INTRODUCTION: The cornea forms the anterior-most barrier of the eye, consisting of a non-keratinized pseudostratified squamous epithelium, a collagen-based stroma, and an endothelium. It is completely avascular, yet the most densely innervated structure in the human body. The sensory nerves project from the ophthalmic branch of the trigeminal cranial nerve into the limbal/stromal interface. From there, the nerves branch and ascend into Bowman’s membrane, a basal lamina delineating the epithelium from the stroma, and project into the epithelium as free nerve endings. Injury to the corneal epithelium can potentially lead to impaired vision if the wound healing process is not properly initiated. Immediately after injury, nucleotides such as ATP are released and bind to purinergic receptors known to be located in epithelial cell membranes, thereby initiating epithelial cell migration to close the wound. Malfunctions in the interactions between the corneal nerves and their epithelial counterparts during the wound healing process are thought to contribute to the attenuated wound healing characteristic of diabetes. However, the precise nature of these interactions, how they facilitate wound healing, and how they are impaired in diabetes, is not well understood. OBJECTIVES: Previously, our lab has shown that a member of purinergic family receptors (P2X7) is localized in the basal epithelial cells and becomes relocated to the leading edge of the wound after injury. When the relocation is inhibited, migration is attenuated. Additionally, it is known that diabetic mouse models display slower wound healing rates. The present study has three aims: (1) to replicate the characteristic sub-basal whorl organization of the corneal nerves in organ-cultured corneas; (2) to elucidate the connections between patterns of corneal innervation and purinergic receptor expression; and (3) to understand how these patterns interact to facilitate normal wound healing and how these interactions are disrupted in a diabetic model. METHODS: Our approach was to use immunohistochemistry of dissected mouse and to visualize the tissue using confocal microscopy. Sensory innervation profiles from diet induced obesity (DIO) mouse corneas and their wildtype C57Bl6 counterparts were compared in unwounded and wounded tissue. To image the nerves a methanol fixation protocol was optimized to examine the sub-basal plexus and the apical nerves. Corneas were dissected, stained with beta III-tubulin, which identifies nerves, and with an antibody to the P2X7 purinergic receptor, which is expressed in the epithelium and nerves. Trephine induced epithelial abrasion injuries were made on separate DIO and control models to compare re-epithelialization and re-innervation between the diseased and healthy states. Corneas were imaged using a Zeiss LSM 700 laser scanning confocal microscope and optical images were taken through the cornea over a distance averaging 115 microns. Corneas were imaged using a macro tiling plugin, stitching 3x3 optical z-stacks into composite images. The 3x3 tiles were created to image the central whorl, as well as the peripheral nerve fibers. Co-localization of P2X7 and betaIII tubulin were determined by thresholding using ImageJ/FIJI software. RESULTS: The elegant organization of the centralized sub-basal whorl of the control mouse was disrupted in the DIO mouse cornea, appearing fragmented and incomplete. Analysis of 7.5 and 15 wk corneas showed the whorl to be present at 7.5 wks. Average apical nerve fiber projection length was decreased in DIO cornea. Yet, analyses at each epithelial layer demonstrated overall increased apical nerve density in the DIO corneas as compared to control while sub-basal nerve density decreased dramatically. Stromal nerves remained equivalent. P2X7 did co-localize to the large stromal nerve fibers but it was difficult to show the localization along the sub-basal nerve plexus. However in cross-section images, P2X7 displayed an intracellular polarity, and was present along the apical surface of the columnar basal epithelial cells lining the basement membrane. This localization may suggest the presence of P2X7 expressing sensory nerves, which may be ideally poised for communication with the basal cells after injury. CONCLUSIONS: These data support the hypothesis that there is indeed a difference between diabetic and control corneal innervation. While wound healing differences due to the interaction between sensory nerves and the localization of P2X7 in epithelium at the leading edge remain to be fully elucidated, the novel finding of P2X7 expression in corneal nerves confirms a potential role of purinergic receptor and nerve coordination in conducting the wound healing response.

