number of cells in the ganglion cell layer. These findings can potentially lead to the development of therapeutic strategies for the preservation or restoration of retinal cells in diseased eyes.

Myopia is a common ocular condition worldwide and the mechanism of myopia is still not clear. A number of animal models of myopia and refractive error development have been proposed. The fact that form deprivation myopia could be induced in tilapia fish, as shown previously in my research, suggests the possibility that tilapia could be a new animal model for myopia research. In the first part of this thesis the tilapia model was perfected and then, based on this model, the effect of systemic hormones (thyroid hormones) associated with eye and body development was investigated during refractive error development. Lastly, the physiological and morphological changes on the retina were further studied with optical coherence tomography (OCT). In these experiments, significant amounts of myopia, and hyperopia were induced within two weeks using goggles with lens inserts as in other higher vertebrate animal models, e.g. chicks. The results from form deprivation treatment also show that the sensitivity of tilapia eyes may be an age related effect during the emmetropization process. The larger the fish, the less hyperopic the fish eye, though the small eye artefact may be a factor. The susceptibility of the refractive development of the eye to the visual environment may be also linked to plasma hormone levels. It was found that induced refractive errors could be shifted in the hyperopic direction with high levels of thyroid hormones. Also, after 2 weeks of treatment with negative or positive lens/goggles, the tilapia retina becomes thinner or thicker, respectively. When the goggles are removed, the thickness of the retina changes within hours and gradually returns to normal. However, the circadian retinomotor movement is a complicating factor since it affects the retinal thickness measurement with OCT at some time points. In conclusion, tilapia represent a good lower vertebrate model for myopia research, suggesting a universal mechanism of myopia development, which may involve systemic hormones and immediate, short term retinal responses.

Myopia is a common ocular condition worldwide and the mechanism of myopia is still not clear. A number of animal models of myopia and refractive error development have been proposed. The fact that form deprivation myopia could be induced in tilapia fish, as shown previously in my research, suggests the possibility that tilapia could be a new animal model for myopia research. In the first part of this thesis the tilapia model was perfected and then, based on this model, the effect of systemic hormones (thyroid hormones) associated with eye and body development was investigated during refractive error development. Lastly, the physiological and morphological changes on the retina were further studied with optical coherence tomography (OCT). In these experiments, significant amounts of myopia, and hyperopia were induced within two weeks using goggles with lens inserts as in other higher vertebrate animal models, e.g. chicks. The results from form deprivation treatment also show that the sensitivity of tilapia eyes may be an age related effect during the emmetropization process. The larger the fish, the less hyperopic the fish eye, though the small eye artefact may be a factor. The susceptibility of the refractive development of the eye to the visual environment may be also linked to plasma hormone levels. It was found that induced refractive errors could be shifted in the hyperopic direction with high levels of thyroid hormones. Also, after 2 weeks of treatment with negative or positive lens/goggles, the tilapia retina becomes thinner or thicker, respectively. When the goggles are removed, the thickness of the retina changes within hours and gradually returns to normal. However, the circadian retinomotor movement is a complicating factor since it affects the retinal thickness measurement with OCT at some time points. In conclusion, tilapia represent a good lower vertebrate model for myopia research, suggesting a universal mechanism of myopia development, which may involve systemic hormones and immediate, short term retinal responses.

Inherited retinal degenerative diseases can affect various components of the retina leading to blindness. Five different mutant strains of chicken have been studied extensively as potential models for inherited retinal degeneration. The Smoky Joe (SJ) chicken is a sixth genetically blind strain of White Leghorns that shows various degrees of blindness at hatch and by 8 weeks post-hatch, have complete blindness for those that are homozygous. The objective of this study was to characterize the retinal degeneration in these birds by histology, both during embryonic and post-hatch development, and to the retinal function using electroretinograms (ERG). For both embryonic and post-hatch development, a significantly lower number of cells were found in the retina of blind birds compared to sighted (both p<0.0001). The significant contributor to cell number decrease was the loss of amacrine cells located in the inner nuclear layer. Photoreceptors were also found to potentially decrease in number, but at a later stage. ERG recordings revealed decreases in amplitudes of b-waves and oscillatory potentials in blind birds, but not in sighted. Both histology and ERG findings support the idea that the inner retinal cells are affected. The results indicate that degeneration in the Smoky Joe retina occurs mostly within the inner nuclear layer affecting amacrine cells. This hampers the functional capacity of the retina, causing blindness.