De par son rôle dans de nombreuses fonctions biologiques, la vitamine A est une molécule majeure et cruciale du développement embryonnaire à l’âge adulte. Celle-ci est aussi, à l’heure actuelle, déjà largement utilisée comme agent thérapeutique pour de nombreuses pathologies affectant principalement la peau et les yeux (cancer, acné, psoriasis,brûlures oculaires) et certains cancers. Son action pro-cicatrisante - bien que largement étudiée grâce à de nombreux modèles humains et animaux - reste encore aujourd’hui mal caractérisée quant aux mécanismes d’action moléculaire (régulation de gènes) et cellulaire(migration, prolifération…) qu’elle utilise.Dans le but d’améliorer et de mieux maîtriser l’utilisation de la vitamine A dans le traitement de lésions telles que les brûlures oculaires, ce travail visait d’une part, à étudier, plus en détail,l’effet de l’acide tout-trans rétinoïque ou ATRA - dérivé le plus actif - sur la cicatrisation de ’épithélium cornéen en utilisant un modèle in vivo de brûlures cornéennes par base chez la souris. Son but était également de déterminer par quels processus cellulaires, l’ATRA agit, en utilisant cette fois-ci un modèle in vitro (cellules épithéliales cornéennes humaines). Enfin,nous nous sommes intéressés à la régulation de gènes cibles par l’ATRA au niveau de la sphère oculaire, connus pour être impliqués dans la dynamique de la MEC.Ainsi, nous avons démontré que l’ATRA permettait la cicatrisation de l’épithélium cornéen en agissant principalement sur la migration cellulaire. Puis nous avons identifié un gène :LOXL4 - membre de la famille des lysyl oxydases - dont l’expression est induite par l’ATRA,et nous avons prouvé que son rôle était essentiel dans la cicatrisation cornéenne, décrivant ainsi pour la première fois un lien génique direct et protéique entre la vitamine A, substance active et son action clinique pro-cicatrisante. / Because of its role in many biological functions, Vitamin A is a major and crucialmolecule from development to adulthood. Currently, it is largely used as therapeutic agent inseveral eye or skin pathologies (acne, psoriasis, ocular burns) and cancers. Its pro-healingproperties have been largely studied in human and animal models but molecular (generegulations) and cellular (migration, proliferation…) mechanisms of the vitamin A actionhave to be more detailed.In order to better improve and control its use in the treatment of lesions such as ocular burns,this work aimed to study, more in details, the effects of atRA (all-trans-retinoic acid), activederivative form of vitamin A, on corneal epithelium healing using an in vivo model of alkaliocular burns in mouse and then, to determine by which cellular process atRA acts, by usingthis time, an in vitro model (human corneal epithelial cells). Finally, we were interested intargeting genes regulation by atRA in the ocular sphere, specially known to be implied in theECM dynamic.First, we demonstrated that atRA improves corneal epithelium wound healing, essentially byacting on migration. Then, we identified a gene, LOXL4, member of the lysyl oxidase family,which expression is induced by atRA and we proved that this one is essential in cornealwound healing, describing for the first time a direct gene and protein link between vitamin A,active substance, and its clinical pro-healing action.

The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells. / Doctor of Philosophy (Ph.D.)

Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.

Aim: This study looks at the effect of orthokeratology on a number of biometric parameters and refractive error in an adult population. Method: Forty three myopic subjects were recruited to a twelve month study into the effects of orthokeratology on ocular biometry and refractive error. Two different back surface lens designs were applied right eye) pentacurve and left eye) aspheric. The aspheric design was chosen to more closely mimic the cornea's natural shape. Anterior and posterior apical radii and p-values; corneal thickness and anterior chamber depth were measured using the Orbscan IIz; together with ocular biometry by IOL Master and a standard clinical refraction. All measurements were repeated at one night, one week, one, three, six and twelve months. Refractive changes were analysed against biometric changes. Results: Twenty seven participants completed one month of lens wear. Twelve subjects completed twelve months of lens wear. Subjects with myopia ≤ -4.00DS were successfully treated with orthokeratology. Both anterior and posterior apical radii and p values were altered by orthokeratology. Corneal thickness changes were in agreement with previously published studies. Axial length and anterior chamber depth were unaffected by the treatment. Conclusion: Orthokeratology should be available as an alternative to laser refractive surgery. It is best restricted to myopes of up to -4.00DS with low levels of with the rule corneal astigmatism. The use of an aspheric back design contact lens did not produce a significant benefit over that of a pentacurve.

Clinical guidelines dictate that frequent blood glucose monitoring in diabetic patients is critical towards proper management of the disease. Although, several different types of glucose monitors are now commercially available, most of these devices are invasive, thereby adversely affecting patient compliance. To this end, optical polarimetric glucose sensing through the eye has been proposed as a potential noninvasive means to aid in the control of diabetes. Arguably, the most critical and limiting factor towards successful application of such a technique is the time varying corneal birefringence due to eye motion artifact. In the first part of this research, we describe a birefringent ocular model along with a geometric ray tracing scheme to serve as a tool towards better understanding of the cornea’s birefringence properties. The simulations show that index-unmatched coupling of light is spatially limited to a smaller range when compared to index-matched situation. Polarimetric measurements on rabbits’ eyes indicate relative agreement between the modeled and experimental values of corneal birefringence. In addition, the observed rotation in the plane of polarized light for multiple wavelengths demonstrates the potential for using a dual-wavelength polarimetric approach to overcome the noise due to time-varying corneal birefringence. These results will ultimately aid in the development of an appropriate eye coupling mechanism for in vivo polarimetric glucose measurements. The latter part of the dissertation focuses on design and development of a dual wavelength optical polarimeter. The described system utilizes real-time closed-loop feedback based on proportional-integral-derivative (PID) control, which effectively reduced the time taken by the system to stabilize while minimizing the effect of motion artifact, which appears as common noise source for both the wavelengths. Glucose measurements performed in both in vitro and ex vivo conditions demonstrate the sensitivity of the current system. Finally, in vivo results in rabbits indicate that dual-wavelength polarimetry has the potential to noninvasively probe glucose through the anterior chamber of the eye.

The cornea is the most powerful refractive component of the eye and as such, subtle changes in corneal shape can cause substantial changes in the optical characteristics of the eye. Monocular diplopia has previously been linked to corneal distortion following near work in various studies but has not been investigated in detail. The work reported in this thesis has investigated the optical effects of corneal distortions caused by eyelid forces and demonstrated that several corneal higher and lower order Zernike wavefront aberrations can change following reading. Measuring subtle changes in corneal topography requires the highest possible instrument accuracy, while software analysis tools should be able to detect and highlight those subtle changes with high reliability. The effect of ocular microfluctuations on the qualitative and quantitative analysis of corneal topography was investigated. A technique was developed to measure tilt, displacement, and cyclotorsion in multiple videokeratographs from the same cornea. This information was used to reposition each videokeratograph according to the average position of a sample of multiple measurements. The corneal topography of ten subjects was measured 20 times each, using videokeratoscopy. The RMSE calculated from difference between single videokeratographs and the average videokeratograph decreased by an average of 24.6 % for the ten subjects' data. The method can improve the precision performance of videokeratoscopy in multiple measurements of corneal topography. A study was undertaken, to investigate whether there are significant changes in corneal topography during accommodation in normal corneas and corneas that are pathologically thinner due to keratoconus. This was done to eliminate the possibility that changes in corneal aberrations associated with near work could be at least partly due to corneal changes caused by the effects of accommodation. A videokeratoscope was modified to present an accommodation stimulus that was coaxial with the instrument's measurement axis. Six subjects with normal corneas and four subjects with keratoconus were studied. In the initial analysis it was found that a number of the subjects showed significant changes in corneal topography as accommodation changed. However further analysis showed a significant group mean excyclotorsion of the topography maps for both accommodation stimuli compared with the 0 D stimulus. When the excyclotorsion was accounted for, no clear evidence of statistically significant changes in corneal topography as a result of accommodation were found. A small ocular excyclotorsion typically accompanies accommodation and this changes the relative orientation of the topography of the cornea. To investigate the effects of eyelid pressure on corneal shape and corneal aberrations during reading, twenty young subjects with normal ocular health were recruited. Cornea1 topography of one eye was measured with a videokeratoscope prior to reading and then again after a 60 minute reading task. Twelve of the twenty corneas showed significant changes in central topography immediately following reading. The location of the changes corresponded closely to the position and angle of the subject's eyelids during reading. Within the central region of the cornea there were significant changes in corneal wavefront Zernike coefficients, the root-mean-square error, overall refractive power and astigmatism. The changes observed in corneal topography appear to be directly related to the force exerted by the eyelids during reading. These findings may have important implications for the definition of refractive status and may also aid in the understanding of the relationship between reading and the development of refractive errors. To study whether corneal distortions after reading significantly differ between refractive error groups, corneal aberrations were measured before and after a period of reading, for a group of ten young progressing myopes and a group of ten young stable emmetropes. The major difference between the two groups was the location and magnitude of the corneal distortions, which had a significantly larger effect on central corneal optics in the myopic group compared to the emmetropic group. A significantly smaller palpebral aperture for the myopic group in the reading gaze position was the cause of this difference. The experiments described in this thesis have shown that numerous corneal characteristics can change due to eyelid forces during near work. The eye was shown to undergo a small cyclotorsion during higher levels of accommodation. There was a shift in direction of against the rule astigmatism of the cornea following reading and a change was found for primary vertical coma and trefoil. The changes in corneal shape following reading appear to be different in myope versus emmetropic refractive error groups. These findings are important for our understanding of the stability of the refractive error of the eye and could have important implications for refractive error development.