A previous study in chickens revealed that myosin light chain kinase (MLCK), f actin, and myosin are found on the crystalline lens. Their polygonal arrangement at the posterior surface resembles a muscle tissue, which suggests that these proteins may have a contractile role in accommodation. The ciliary muscle in chickens is skeletal in nature and, therefore, chickens were used to test the hypothesis that contractile microfilaments play a role in accommodation. Ciliary nerve-induced accommodation was measured in the presence of an MLCK inhibitor 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). Eyes of 6-day old white Leghorn chickens (gallus gallus domesticus) were enucleated in Tyrode’s saline solution while keeping the ciliary nerve intact. One eye was treated with ML-7 and the other eye was treated with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. Two experiments were carried out, one including a (3×10 min) wash and one without. Focal lengths of the vehicle- and ML-7-treated eyes were measured before, during and after accommodation. Immunoblots were used to detect the amount of phosphorylated myosin with and without the inhibitor. Focal lengths for accommodation were shorter than those at rest (p<0.001). In the wash experiment, vehicle-treated eyes had higher accommodative amplitudes compared to ML-7-treated eyes for all three dosage groups. In the no-wash experiment, only the 1 µM group demonstrated the same trend as the wash experiment. For the 10 µM and 100 µM groups, ML-7-treated eyes had higher accommodative amplitudes compared to vehicle-treated eyes. Immunoblots revealed varying amounts of inhibition within pairs of eyes as well as between birds for both experiments. Results from this experiment indicate that ML-7 was not effective at determining whether contractile microfilaments found on the lens contribute to accommodation.

A previous study in chickens revealed that myosin light chain kinase (MLCK), f actin, and myosin are found on the crystalline lens. Their polygonal arrangement at the posterior surface resembles a muscle tissue, which suggests that these proteins may have a contractile role in accommodation. The ciliary muscle in chickens is skeletal in nature and, therefore, chickens were used to test the hypothesis that contractile microfilaments play a role in accommodation. Ciliary nerve-induced accommodation was measured in the presence of an MLCK inhibitor 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). Eyes of 6-day old white Leghorn chickens (gallus gallus domesticus) were enucleated in Tyrode’s saline solution while keeping the ciliary nerve intact. One eye was treated with ML-7 and the other eye was treated with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. Two experiments were carried out, one including a (3×10 min) wash and one without. Focal lengths of the vehicle- and ML-7-treated eyes were measured before, during and after accommodation. Immunoblots were used to detect the amount of phosphorylated myosin with and without the inhibitor. Focal lengths for accommodation were shorter than those at rest (p<0.001). In the wash experiment, vehicle-treated eyes had higher accommodative amplitudes compared to ML-7-treated eyes for all three dosage groups. In the no-wash experiment, only the 1 µM group demonstrated the same trend as the wash experiment. For the 10 µM and 100 µM groups, ML-7-treated eyes had higher accommodative amplitudes compared to vehicle-treated eyes. Immunoblots revealed varying amounts of inhibition within pairs of eyes as well as between birds for both experiments. Results from this experiment indicate that ML-7 was not effective at determining whether contractile microfilaments found on the lens contribute to accommodation.

Inherited retinal degenerative diseases can affect various components of the retina leading to blindness. Five different mutant strains of chicken have been studied extensively as potential models for inherited retinal degeneration. The Smoky Joe (SJ) chicken is a sixth genetically blind strain of White Leghorns that shows various degrees of blindness at hatch and by 8 weeks post-hatch, have complete blindness for those that are homozygous. The objective of this study was to characterize the retinal degeneration in these birds by histology, both during embryonic and post-hatch development, and to the retinal function using electroretinograms (ERG). For both embryonic and post-hatch development, a significantly lower number of cells were found in the retina of blind birds compared to sighted (both p<0.0001). The significant contributor to cell number decrease was the loss of amacrine cells located in the inner nuclear layer. Photoreceptors were also found to potentially decrease in number, but at a later stage. ERG recordings revealed decreases in amplitudes of b-waves and oscillatory potentials in blind birds, but not in sighted. Both histology and ERG findings support the idea that the inner retinal cells are affected. The results indicate that degeneration in the Smoky Joe retina occurs mostly within the inner nuclear layer affecting amacrine cells. This hampers the functional capacity of the retina, causing blindness.