The human eye suffers various optical aberrations that degrade the retinal image. These aberrations include defocus and astigmatism, as well as the higher order aberrations that also play an important role in our vision. The optics of the eye are not static, but are continuously fluctuating. The work reported in this thesis has studied the nature of the microfluctuations of the wavefront aberrations of the eye and has investigated factors that influence the microfluctuations. The fluctuations in the ocular surface of the eye were investigated using high speed videokeratoscopy which measures the dynamics of the ocular surface topography. Ocular surface height difference maps were computed to illustrate the changes in the tear film in the inter-blink interval. The videokeratoscopy data was used to derive the ocular surface wavefront aberrations up to the 4th radial order of the Zernike polynomial expension. We examined the ocular surface dynamics and temporal changes in the ocular surface wavefront aberrations in the inter-blink interval. During the first 0.5 sec following a blink, the tear thickness at the upper edge of the topography map appeared to thicken by about 2 microns. The influence of pulse and instantaneous pulse rate on the microfluctuations in the corneal wavefront aberrations was also investigated. The fluctuations in ocular surface wavefront aberrations were found to be uncorrelated with the pulse and instantaneous heart rates. In the clinical measurement of the ocular surface topography using videokeratoscopy, capturing images 2 to 3 seconds after a blink will result in more consistent results. To investigate fluctuations in the wavefront aberrations of the eye and their relation to pulse and respiration frequencies we used a wavefront sensor to measure the dynamics of the aberrations up to the Zernike polynomial 4th radial order. Simultaneously, the subject's pulse rate was measured, from which the instantaneous heart rate was derived. An auto-regressive process was used to derive the power spectra of the Zernike aberration signals, as well as pulse and instantaneous heart rate signals. Linear regression analysis was performed between the frequency components of Zernike aberrations and the pulse and instantaneous heart rate frequencies. Cross spectrum density and coherence analyses were also applied to investigate the relation between fluctuations of wavefront aberrations and pulse and instantaneous heart rate. The correlations between fluctuations of individual Zernike aberrations were also determined. A frequency component of all Zernike aberrations up to the 4th radial order was found to be significantly correlated with the pulse frequency (all > 2R0.51, p<0.02), and a frequency component of 9 out of 12 Zernike aberrations was also significantly correlated with instantaneous heart rate frequency (all>2R0.46, p<0.05). The major correlations among Zernike aberrations occurred between second order and fourth order aberrations with the same angular frequencies. Higher order aberrations appear to be related to the cardiopulmonary system in a similar way to that reported for the accommodation signal and pupil fluctuations. A wavefront sensor and high speed videokeratoscopy were used to investigate the contribution of the ocular surface, the effect of stimulus vergence, and refractive error on the microfluctuations of the wavefront aberrations of the eye. The fluctuations of the Zernike wavefront aberrations were quantified by their variations around the mean and using power spectrum analysis. Integrated power was determined in two regions: 0.1 Hz ─ 0.7 Hz (low frequencies) and 0.8 Hz ─ 1.8 Hz (high frequencies). Changes in the ocular surface topography were measured using high speed videokeratoscopy and variations in the ocular wavefront aberrations were calculated. The microfluctuations of wavefront aberrations in the ocular surface were found to be small compared with the microfluctuations of the wavefront aberrations in the total eye. The variations in defocus while viewing a closer target at 2 D and 4 D stimulus vergence were found to be significantly greater than variations in defocus when viewing a far target. This increase in defocus fluctuations occurred in both the low and high frequency regions (all p<0.001) of the power spectra. The microfluctuations in astigmatism and most of the 3rd order and 4th order Zernike wavefront aberrations of the total eye were found to significantly increase with the magnitude of myopia. The experiments reported in this thesis have demonstrated the characteristics of the microfluctuations of the wavefront aberrations of the eye and have shown some of the factors that can influence the fluctuations. Major fluctuation frequencies of the eye's wavefront aberrations were shown to be significantly correlated with the pulse and instantaneous heart rate frequencies. Fluctuations in the ocular surface wavefront aberrations made a small contribution to those of the total eye. Changing stimulus vergence primarily affected the fluctuations of defocus in both low and high frequency components. Variations in astigmatism and most 3rd and 4th order aberrations were associated with refractive error magnitude. These findings will aid our fundamental understanding of the complex visual optics of the human eye and may allow the opportunity for better dynamic correction of the aberrations with adaptive optics.

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 15, 2008). Includes bibliographical references (p. 33-37).

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.